Colloidosome like structures: self-assembly of silica microrods

DOE PAGES

Datskos, P.; Polizos, G.; Bhandari, M.; ...

2016-03-07

Self-assembly of one-dimensional structures is attracting a great deal of interest because assembled structures can provide better properties compared to individual building blocks. We demonstrate silica microrod self-assembly by exploiting Pickering emulsion based strategy. Micron-sized silica rods were synthesized employing previously reported methods based on polyvinylpyrrolidone/ pentanol emulsion droplets. Moreover, rods self-assembled to make structures in the range of z10 40 mm. Smooth rods assembled better than segmented rods. Finally, the assembled structures were bonded by weak van der Waals forces.

Self-assembly of tetravalent Goldberg polyhedra from 144 small components

NASA Astrophysics Data System (ADS)

Fujita, Daishi; Ueda, Yoshihiro; Sato, Sota; Mizuno, Nobuhiro; Kumasaka, Takashi; Fujita, Makoto

2016-12-01

Rational control of the self-assembly of large structures is one of the key challenges in chemistry, and is believed to become increasingly difficult and ultimately impossible as the number of components involved increases. So far, it has not been possible to design a self-assembled discrete molecule made up of more than 100 components. Such molecules—for example, spherical virus capsids—are prevalent in nature, which suggests that the difficulty in designing these very large self-assembled molecules is due to a lack of understanding of the underlying design principles. For example, the targeted assembly of a series of large spherical structures containing up to 30 palladium ions coordinated by up to 60 bent organic ligands was achieved by considering their topologies. Here we report the self-assembly of a spherical structure that also contains 30 palladium ions and 60 bent ligands, but belongs to a shape family that has not previously been observed experimentally. The new structure consists of a combination of 8 triangles and 24 squares, and has the symmetry of a tetravalent Goldberg polyhedron. Platonic and Archimedean solids have previously been prepared through self-assembly, as have trivalent Goldberg polyhedra, which occur naturally in the form of virus capsids and fullerenes. But tetravalent Goldberg polyhedra have not previously been reported at the molecular level, although their topologies have been predicted using graph theory. We use graph theory to predict the self-assembly of even larger tetravalent Goldberg polyhedra, which should be more stable, enabling another member of this polyhedron family to be assembled from 144 components: 48 palladium ions and 96 bent ligands.

Glia initiate brain assembly through non-canonical Chimaerin/Furin axon guidance in C. elegans

PubMed Central

Rapti, Georgia; Li, Chang; Shan, Alan; Lu, Yun; Shaham, Shai

2017-01-01

Brain assembly is hypothesized to begin when pioneer axons extend over non-neuronal cells, forming tracts guiding follower axons. Yet pioneer-neuron identities, their guidance substrates, and their interactions, are not well understood. Here, using time-lapse embryonic imaging, genetics, protein-interaction, and functional studies, we uncover the early events of C. elegans brain assembly. We demonstrate that C. elegans glia are key for assembly initiation, guiding pioneer and follower axons using distinct signals. Pioneer sublateral neurons, with unique growth properties, anatomy, and innervation, cooperate with glia to mediate follower-axon guidance. We further identify a CHIN-1/Chimaerin-KPC-1/Furin double mutant that severely disrupts assembly. CHIN-1/Chimaerin and KPC-1/Furin function non-canonically in glia and pioneer neurons for guidance-cue trafficking. We exploit this bottleneck to define roles for glial Netrin and Semaphorin in pioneer- and follower-axon guidance, respectively, and for glial and pioneer-neuron Flamingo/CELSR in follower-axon navigation. Altogether, our studies reveal previously-unknown glial roles in pioneer-axon guidance, suggesting conserved brain-assembly principles. PMID:28846083

ATP-dependent human RISC assembly pathways.

PubMed

Yoda, Mayuko; Kawamata, Tomoko; Paroo, Zain; Ye, Xuecheng; Iwasaki, Shintaro; Liu, Qinghua; Tomari, Yukihide

2010-01-01

The assembly of RNA-induced silencing complex (RISC) is a key process in small RNA-mediated gene silencing. In humans, small interfering RNAs (siRNAs) and microRNAs (miRNAs) are incorporated into RISCs containing the Argonaute (AGO) subfamily proteins Ago1-4. Previous studies have proposed that, unlike Drosophila melanogaster RISC assembly pathways, human RISC assembly is coupled with dicing and is independent of ATP. Here we show by careful reexamination that, in humans, RISC assembly and dicing are uncoupled, and ATP greatly facilitates RISC loading of small-RNA duplexes. Moreover, all four human AGO proteins show remarkably similar structural preferences for small-RNA duplexes: central mismatches promote RISC loading, and seed or 3'-mid (guide position 12-15) mismatches facilitate unwinding. All these features of human AGO proteins are highly reminiscent of fly Ago1 but not fly Ago2.

The Autographa californica Multiple Nucleopolyhedrovirus ac83 Gene Contains a cis-Acting Element That Is Essential for Nucleocapsid Assembly.

PubMed

Huang, Zhihong; Pan, Mengjia; Zhu, Silei; Zhang, Hao; Wu, Wenbi; Yuan, Meijin; Yang, Kai

2017-03-01

Baculoviridae is a family of insect-specific viruses that have a circular double-stranded DNA genome packaged within a rod-shaped capsid. The mechanism of baculovirus nucleocapsid assembly remains unclear. Previous studies have shown that deletion of the ac83 gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) blocks viral nucleocapsid assembly. Interestingly, the ac83 -encoded protein Ac83 is not a component of the nucleocapsid, implying a particular role for ac83 in nucleocapsid assembly that may be independent of its protein product. To examine this possibility, Ac83 synthesis was disrupted by insertion of a chloramphenicol resistance gene into its coding sequence or by deleting its promoter and translation start codon. Both mutants produced progeny viruses normally, indicating that the Ac83 protein is not required for nucleocapsid assembly. Subsequently, complementation assays showed that the production of progeny viruses required the presence of ac83 in the AcMNPV genome instead of its presence in trans Therefore, we reasoned that ac83 is involved in nucleocapsid assembly via an internal cis -acting element, which we named the nucleocapsid assembly-essential element (NAE). The NAE was identified to lie within nucleotides 1651 to 1850 of ac83 and had 8 conserved A/T-rich regions. Sequences homologous to the NAE were found only in alphabaculoviruses and have a conserved positional relationship with another essential cis -acting element that was recently identified. The identification of the NAE may help to connect the data of viral cis -acting elements and related proteins in the baculovirus nucleocapsid assembly, which is important for elucidating DNA-protein interaction events during this process. IMPORTANCE Virus nucleocapsid assembly usually requires specific cis -acting elements in the viral genome for various processes, such as the selection of the viral genome from the cellular nucleic acids, the cleavage of concatemeric viral genome replication intermediates, and the encapsidation of the viral genome into procapsids. In linear DNA viruses, such elements generally locate at the ends of the viral genome; however, most of these elements remain unidentified in circular DNA viruses (including baculovirus) due to their circular genomic conformation. Here, we identified a nucleocapsid assembly-essential element in the AcMNPV (the archetype of baculovirus) genome. This finding provides an important reference for studies of nucleocapsid assembly-related elements in baculoviruses and other circular DNA viruses. Moreover, as most of the previous studies of baculovirus nucleocapsid assembly have been focused on viral proteins, our study provides a novel entry point to investigate this mechanism via cis -acting elements in the viral genome. Copyright © 2017 American Society for Microbiology.

Solving Assembly Sequence Planning using Angle Modulated Simulated Kalman Filter

NASA Astrophysics Data System (ADS)

Mustapa, Ainizar; Yusof, Zulkifli Md.; Adam, Asrul; Muhammad, Badaruddin; Ibrahim, Zuwairie

2018-03-01

This paper presents an implementation of Simulated Kalman Filter (SKF) algorithm for optimizing an Assembly Sequence Planning (ASP) problem. The SKF search strategy contains three simple steps; predict-measure-estimate. The main objective of the ASP is to determine the sequence of component installation to shorten assembly time or save assembly costs. Initially, permutation sequence is generated to represent each agent. Each agent is then subjected to a precedence matrix constraint to produce feasible assembly sequence. Next, the Angle Modulated SKF (AMSKF) is proposed for solving ASP problem. The main idea of the angle modulated approach in solving combinatorial optimization problem is to use a function, g(x), to create a continuous signal. The performance of the proposed AMSKF is compared against previous works in solving ASP by applying BGSA, BPSO, and MSPSO. Using a case study of ASP, the results show that AMSKF outperformed all the algorithms in obtaining the best solution.

A KiDS weak lensing analysis of assembly bias in GAMA galaxy groups

NASA Astrophysics Data System (ADS)

Dvornik, Andrej; Cacciato, Marcello; Kuijken, Konrad; Viola, Massimo; Hoekstra, Henk; Nakajima, Reiko; van Uitert, Edo; Brouwer, Margot; Choi, Ami; Erben, Thomas; Fenech Conti, Ian; Farrow, Daniel J.; Herbonnet, Ricardo; Heymans, Catherine; Hildebrandt, Hendrik; Hopkins, Andrew M.; McFarland, John; Norberg, Peder; Schneider, Peter; Sifón, Cristóbal; Valentijn, Edwin; Wang, Lingyu

2017-07-01

We investigate possible signatures of halo assembly bias for spectroscopically selected galaxy groups from the Galaxy And Mass Assembly (GAMA) survey using weak lensing measurements from the spatially overlapping regions of the deeper, high-imaging-quality photometric Kilo-Degree Survey. We use GAMA groups with an apparent richness larger than 4 to identify samples with comparable mean host halo masses but with a different radial distribution of satellite galaxies, which is a proxy for the formation time of the haloes. We measure the weak lensing signal for groups with a steeper than average and with a shallower than average satellite distribution and find no sign of halo assembly bias, with the bias ratio of 0.85^{+0.37}_{-0.25}, which is consistent with the Λ cold dark matter prediction. Our galaxy groups have typical masses of 1013 M⊙ h-1, naturally complementing previous studies of halo assembly bias on galaxy cluster scales.

Quantitative characterization of all single amino acid variants of a viral capsid-based drug delivery vehicle.

PubMed

Hartman, Emily C; Jakobson, Christopher M; Favor, Andrew H; Lobba, Marco J; Álvarez-Benedicto, Ester; Francis, Matthew B; Tullman-Ercek, Danielle

2018-04-11

Self-assembling proteins are critical to biological systems and industrial technologies, but predicting how mutations affect self-assembly remains a significant challenge. Here, we report a technique, termed SyMAPS (Systematic Mutation and Assembled Particle Selection), that can be used to characterize the assembly competency of all single amino acid variants of a self-assembling viral structural protein. SyMAPS studies on the MS2 bacteriophage coat protein revealed a high-resolution fitness landscape that challenges some conventional assumptions of protein engineering. An additional round of selection identified a previously unknown variant (CP[T71H]) that is stable at neutral pH but less tolerant to acidic conditions than the wild-type coat protein. The capsids formed by this variant could be more amenable to disassembly in late endosomes or early lysosomes-a feature that is advantageous for delivery applications. In addition to providing a mutability blueprint for virus-like particles, SyMAPS can be readily applied to other self-assembling proteins.

Assembling the Setaria italica L. Beauv. genome into nine chromosomes and insights into regions affecting growth and drought tolerance

PubMed Central

Tsai, Kevin J.; Lu, Mei-Yeh Jade; Yang, Kai-Jung; Li, Mengyun; Teng, Yuchuan; Chen, Shihmay; Ku, Maurice S. B.; Li, Wen-Hsiung

2016-01-01

The diploid C4 plant foxtail millet (Setaria italica L. Beauv.) is an important crop in many parts of Africa and Asia for the vast consumption of its grain and ability to grow in harsh environments, but remains understudied in terms of complete genomic architecture. To date, there have been only two genome assembly and annotation efforts with neither assembly reaching over 86% of the estimated genome size. We have combined de novo assembly with custom reference-guided improvements on a popular cultivar of foxtail millet and have achieved a genome assembly of 477 Mbp in length, which represents over 97% of the estimated 490 Mbp. The assembly anchors over 98% of the predicted genes to the nine assembled nuclear chromosomes and contains more functional annotation gene models than previous assemblies. Our annotation has identified a large number of unique gene ontology terms related to metabolic activities, a region of chromosome 9 with several growth factor proteins, and regions syntenic with pearl millet or maize genomic regions that have been previously shown to affect growth. The new assembly and annotation for this important species can be used for detailed investigation and future innovations in growth for millet and other grains. PMID:27734962

Assembling the Setaria italica L. Beauv. genome into nine chromosomes and insights into regions affecting growth and drought tolerance.

PubMed

Tsai, Kevin J; Lu, Mei-Yeh Jade; Yang, Kai-Jung; Li, Mengyun; Teng, Yuchuan; Chen, Shihmay; Ku, Maurice S B; Li, Wen-Hsiung

2016-10-13

The diploid C 4 plant foxtail millet (Setaria italica L. Beauv.) is an important crop in many parts of Africa and Asia for the vast consumption of its grain and ability to grow in harsh environments, but remains understudied in terms of complete genomic architecture. To date, there have been only two genome assembly and annotation efforts with neither assembly reaching over 86% of the estimated genome size. We have combined de novo assembly with custom reference-guided improvements on a popular cultivar of foxtail millet and have achieved a genome assembly of 477 Mbp in length, which represents over 97% of the estimated 490 Mbp. The assembly anchors over 98% of the predicted genes to the nine assembled nuclear chromosomes and contains more functional annotation gene models than previous assemblies. Our annotation has identified a large number of unique gene ontology terms related to metabolic activities, a region of chromosome 9 with several growth factor proteins, and regions syntenic with pearl millet or maize genomic regions that have been previously shown to affect growth. The new assembly and annotation for this important species can be used for detailed investigation and future innovations in growth for millet and other grains.

Space Station Freedom assembly and operation at a 51.6 degree inclination orbit

NASA Technical Reports Server (NTRS)

Troutman, Patrick A.; Brewer, Laura M.; Heck, Michael L.; Kumar, Renjith R.

1993-01-01

This study examines the implications of assembling and operating Space Station Freedom at a 51.6 degree inclination orbit utilizing an enhanced lift Space Shuttle. Freedom assembly is currently baselined at a 220 nautical mile high, 28.5 degree inclination orbit. Some of the reasons for increasing the orbital inclination are (1) increased ground coverage for Earth observations, (2) greater accessibility from Russian and other international launch sites, and (3) increased number of Assured Crew Return Vehicle (ACRV) landing sites. Previous studies have looked at assembling Freedom at a higher inclination using both medium and heavy lift expendable launch vehicles (such as Shuttle-C and Energia). The study assumes that the shuttle is used exclusively for delivering the station to orbit and that it can gain additional payload capability from design changes such as a lighter external tank that somewhat offsets the performance decrease that occurs when the shuttle is launched to a 51.6 degree inclination orbit.

Architecture and assembly of the Bacillus subtilis spore coat.

PubMed

Plomp, Marco; Carroll, Alicia Monroe; Setlow, Peter; Malkin, Alexander J

2014-01-01

Bacillus spores are encased in a multilayer, proteinaceous self-assembled coat structure that assists in protecting the bacterial genome from stresses and consists of at least 70 proteins. The elucidation of Bacillus spore coat assembly, architecture, and function is critical to determining mechanisms of spore pathogenesis, environmental resistance, immune response, and physicochemical properties. Recently, genetic, biochemical and microscopy methods have provided new insight into spore coat architecture, assembly, structure and function. However, detailed spore coat architecture and assembly, comprehensive understanding of the proteomic composition of coat layers, and specific roles of coat proteins in coat assembly and their precise localization within the coat remain in question. In this study, atomic force microscopy was used to probe the coat structure of Bacillus subtilis wild type and cotA, cotB, safA, cotH, cotO, cotE, gerE, and cotE gerE spores. This approach provided high-resolution visualization of the various spore coat structures, new insight into the function of specific coat proteins, and enabled the development of a detailed model of spore coat architecture. This model is consistent with a recently reported four-layer coat assembly and further adds several coat layers not reported previously. The coat is organized starting from the outside into an outermost amorphous (crust) layer, a rodlet layer, a honeycomb layer, a fibrous layer, a layer of "nanodot" particles, a multilayer assembly, and finally the undercoat/basement layer. We propose that the assembly of the previously unreported fibrous layer, which we link to the darkly stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose that the inner coat multilayer structure is crystalline with its apparent two-dimensional (2D) nuclei being the first example of a non-mineral 2D nucleation crystallization pattern in a biological organism.

Architecture and Assembly of the Bacillus subtilis Spore Coat

PubMed Central

Plomp, Marco; Carroll, Alicia Monroe; Setlow, Peter; Malkin, Alexander J.

2014-01-01

Bacillus spores are encased in a multilayer, proteinaceous self-assembled coat structure that assists in protecting the bacterial genome from stresses and consists of at least 70 proteins. The elucidation of Bacillus spore coat assembly, architecture, and function is critical to determining mechanisms of spore pathogenesis, environmental resistance, immune response, and physicochemical properties. Recently, genetic, biochemical and microscopy methods have provided new insight into spore coat architecture, assembly, structure and function. However, detailed spore coat architecture and assembly, comprehensive understanding of the proteomic composition of coat layers, and specific roles of coat proteins in coat assembly and their precise localization within the coat remain in question. In this study, atomic force microscopy was used to probe the coat structure of Bacillus subtilis wild type and cotA, cotB, safA, cotH, cotO, cotE, gerE, and cotE gerE spores. This approach provided high-resolution visualization of the various spore coat structures, new insight into the function of specific coat proteins, and enabled the development of a detailed model of spore coat architecture. This model is consistent with a recently reported four-layer coat assembly and further adds several coat layers not reported previously. The coat is organized starting from the outside into an outermost amorphous (crust) layer, a rodlet layer, a honeycomb layer, a fibrous layer, a layer of “nanodot” particles, a multilayer assembly, and finally the undercoat/basement layer. We propose that the assembly of the previously unreported fibrous layer, which we link to the darkly stained outer coat seen by electron microscopy, and the nanodot layer are cotH- and cotE- dependent and cotE-specific respectively. We further propose that the inner coat multilayer structure is crystalline with its apparent two-dimensional (2D) nuclei being the first example of a non-mineral 2D nucleation crystallization pattern in a biological organism. PMID:25259857

Fatigue FEM analysis in the case of brazed aluminium alloy 3L59 used in aeronautical industry

NASA Astrophysics Data System (ADS)

Dimitrescu, A.; Amza, Gh; Niţoi, D. F.; Amza, C. Gh; Apostolescu, Z.

2016-08-01

The use, on a larger scale, of brazed aluminum alloys in the aerospace industry led to the need for a detailed study of the assemblies behavior. These are built from 6061 aluminum aloy (3L59) brazed with aluminum aloy A103. Therefore, a finit element simulation (FEM) of durability is necessary, that consists in the observation of gradual deterioration until failure. These studies are required and are previous to the stage of the producing the assembly and test it by traditional methods.

Improvement of the Threespine Stickleback Genome Using a Hi-C-Based Proximity-Guided Assembly.

PubMed

Peichel, Catherine L; Sullivan, Shawn T; Liachko, Ivan; White, Michael A

2017-09-01

Scaffolding genomes into complete chromosome assemblies remains challenging even with the rapidly increasing sequence coverage generated by current next-generation sequence technologies. Even with scaffolding information, many genome assemblies remain incomplete. The genome of the threespine stickleback (Gasterosteus aculeatus), a fish model system in evolutionary genetics and genomics, is not completely assembled despite scaffolding with high-density linkage maps. Here, we first test the ability of a Hi-C based proximity-guided assembly (PGA) to perform a de novo genome assembly from relatively short contigs. Using Hi-C based PGA, we generated complete chromosome assemblies from a distribution of short contigs (20-100 kb). We found that 96.40% of contigs were correctly assigned to linkage groups (LGs), with ordering nearly identical to the previous genome assembly. Using available bacterial artificial chromosome (BAC) end sequences, we provide evidence that some of the few discrepancies between the Hi-C assembly and the existing assembly are due to structural variation between the populations used for the 2 assemblies or errors in the existing assembly. This Hi-C assembly also allowed us to improve the existing assembly, assigning over 60% (13.35 Mb) of the previously unassigned (~21.7 Mb) contigs to LGs. Together, our results highlight the potential of the Hi-C based PGA method to be used in combination with short read data to perform relatively inexpensive de novo genome assemblies. This approach will be particularly useful in organisms in which it is difficult to perform linkage mapping or to obtain high molecular weight DNA required for other scaffolding methods. © The American Genetic Association 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Oral microbial community assembly under the influence of periodontitis.

PubMed

Chen, Hongju; Peng, Shuting; Dai, Lin; Zou, Quan; Yi, Bin; Yang, Xianghong; Ma, Zhanshan Sam

2017-01-01

Several ecological hypotheses (e.g., specific plaque, non-specific plaque and keystone pathogen) regarding the etiology of periodontitis have been proposed since the 1990s, most of which have been centered on the concept of dysbiosis associated with periodontitis. Nevertheless, none of the existing hypotheses have presented mechanistic interpretations on how and why dysbiosis actually occurs. Hubbell's neutral theory of biodiversity offers a powerful null model to test hypothesis regarding the mechanism of community assembly and diversity maintenance from the metagenomic sequencing data, which can help to understand the forces that shape the community dynamics such as dysbiosis. Here we reanalyze the dataset from Abusleme et al.'s comparative study of the oral microbial communities from periodontitis patients and healthy individuals. Our study demonstrates that 14 out of 61 communities (23%) passed the neutrality test, a percentage significantly higher than the previous reported neutrality rate of 1% in human microbiome (Li & Ma 2016, Scientific Reports). This suggests that, while the niche selection may play a predominant role in the assembly and diversity maintenance in oral microbiome, the effect of neutral dynamics may not be ignored. However, no statistically significant differences in the neutrality passing rates were detected between the periodontitis and healthy treatments with Fisher's exact probability test and multiple testing corrections, suggesting that the mechanism of community assembly is robust against disturbances such as periodontitis. In addition, our study confirmed previous finding that periodontitis patients exhibited higher biodiversity. These findings suggest that while periodontitis may significantly change the community composition measured by diversity (i.e., the exhibition or 'phenotype' of community assembly), it does not seem to cause the 'mutation' of the 'genotype" (mechanism) of community assembly. We argue that the 'phenotypic' changes explain the observed link (not necessarily causal) between periodontitis and community dysbiosis, which is certainly worthy of further investigation.

Oral microbial community assembly under the influence of periodontitis

PubMed Central

Chen, Hongju; Peng, Shuting; Dai, Lin; Zou, Quan; Yi, Bin; Yang, Xianghong

2017-01-01

Several ecological hypotheses (e.g., specific plaque, non-specific plaque and keystone pathogen) regarding the etiology of periodontitis have been proposed since the 1990s, most of which have been centered on the concept of dysbiosis associated with periodontitis. Nevertheless, none of the existing hypotheses have presented mechanistic interpretations on how and why dysbiosis actually occurs. Hubbell’s neutral theory of biodiversity offers a powerful null model to test hypothesis regarding the mechanism of community assembly and diversity maintenance from the metagenomic sequencing data, which can help to understand the forces that shape the community dynamics such as dysbiosis. Here we reanalyze the dataset from Abusleme et al.’s comparative study of the oral microbial communities from periodontitis patients and healthy individuals. Our study demonstrates that 14 out of 61 communities (23%) passed the neutrality test, a percentage significantly higher than the previous reported neutrality rate of 1% in human microbiome (Li & Ma 2016, Scientific Reports). This suggests that, while the niche selection may play a predominant role in the assembly and diversity maintenance in oral microbiome, the effect of neutral dynamics may not be ignored. However, no statistically significant differences in the neutrality passing rates were detected between the periodontitis and healthy treatments with Fisher’s exact probability test and multiple testing corrections, suggesting that the mechanism of community assembly is robust against disturbances such as periodontitis. In addition, our study confirmed previous finding that periodontitis patients exhibited higher biodiversity. These findings suggest that while periodontitis may significantly change the community composition measured by diversity (i.e., the exhibition or ‘phenotype’ of community assembly), it does not seem to cause the ‘mutation’ of the ‘genotype” (mechanism) of community assembly. We argue that the ‘phenotypic’ changes explain the observed link (not necessarily causal) between periodontitis and community dysbiosis, which is certainly worthy of further investigation. PMID:28813450

Transcriptome Assembly, Gene Annotation and Tissue Gene Expression Atlas of the Rainbow Trout

PubMed Central

Salem, Mohamed; Paneru, Bam; Al-Tobasei, Rafet; Abdouni, Fatima; Thorgaard, Gary H.; Rexroad, Caird E.; Yao, Jianbo

2015-01-01

Efforts to obtain a comprehensive genome sequence for rainbow trout are ongoing and will be complemented by transcriptome information that will enhance genome assembly and annotation. Previously, transcriptome reference sequences were reported using data from different sources. Although the previous work added a great wealth of sequences, a complete and well-annotated transcriptome is still needed. In addition, gene expression in different tissues was not completely addressed in the previous studies. In this study, non-normalized cDNA libraries were sequenced from 13 different tissues of a single doubled haploid rainbow trout from the same source used for the rainbow trout genome sequence. A total of ~1.167 billion paired-end reads were de novo assembled using the Trinity RNA-Seq assembler yielding 474,524 contigs > 500 base-pairs. Of them, 287,593 had homologies to the NCBI non-redundant protein database. The longest contig of each cluster was selected as a reference, yielding 44,990 representative contigs. A total of 4,146 contigs (9.2%), including 710 full-length sequences, did not match any mRNA sequences in the current rainbow trout genome reference. Mapping reads to the reference genome identified an additional 11,843 transcripts not annotated in the genome. A digital gene expression atlas revealed 7,678 housekeeping and 4,021 tissue-specific genes. Expression of about 16,000–32,000 genes (35–71% of the identified genes) accounted for basic and specialized functions of each tissue. White muscle and stomach had the least complex transcriptomes, with high percentages of their total mRNA contributed by a small number of genes. Brain, testis and intestine, in contrast, had complex transcriptomes, with a large numbers of genes involved in their expression patterns. This study provides comprehensive de novo transcriptome information that is suitable for functional and comparative genomics studies in rainbow trout, including annotation of the genome. PMID:25793877

A high quality assembly of the Nile Tilapia (Oreochromis niloticus) genome reveals the structure of two sex determination regions.

PubMed

Conte, Matthew A; Gammerdinger, William J; Bartie, Kerry L; Penman, David J; Kocher, Thomas D

2017-05-02

Tilapias are the second most farmed fishes in the world and a sustainable source of food. Like many other fish, tilapias are sexually dimorphic and sex is a commercially important trait in these fish. In this study, we developed a significantly improved assembly of the tilapia genome using the latest genome sequencing methods and show how it improves the characterization of two sex determination regions in two tilapia species. A homozygous clonal XX female Nile tilapia (Oreochromis niloticus) was sequenced to 44X coverage using Pacific Biosciences (PacBio) SMRT sequencing. Dozens of candidate de novo assemblies were generated and an optimal assembly (contig NG50 of 3.3Mbp) was selected using principal component analysis of likelihood scores calculated from several paired-end sequencing libraries. Comparison of the new assembly to the previous O. niloticus genome assembly reveals that recently duplicated portions of the genome are now well represented. The overall number of genes in the new assembly increased by 27.3%, including a 67% increase in pseudogenes. The new tilapia genome assembly correctly represents two recent vasa gene duplication events that have been verified with BAC sequencing. At total of 146Mbp of additional transposable element sequence are now assembled, a large proportion of which are recent insertions. Large centromeric satellite repeats are assembled and annotated in cichlid fish for the first time. Finally, the new assembly identifies the long-range structure of both a ~9Mbp XY sex determination region on LG1 in O. niloticus, and a ~50Mbp WZ sex determination region on LG3 in the related species O. aureus. This study highlights the use of long read sequencing to correctly assemble recent duplications and to characterize repeat-filled regions of the genome. The study serves as an example of the need for high quality genome assemblies and provides a framework for identifying sex determining genes in tilapia and related fish species.

Roles of Gag-RNA interactions in HIV-1 virus assembly deciphered by single-molecule localization microscopy.

PubMed

Yang, Yantao; Qu, Na; Tan, Jie; Rushdi, Muaz N; Krueger, Christopher J; Chen, Antony K

2018-06-11

During HIV-1 assembly, the retroviral structural protein Gag forms an immature capsid, containing thousands of Gag molecules, at the plasma membrane (PM). Interactions between Gag nucleocapsid (NC) and viral RNA (vRNA) are thought to drive assembly, but the exact roles of these interactions have remained poorly understood. Since previous studies have shown that Gag dimer- or trimer-forming mutants (Gag ZiL ) lacking an NC domain can form immature capsids independent of RNA binding, it is often hypothesized that vRNA drives Gag assembly by inducing Gag to form low-ordered multimers, but is dispensable for subsequent assembly. In this study, we examined the role of vRNA in HIV-1 assembly by characterizing the distribution and mobility of Gag and Gag NC mutants at the PM using photoactivated localization microscopy (PALM) and single-particle tracking PALM (spt-PALM). We showed that both Gag and Gag ZiL assembly involve a similar basic assembly unit, as expected. Unexpectedly, the two proteins underwent different subsequent assembly pathways, with Gag cluster density increasing asymptotically, while Gag ZiL cluster density increased linearly. Additionally, the directed movement of Gag, but not Gag ZiL , was maintained at a constant speed, suggesting that the two proteins experience different external driving forces. Assembly was abolished when Gag was rendered monomeric by NC deletion. Collectively, these results suggest that, beyond inducing Gag to form low-ordered multimer basic assembly units, vRNA is essential in scaffolding and maintaining the stability of the subsequent assembly process. This finding should advance the current understanding of HIV-1 and, potentially, other retroviruses. Copyright © 2018 the Author(s). Published by PNAS.

Essential Assembly Factor Rpf2 Forms Novel Interactions within the 5S RNP in Trypanosoma brucei

PubMed Central

Kamina, Anyango D.; Jaremko, Daniel; Christen, Linda

2017-01-01

ABSTRACT Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in Trypanosoma brucei, the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the T. brucei homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. T. brucei Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the T. brucei 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T. brucei. IMPORTANCE Trypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the regulatory checkpoints during ribosome biogenesis. We have previously characterized the 5S RNP in T. brucei and showed that trypanosome-specific proteins P34 and P37 are part of this complex. In this study, we characterize for the first time the interactions of the homolog of the assembly factor Rpf2 with members of the 5S RNP in another organism besides fungi. Our studies show that Rpf2 is essential in T. brucei and that it forms unique interactions within the 5S RNP, particularly with P34 and P37. These studies have identified parasite-specific interactions that can potentially function as new therapeutic targets against sleeping sickness. PMID:29062898

Essential Assembly Factor Rpf2 Forms Novel Interactions within the 5S RNP in Trypanosoma brucei.

PubMed

Kamina, Anyango D; Jaremko, Daniel; Christen, Linda; Williams, Noreen

2017-01-01

Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in Trypanosoma brucei , the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the T. brucei homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. T . brucei Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the T. brucei 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in T. brucei . IMPORTANCE Trypanosoma brucei is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the regulatory checkpoints during ribosome biogenesis. We have previously characterized the 5S RNP in T. brucei and showed that trypanosome-specific proteins P34 and P37 are part of this complex. In this study, we characterize for the first time the interactions of the homolog of the assembly factor Rpf2 with members of the 5S RNP in another organism besides fungi. Our studies show that Rpf2 is essential in T. brucei and that it forms unique interactions within the 5S RNP, particularly with P34 and P37. These studies have identified parasite-specific interactions that can potentially function as new therapeutic targets against sleeping sickness.

Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly

PubMed Central

Cook, Jeremy D.; Kondapalli, Kalyan C.; Rawat, Swati; Childs, William C.; Murugesan, Yogapriya; Dancis, Andrew; Stemmler, Timothy L.

2010-01-01

Frataxin, a conserved nuclear encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich’s ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two: Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone n the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to better understand the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry (ITC). Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into a Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly. PMID:20815377

Genome assembly and annotation of Arabidopsis halleri, a model for heavy metal hyperaccumulation and evolutionary ecology.

PubMed

Briskine, Roman V; Paape, Timothy; Shimizu-Inatsugi, Rie; Nishiyama, Tomoaki; Akama, Satoru; Sese, Jun; Shimizu, Kentaro K

2017-09-01

The self-incompatible species Arabidopsis halleri is a close relative of the self-compatible model plant Arabidopsis thaliana. The broad European and Asian distribution and heavy metal hyperaccumulation ability make A. halleri a useful model for ecological genomics studies. We used long-insert mate-pair libraries to improve the genome assembly of the A. halleri ssp. gemmifera Tada mine genotype (W302) collected from a site with high contamination by heavy metals in Japan. After five rounds of forced selfing, heterozygosity was reduced to 0.04%, which facilitated subsequent genome assembly. Our assembly now covers 196 Mb or 78% of the estimated genome size and achieved scaffold N50 length of 712 kb. To validate assembly and annotation, we used synteny of A. halleri Tada mine with a previously published high-quality reference assembly of a closely related species, Arabidopsis lyrata. Further validation of the assembly quality comes from synteny and phylogenetic analysis of the HEAVY METAL ATPASE4 (HMA4) and METAL TOLERANCE PROTEIN1 (MTP1) regions using published sequences from European A. halleri for comparison. Three tandemly duplicated copies of HMA4, key gene involved in cadmium and zinc hyperaccumulation, were assembled on a single scaffold. The assembly will enhance the genomewide studies of A. halleri as well as the allopolyploid Arabidopsis kamchatica derived from A. lyrata and A. halleri. © 2016 The Authors. Molecular Ecology Resources Published by John Wiley & Sons Ltd.

Molecular Details of the Yeast Frataxin-Isu1 Interaction during Mitochondrial Fe-S Cluster Assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cook, J.; Kondapalli, K; Rawat, S

2010-01-01

Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural modulemore » to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.« less

Molecular details of the yeast frataxin-Isu1 interaction during mitochondrial Fe-S cluster assembly.

PubMed

Cook, Jeremy D; Kondapalli, Kalyan C; Rawat, Swati; Childs, William C; Murugesan, Yogapriya; Dancis, Andrew; Stemmler, Timothy L

2010-10-12

Frataxin, a conserved nuclear-encoded mitochondrial protein, plays a direct role in iron-sulfur cluster biosynthesis within the ISC assembly pathway. Humans with frataxin deficiency have Friedreich's ataxia, a neurodegenerative disorder characterized by mitochondrial iron overload and disruption in Fe-S cluster synthesis. Biochemical and genetic studies have shown frataxin interacts with the iron-sulfur cluster assembly scaffold protein (in yeast, there are two, Isu1 and Isu2), indicating frataxin plays a direct role in cluster assembly, possibly by serving as an iron chaperone in the assembly pathway. Here we provide molecular details of how yeast frataxin (Yfh1) interacts with Isu1 as a structural module to improve our understanding of the multiprotein complex assembly that completes Fe-S cluster assembly; this complex also includes the cysteine desulfurase (Nfs1 in yeast) and the accessory protein (Isd11), together in the mitochondria. Thermodynamic binding parameters for protein partner and iron binding were measured for the yeast orthologs using isothermal titration calorimetry. Nuclear magnetic resonance spectroscopy was used to provide the molecular details to understand how Yfh1 interacts with Isu1. X-ray absorption studies were used to electronically and structurally characterize how iron is transferred to Isu1 and then incorporated into an Fe-S cluster. These results were combined with previously published data to generate a structural model for how the Fe-S cluster protein assembly complex can come together to accomplish Fe-S cluster assembly.

A New Chicken Genome Assembly Provides Insight into Avian Genome Structure.

PubMed

Warren, Wesley C; Hillier, LaDeana W; Tomlinson, Chad; Minx, Patrick; Kremitzki, Milinn; Graves, Tina; Markovic, Chris; Bouk, Nathan; Pruitt, Kim D; Thibaud-Nissen, Francoise; Schneider, Valerie; Mansour, Tamer A; Brown, C Titus; Zimin, Aleksey; Hawken, Rachel; Abrahamsen, Mitch; Pyrkosz, Alexis B; Morisson, Mireille; Fillon, Valerie; Vignal, Alain; Chow, William; Howe, Kerstin; Fulton, Janet E; Miller, Marcia M; Lovell, Peter; Mello, Claudio V; Wirthlin, Morgan; Mason, Andrew S; Kuo, Richard; Burt, David W; Dodgson, Jerry B; Cheng, Hans H

2017-01-05

The importance of the Gallus gallus (chicken) as a model organism and agricultural animal merits a continuation of sequence assembly improvement efforts. We present a new version of the chicken genome assembly (Gallus_gallus-5.0; GCA_000002315.3), built from combined long single molecule sequencing technology, finished BACs, and improved physical maps. In overall assembled bases, we see a gain of 183 Mb, including 16.4 Mb in placed chromosomes with a corresponding gain in the percentage of intact repeat elements characterized. Of the 1.21 Gb genome, we include three previously missing autosomes, GGA30, 31, and 33, and improve sequence contig length 10-fold over the previous Gallus_gallus-4.0. Despite the significant base representation improvements made, 138 Mb of sequence is not yet located to chromosomes. When annotated for gene content, Gallus_gallus-5.0 shows an increase of 4679 annotated genes (2768 noncoding and 1911 protein-coding) over those in Gallus_gallus-4.0. We also revisited the question of what genes are missing in the avian lineage, as assessed by the highest quality avian genome assembly to date, and found that a large fraction of the original set of missing genes are still absent in sequenced bird species. Finally, our new data support a detailed map of MHC-B, encompassing two segments: one with a highly stable gene copy number and another in which the gene copy number is highly variable. The chicken model has been a critical resource for many other fields of study, and this new reference assembly will substantially further these efforts. Copyright © 2017 Warren et al.

Adaptation of a Filter Assembly to Assess Microbial Bioburden of Pressurant Within a Propulsion System

NASA Technical Reports Server (NTRS)

Benardini, James N.; Koukol, Robert C.; Schubert, Wayne W.; Morales, Fabian; Klatte, Marlin F.

2012-01-01

A report describes an adaptation of a filter assembly to enable it to be used to filter out microorganisms from a propulsion system. The filter assembly has previously been used for particulates greater than 2 micrometers. Projects that utilize large volumes of nonmetallic materials of planetary protection concern pose a challenge to their bioburden budget, as a conservative specification value of 30 spores per cubic centimeter is typically used. Helium was collected utilizing an adapted filtration approach employing an existing Millipore filter assembly apparatus used by the propulsion team for particulate analysis. The filter holder on the assembly has a 47-mm diameter, and typically a 1.2-5 micrometer pore-size filter is used for particulate analysis making it compatible with commercially available sterilization filters (0.22 micrometers) that are necessary for biological sampling. This adaptation to an existing technology provides a proof-of-concept and a demonstration of successful use in a ground equipment system. This adaptation has demonstrated that the Millipore filter assembly can be utilized to filter out microorganisms from a propulsion system, whereas in previous uses the filter assembly was utilized for particulates greater than 2 micrometers.

An anisotropic numerical model for thermal hydraulic analyses: application to liquid metal flow in fuel assemblies

NASA Astrophysics Data System (ADS)

Vitillo, F.; Vitale Di Maio, D.; Galati, C.; Caruso, G.

2015-11-01

A CFD analysis has been carried out to study the thermal-hydraulic behavior of liquid metal coolant in a fuel assembly of triangular lattice. In order to obtain fast and accurate results, the isotropic two-equation RANS approach is often used in nuclear engineering applications. A different approach is provided by Non-Linear Eddy Viscosity Models (NLEVM), which try to take into account anisotropic effects by a nonlinear formulation of the Reynolds stress tensor. This approach is very promising, as it results in a very good numerical behavior and in a potentially better fluid flow description than classical isotropic models. An Anisotropic Shear Stress Transport (ASST) model, implemented into a commercial software, has been applied in previous studies, showing very trustful results for a large variety of flows and applications. In the paper, the ASST model has been used to perform an analysis of the fluid flow inside the fuel assembly of the ALFRED lead cooled fast reactor. Then, a comparison between the results of wall-resolved conjugated heat transfer computations and the results of a decoupled analysis using a suitable thermal wall-function previously implemented into the solver has been performed and presented.

Huntingtin interacting protein 1 (HIP1) regulates clathrin assembly through direct binding to the regulatory region of the clathrin light chain.

PubMed

Legendre-Guillemin, Valerie; Metzler, Martina; Lemaire, Jean-Francois; Philie, Jacynthe; Gan, Lu; Hayden, Michael R; McPherson, Peter S

2005-02-18

Huntingtin interacting protein 1 (HIP1) is a component of clathrin coats. We previously demonstrated that HIP1 promotes clathrin assembly through its central helical domain, which binds directly to clathrin light chains (CLCs). To better understand the relationship between CLC binding and clathrin assembly we sought to dissect this interaction. Using C-terminal deletion constructs of the HIP1 helical domain, we identified a region between residues 450 and 456 that is required for CLC binding. Within this region, point mutations showed the importance of residues Leu-451, Leu-452, and Arg-453. Mutants that fail to bind CLC are unable to promote clathrin assembly in vitro but still mediate HIP1 homodimerization and heterodimerization with the family member HIP12/HIP1R. Moreover, HIP1 binding to CLC is necessary for HIP1 targeting to clathrin-coated pits and clathrin-coated vesicles. Interestingly, HIP1 binds to a highly conserved region of CLC previously demonstrated to regulate clathrin assembly. These results suggest a role for HIP1/CLC interactions in the regulation of clathrin assembly.

Assembly and diploid architecture of an individual human genome via single-molecule technologies

PubMed Central

Pendleton, Matthew; Sebra, Robert; Pang, Andy Wing Chun; Ummat, Ajay; Franzen, Oscar; Rausch, Tobias; Stütz, Adrian M; Stedman, William; Anantharaman, Thomas; Hastie, Alex; Dai, Heng; Fritz, Markus Hsi-Yang; Cao, Han; Cohain, Ariella; Deikus, Gintaras; Durrett, Russell E; Blanchard, Scott C; Altman, Roger; Chin, Chen-Shan; Guo, Yan; Paxinos, Ellen E; Korbel, Jan O; Darnell, Robert B; McCombie, W Richard; Kwok, Pui-Yan; Mason, Christopher E; Schadt, Eric E; Bashir, Ali

2015-01-01

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality. PMID:26121404

Assembly and diploid architecture of an individual human genome via single-molecule technologies.

PubMed

Pendleton, Matthew; Sebra, Robert; Pang, Andy Wing Chun; Ummat, Ajay; Franzen, Oscar; Rausch, Tobias; Stütz, Adrian M; Stedman, William; Anantharaman, Thomas; Hastie, Alex; Dai, Heng; Fritz, Markus Hsi-Yang; Cao, Han; Cohain, Ariella; Deikus, Gintaras; Durrett, Russell E; Blanchard, Scott C; Altman, Roger; Chin, Chen-Shan; Guo, Yan; Paxinos, Ellen E; Korbel, Jan O; Darnell, Robert B; McCombie, W Richard; Kwok, Pui-Yan; Mason, Christopher E; Schadt, Eric E; Bashir, Ali

2015-08-01

We present the first comprehensive analysis of a diploid human genome that combines single-molecule sequencing with single-molecule genome maps. Our hybrid assembly markedly improves upon the contiguity observed from traditional shotgun sequencing approaches, with scaffold N50 values approaching 30 Mb, and we identified complex structural variants (SVs) missed by other high-throughput approaches. Furthermore, by combining Illumina short-read data with long reads, we phased both single-nucleotide variants and SVs, generating haplotypes with over 99% consistency with previous trio-based studies. Our work shows that it is now possible to integrate single-molecule and high-throughput sequence data to generate de novo assembled genomes that approach reference quality.

The different time courses of reading different levels of Chinese characters: an ERP study.

PubMed

Lu, Qilin; Tang, Yi-Yuan; Zhou, Li; Yu, Qingbao

2011-07-12

The dual route processing was generally accepted in the reading of alphabetic languages, which suggests alphabetic words can be read by either addressed pathway or assembled pathway. However, it was still unclear whether there was a particular 'dual route mechanism' during reading Chinese characters. In our previous fMRI study, the result showed that there might be a particular 'dual route mechanism', and its addressed pathway was similar between Chinese and English, whereas for the need of spatial analysis, the assembled pathway of Chinese was different from that of English which involved grapheme-to-phoneme correspondences. The present study, using event-related potential, which provide more temporal information, aimed to further support our previous view, and peered inside the different time courses of reading different types of Chinese characters. It was found that reading high frequency Chinese characters increased the N170 component which was believed to enhance attention to the addressed pathway in the left occipital-temporal area. Pseudo Chinese characters could be read by a particular assembled pathway, which caused the largest amplitude of P320 component in the right occipital-temporal area, which considered as a key brain area for radical analysis. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

A highly flexible platform for nanowire sensor assembly using a combination of optically induced and conventional dielectrophoresis.

PubMed

Lin, Yen-Heng; Ho, Kai-Siang; Yang, Chin-Tien; Wang, Jung-Hao; Lai, Chao-Sung

2014-06-02

The number and position of assembled nanowires cannot be controlled using most nanowire sensor assembling methods. In this paper, we demonstrate a high-yield, highly flexible platform for nanowire sensor assembly using a combination of optically induced dielectrophoresis (ODEP) and conventional dielectrophoresis (DEP). With the ODEP platform, optical images can be used as virtual electrodes to locally turn on a non-contact DEP force and manipulate a micron- or nano-scale substance suspended in fluid. Nanowires were first moved next to the previously deposited metal electrodes using optical images and, then, were attracted to and arranged in the gap between two electrodes through DEP forces generated by switching on alternating current signals to the metal electrodes. A single nanowire can be assembled within 24 seconds using this approach. In addition, the number of nanowires in a single nanowire sensor can be controlled, and the assembly of a single nanowire on each of the adjacent electrodes can also be achieved. The electrical properties of the assembled nanowires were characterized by IV curve measurement. Additionally, the contact resistance between the nanowires and electrodes and the stickiness between the nanowires and substrates were further investigated in this study.

Self-Assembly of Telechelic Tyrosine End-Capped PEO Star Polymers in Aqueous Solution.

PubMed

Edwards-Gayle, Charlotte J C; Greco, Francesca; Hamley, Ian W; Rambo, Robert P; Reza, Mehedi; Ruokolainen, Janne; Skoulas, Dimitrios; Iatrou, Hermis

2018-01-08

We investigate the self-assembly of two telechelic star polymer-peptide conjugates based on poly(ethylene oxide) (PEO) four-arm star polymers capped with oligotyrosine. The conjugates were prepared via N-carboxy anhydride-mediated ring-opening polymerization from PEO star polymer macroinitiators. Self-assembly occurs above a critical aggregation concentration determined via fluorescence probe assays. Peptide conformation was examined using circular dichroism spectroscopy. The structure of self-assembled aggregates was probed using small-angle X-ray scattering and cryogenic transmission electron microscopy. In contrast to previous studies on linear telechelic PEO-oligotyrosine conjugates that show self-assembly into β-sheet fibrils, the star architecture suppresses fibril formation and micelles are generally observed instead, a small population of fibrils only being observed upon pH adjustment. Hydrogelation is also suppressed by the polymer star architecture. These peptide-functionalized star polymer solutions are cytocompatible at sufficiently low concentration. These systems present tyrosine at high density and may be useful in the development of future enzyme or pH-responsive biomaterials.

A biophysical study of clathrin utilizing light scattering, neutron scattering and structure based computer modeling

NASA Astrophysics Data System (ADS)

Ferguson, Matthew Lee

A principal component in the protein coats of certain post-golgi and endocytic vesicles is clathrin, which appears as a three-legged heteropolymer (known as a triskelion) that assembles into polyhedral baskets principally made up of pentagonal and hexagonal faces. In vitro, this assembly depends on the pH, with baskets forming more readily at low pH and less readily at high pH. We have developed procedures, based on static and dynamic light scattering, to determine the radius of gyration, Rg, and hydrodynamic radius, RH, of isolated triskelia under conditions where basket assembly occurs. Calculations based on rigid molecular bead models of a triskelion show that the measured values can be accounted for by bending of the legs and a puckering at the vertex. We also show that the values of Rg and R H measured for clathrin triskelia in solution are qualitatively consistent with the conformation of an individual triskelion that is part of a "D6 barrel" basket assembly measured by cryo-EM tomography. We extended this study by performing small angle neutron scattering (SANS) experiments on isolated triskelia in solution under conditions where baskets do not assemble. SANS experiments were consistent with previous static light scattering experiments but showed a shoulder in the scattering function at intermediate q-values just beyond the central diffraction peak (the Guinier regime). Theoretical calculations based on rigid bead models of a triskelion showed well-defined features in this region different from the experiment. A flexible bead-spring model of a triskelion and Brownian dynamics simulations were used to generate a time averaged scattering function. This model adequately described the experimental data for flexibilities close to previous estimates from the analysis of electron micrographs.

Spherical: an iterative workflow for assembling metagenomic datasets.

PubMed

Hitch, Thomas C A; Creevey, Christopher J

2018-01-24

The consensus emerging from the study of microbiomes is that they are far more complex than previously thought, requiring better assemblies and increasingly deeper sequencing. However, current metagenomic assembly techniques regularly fail to incorporate all, or even the majority in some cases, of the sequence information generated for many microbiomes, negating this effort. This can especially bias the information gathered and the perceived importance of the minor taxa in a microbiome. We propose a simple but effective approach, implemented in Python, to address this problem. Based on an iterative methodology, our workflow (called Spherical) carries out successive rounds of assemblies with the sequencing reads not yet utilised. This approach also allows the user to reduce the resources required for very large datasets, by assembling random subsets of the whole in a "divide and conquer" manner. We demonstrate the accuracy of Spherical using simulated data based on completely sequenced genomes and the effectiveness of the workflow at retrieving lost information for taxa in three published metagenomics studies of varying sizes. Our results show that Spherical increased the amount of reads utilized in the assembly by up to 109% compared to the base assembly. The additional contigs assembled by the Spherical workflow resulted in a significant (P < 0.05) changes in the predicted taxonomic profile of all datasets analysed. Spherical is implemented in Python 2.7 and freely available for use under the MIT license. Source code and documentation is hosted publically at: https://github.com/thh32/Spherical .

GAAP: Genome-organization-framework-Assisted Assembly Pipeline for prokaryotic genomes.

PubMed

Yuan, Lina; Yu, Yang; Zhu, Yanmin; Li, Yulai; Li, Changqing; Li, Rujiao; Ma, Qin; Siu, Gilman Kit-Hang; Yu, Jun; Jiang, Taijiao; Xiao, Jingfa; Kang, Yu

2017-01-25

Next-generation sequencing (NGS) technologies have greatly promoted the genomic study of prokaryotes. However, highly fragmented assemblies due to short reads from NGS are still a limiting factor in gaining insights into the genome biology. Reference-assisted tools are promising in genome assembly, but tend to result in false assembly when the assigned reference has extensive rearrangements. Herein, we present GAAP, a genome assembly pipeline for scaffolding based on core-gene-defined Genome Organizational Framework (cGOF) described in our previous study. Instead of assigning references, we use the multiple-reference-derived cGOFs as indexes to assist in order and orientation of the scaffolds and build a skeleton structure, and then use read pairs to extend scaffolds, called local scaffolding, and distinguish between true and chimeric adjacencies in the scaffolds. In our performance tests using both empirical and simulated data of 15 genomes in six species with diverse genome size, complexity, and all three categories of cGOFs, GAAP outcompetes or achieves comparable results when compared to three other reference-assisted programs, AlignGraph, Ragout and MeDuSa. GAAP uses both cGOF and pair-end reads to create assemblies in genomic scale, and performs better than the currently available reference-assisted assembly tools as it recovers more assemblies and makes fewer false locations, especially for species with extensive rearranged genomes. Our method is a promising solution for reconstruction of genome sequence from short reads of NGS.

Seven Liberal Outcomes of Professional Study.

ERIC Educational Resources Information Center

Stark, Joan S.

A group of faculty members and accrediting representatives was assembled to explore ways to better integrate liberal and professional study during the undergraduate years. The group based its discussions on educational outcomes previously identified by the project directors as important for all college-educated professionals. The project involved…

Microstructure of β-Sitosterol:γ-Oryzanol Edible Organogels.

PubMed

Matheson, Andrew B; Koutsos, Vasileios; Dalkas, Georgios; Euston, Stephen; Clegg, Paul

2017-05-09

Rheology and atomic force microscopy (AFM) were employed to examine the microstructure of β-sitosterol:γ-oryzanol organogels in sunflower oil. Using time-resolved rheology, we followed gel formation, paying specific attention to the fibril aggregation process, which had not been studied in detail previously for this system. Using AFM, we observed gel structures directly and obtained detailed information on the gel structure, far exceeding previous studies. Our analysis suggests that though gels are formed by the self-assembly and aggregation of one-dimensional fibrils, the manner in which these fibrils aggregate into ribbons results in complex structures of higher dimensionality. We emphasize that it is a surprise to find ribbons and not twisted strands. Comparing AFM images of 10% w/w and 20% w/w gelator systems, we observed differences in the degree of branching which are consistent with the rheology. We also observed the individual self-assembled fibrils which make up these gels with much greater clarity than in previous microscopy studies, and the fibril diameters of ∼9.8 nm we measured agree excellently with those obtained from existing small-angle neutron scattering data. These results provide new insight into the structure and formation kinetics of this important organogel system.

DNA packaging and the pathway of bacteriophage T4 head assembly.

PubMed Central

Hsiao, C L; Black, L W

1977-01-01

A cold-sensitive mutation in the structural gene for a minor phage T4 capsid protein (p20) leads to formation of heads containing p20 and cleaved head proteins and empty of DNA. Such heads can be filled with DNA and converted to active phages in vivo uponshift to high temperature. It appears that p20 has two distinct roles in head assembly: first, in construction of the prehead shell (blocked by ts and am mutation) and, second,in DNA packaging (blocked by cs mutation). The latter function is closely associated with gene 17 product, previously known to be required for DNA packagaing. Temperature shift studies of cs-ts double mutants and other observations allow determination of phage function required for DNA packaging. Contrary to previous proposals, we find that T4 DNA packaging is not directly coupled to and can follow DNA synthesis, protein cleavage, prehead core removal, and gene 21-mediated cleavage-induced increase in head volume. Our evidence suggests that an altered head assembly pathway exists and that DNA packaging is probably initiated by DNA-capsid (p20) interaction. Images PMID:269421

Mutations in the putative calcium-binding domain of polyomavirus VP1 affect capsid assembly

NASA Technical Reports Server (NTRS)

Haynes, J. I. 2nd; Chang, D.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

Calcium ions appear to play a major role in maintaining the structural integrity of the polyomavirus and are likely involved in the processes of viral uncoating and assembly. Previous studies demonstrated that a VP1 fragment extending from Pro-232 to Asp-364 has calcium-binding capabilities. This fragment contains an amino acid stretch from Asp-266 to Glu-277 which is quite similar in sequence to the amino acids that make up the calcium-binding EF hand structures found in many proteins. To assess the contribution of this domain to polyomavirus structural integrity, the effects of mutations in this region were examined by transfecting mutated viral DNA into susceptible cells. Immunofluorescence studies indicated that although viral protein synthesis occurred normally, infective viral progeny were not produced in cells transfected with polyomavirus genomes encoding either a VP1 molecule lacking amino acids Thr-262 through Gly-276 or a VP1 molecule containing a mutation of Asp-266 to Ala. VP1 molecules containing the deletion mutation were unable to bind 45Ca in an in vitro assay. Upon expression in Escherichia coli and purification by immunoaffinity chromatography, wild-type VP1 was isolated as pentameric, capsomere-like structures which could be induced to form capsid-like structures upon addition of CaCl2, consistent with previous studies. However, although VP1 containing the point mutation was isolated as pentamers which were indistinguishable from wild-type VP1 pentamers, addition of CaCl2 did not result in their assembly into capsid-like structures. Immunogold labeling and electron microscopy studies of transfected mammalian cells provided in vivo evidence that a mutation in this region affects the process of viral assembly.

High-power fused assemblies enabled by advances in fiber-processing technologies

NASA Astrophysics Data System (ADS)

Wiley, Robert; Clark, Brett

2011-02-01

The power handling capabilities of fiber lasers are limited by the technologies available to fabricate and assemble the key optical system components. Previous tools for the assembly, tapering, and fusion of fiber laser elements have had drawbacks with regard to temperature range, alignment capability, assembly flexibility and surface contamination. To provide expanded capabilities for fiber laser assembly, a wide-area electrical plasma heat source was used in conjunction with an optimized image analysis method and a flexible alignment system, integrated according to mechatronic principles. High-resolution imaging and vision-based measurement provided feedback to adjust assembly, fusion, and tapering process parameters. The system was used to perform assembly steps including dissimilar-fiber splicing, tapering, bundling, capillary bundling, and fusion of fibers to bulk optic devices up to several mm in diameter. A wide range of fiber types and diameters were tested, including extremely large diameters and photonic crystal fibers. The assemblies were evaluated for conformation to optical and mechanical design criteria, such as taper geometry and splice loss. The completed assemblies met the performance targets and exhibited reduced surface contamination compared to assemblies prepared on previously existing equipment. The imaging system and image analysis algorithms provided in situ fiber geometry measurement data that agreed well with external measurement. The ability to adjust operating parameters dynamically based on imaging was shown to provide substantial performance benefits, particularly in the tapering of fibers and bundles. The integrated design approach was shown to provide sufficient flexibility to perform all required operations with a minimum of reconfiguration.

Novel transcriptome assembly and improved annotation of the whiteleg shrimp (Litopenaeus vannamei), a dominant crustacean in global seafood mariculture.

PubMed

Ghaffari, Noushin; Sanchez-Flores, Alejandro; Doan, Ryan; Garcia-Orozco, Karina D; Chen, Patricia L; Ochoa-Leyva, Adrian; Lopez-Zavala, Alonso A; Carrasco, J Salvador; Hong, Chris; Brieba, Luis G; Rudiño-Piñera, Enrique; Blood, Philip D; Sawyer, Jason E; Johnson, Charles D; Dindot, Scott V; Sotelo-Mundo, Rogerio R; Criscitiello, Michael F

2014-11-25

We present a new transcriptome assembly of the Pacific whiteleg shrimp (Litopenaeus vannamei), the species most farmed for human consumption. Its functional annotation, a substantial improvement over previous ones, is provided freely. RNA-Seq with Illumina HiSeq technology was used to analyze samples extracted from shrimp abdominal muscle, hepatopancreas, gills and pleopods. We used the Trinity and Trinotate software suites for transcriptome assembly and annotation, respectively. The quality of this assembly and the affiliated targeted homology searches greatly enrich the curated transcripts currently available in public databases for this species. Comparison with the model arthropod Daphnia allows some insights into defining characteristics of decapod crustaceans. This large-scale gene discovery gives the broadest depth yet to the annotated transcriptome of this important species and should be of value to ongoing genomics and immunogenetic resistance studies in this shrimp of paramount global economic importance.

A System for the Automatic Assembly of Test Questions Using a No-SQL Database

ERIC Educational Resources Information Center

Shin, Sanggyu; Hashimoto, Hiroshi

2014-01-01

We describe a system that automatically assembles test questions from a set of examples. Our system can create test questions appropriate for each user's level at low cost. In particular, when a user review their lesson, our system provides new test questions which are assembled based on their previous test results and past mistakes, rather than a…

Self-assembly of Spherical Macroions in Solution: A Coarse-grained Molecular Dynamics Study

NASA Astrophysics Data System (ADS)

Liu, Zhuonan; Liu, Tianbo; Tsige, Mesfin

2015-03-01

Macroions (such as polyoxometalates) in solution can form a stable hollow spherical super-molecular structure called blackberry when they have moderate surface charge density and size (1-10 nm). Depending on the surface charge density of macroions, the size of the blackberry can be from 20 to more than 100 nm. Other macroions such as dendrimers can also self-assemble into similar super-molecular structure in solution. Existing theories such as Debye-Hückel and DLVO theories cannot explain this phenomenon and we are not aware of any other theory that can explain this. Previous studies using all-atom Molecular Dynamics simulations have shown identical macroions forming oligomers mediated by counterions. Due to the limitations in all-atom simulation and available computational capabilities, these studies handled only small systems with simple macroions, leading to less conclusive but still relevant results on the self-assembly behavior. To overcome these limitations, in this work large-scale coarse-grained modeling of macroions in solution is used. In order to understand the origin of the attractive force that is responsible for the self-assembly of macroions, different types of macroions in different solution conditions are studied. This work was supported by NSF Grant DMR0847580.

preAssemble: a tool for automatic sequencer trace data processing.

PubMed

Adzhubei, Alexei A; Laerdahl, Jon K; Vlasova, Anna V

2006-01-17

Trace or chromatogram files (raw data) are produced by automatic nucleic acid sequencing equipment or sequencers. Each file contains information which can be interpreted by specialised software to reveal the sequence (base calling). This is done by the sequencer proprietary software or publicly available programs. Depending on the size of a sequencing project the number of trace files can vary from just a few to thousands of files. Sequencing quality assessment on various criteria is important at the stage preceding clustering and contig assembly. Two major publicly available packages--Phred and Staden are used by preAssemble to perform sequence quality processing. The preAssemble pre-assembly sequence processing pipeline has been developed for small to large scale automatic processing of DNA sequencer chromatogram (trace) data. The Staden Package Pregap4 module and base-calling program Phred are utilized in the pipeline, which produces detailed and self-explanatory output that can be displayed with a web browser. preAssemble can be used successfully with very little previous experience, however options for parameter tuning are provided for advanced users. preAssemble runs under UNIX and LINUX operating systems. It is available for downloading and will run as stand-alone software. It can also be accessed on the Norwegian Salmon Genome Project web site where preAssemble jobs can be run on the project server. preAssemble is a tool allowing to perform quality assessment of sequences generated by automatic sequencing equipment. preAssemble is flexible since both interactive jobs on the preAssemble server and the stand alone downloadable version are available. Virtually no previous experience is necessary to run a default preAssemble job, on the other hand options for parameter tuning are provided. Consequently preAssemble can be used as efficiently for just several trace files as for large scale sequence processing.

Self-assembly of cationic multidomain peptide hydrogels: supramolecular nanostructure and rheological properties dictate antimicrobial activity

NASA Astrophysics Data System (ADS)

Jiang, Linhai; Xu, Dawei; Sellati, Timothy J.; Dong, He

2015-11-01

Hydrogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. The biological performance of hydrogels, particularly those used as wound dressing could be greatly advanced if imbued with inherent antimicrobial activity capable of staving off colonization of the wound site by opportunistic bacterial pathogens. Possessing such antimicrobial properties would also protect the hydrogel itself from being adversely affected by microbial attachment to its surface. We have previously demonstrated the broad-spectrum antimicrobial activity of supramolecular assemblies of cationic multi-domain peptides (MDPs) in solution. Here, we extend the 1-D soluble supramolecular assembly to 3-D hydrogels to investigate the effect of the supramolecular nanostructure and its rheological properties on the antimicrobial activity of self-assembled hydrogels. Among designed MDPs, the bactericidal activity of peptide hydrogels was found to follow an opposite trend to that in solution. Improved antimicrobial activity of self-assembled peptide hydrogels is dictated by the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. The structure-property-activity relationship developed through this study will provide important guidelines for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications.Hydrogels are an important class of biomaterials that have been widely utilized for a variety of biomedical/medical applications. The biological performance of hydrogels, particularly those used as wound dressing could be greatly advanced if imbued with inherent antimicrobial activity capable of staving off colonization of the wound site by opportunistic bacterial pathogens. Possessing such antimicrobial properties would also protect the hydrogel itself from being adversely affected by microbial attachment to its surface. We have previously demonstrated the broad-spectrum antimicrobial activity of supramolecular assemblies of cationic multi-domain peptides (MDPs) in solution. Here, we extend the 1-D soluble supramolecular assembly to 3-D hydrogels to investigate the effect of the supramolecular nanostructure and its rheological properties on the antimicrobial activity of self-assembled hydrogels. Among designed MDPs, the bactericidal activity of peptide hydrogels was found to follow an opposite trend to that in solution. Improved antimicrobial activity of self-assembled peptide hydrogels is dictated by the combined effect of supramolecular surface chemistry and storage modulus of the bulk materials, rather than the ability of individual peptides/peptide assemblies to penetrate bacterial cell membrane as observed in solution. The structure-property-activity relationship developed through this study will provide important guidelines for designing biocompatible peptide hydrogels with built-in antimicrobial activity for various biomedical applications. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05233e

Reactive oxygen species promote heat shock protein 90-mediated HBV capsid assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Yoon Sik, E-mail: yumshak@naver.com; Seo, Hyun Wook, E-mail: suruk@naver.com; Jung, Guhung, E-mail: drjung@snu.ac.kr

2015-02-13

Hepatitis B virus (HBV) infection induces reactive oxygen species (ROS) production and has been associated with the development of hepatocellular carcinoma (HCC). ROS are also an important factor in HCC because the accumulated ROS leads to abnormal cell proliferation and chromosome mutation. In oxidative stress, heat shock protein 90 (Hsp90) and glutathione (GSH) function as part of the defense mechanism. Hsp90 prevents cellular component from oxidative stress, and GSH acts as antioxidants scavenging ROS in the cell. However, it is not known whether molecules regulated by oxidative stress are involved in HBV capsid assembly. Based on the previous study thatmore » Hsp90 facilitates HBV capsid assembly, which is an important step for the packing of viral particles, here, we show that ROS enrich Hsp90-driven HBV capsid formation. In cell-free system, HBV capsid assembly was facilitated by ROS with Hsp90, whereas it was decreased without Hsp90. In addition, GSH inhibited the function of Hsp90 to decrease HBV capsid assembly. Consistent with the result of cell-free system, ROS and buthionine sulfoximine (BS), an inhibitor of GSH synthesis, increased HBV capsid formation in HepG2.2.15 cells. Thus, our study uncovers the interplay between ROS and Hsp90 during HBV capsid assembly. - Highlights: • We examined H{sub 2}O{sub 2} and GSH modulate HBV capsid assembly. • H{sub 2}O{sub 2} facilitates HBV capsid assembly in the presence of Hsp90. • GSH inhibits function of Hsp90 in facilitating HBV capsid assembly. • H{sub 2}O{sub 2} and GSH induce conformation change of Hsp90.« less

Abnormal assembly of annulate lamellae and nuclear pore complexes coincides with fertilization arrest at the pronuclear stage of human zygotic development.

PubMed

Rawe, V Y; Olmedo, S Brugo; Nodar, F N; Ponzio, R; Sutovsky, P

2003-03-01

The assembly of nuclear pore complexes (NPC) and their cytoplasmic stacks, annulate lamellae (AL), promote normal nucleocytoplasmic trafficking and accompany pronuclear development within the mammalian zygote. Previous studies showed that a percentage of human oocytes fertilized in vitro failed to develop normal pronuclei and cleave within 40-48 h post insemination. We hypothesized that an aberrant recruitment of NPC proteins, nucleoporins and/or NPC preassembled into AL, might accompany human fertilization arrest. We explored NPC and AL assembly in unfertilized human oocytes, and fertilized and arrested zygotes by immunofluorescence with an NPC- and AL-specific antibody, mAb 414, and by transmission electron microscopy. Major NPC or AL assembly was not observed in the unfertilized human oocytes. Once fertilization took place, the formation of AL was observed throughout the cytoplasm and near the developing pronuclei with NPC. On the contrary, NPC assembly was disrupted in the arrested zygotes, whereas AL were clustered into large sheaths. This was accompanied by the lack of NPC incorporation into the nuclear envelopes. We conclude that the aberrant assembly of NPC and AL coincides with early developmental failure in humans.

TIMMDC1/C3orf1 functions as a membrane-embedded mitochondrial complex I assembly factor through association with the MCIA complex.

PubMed

Guarani, Virginia; Paulo, Joao; Zhai, Bo; Huttlin, Edward L; Gygi, Steven P; Harper, J Wade

2014-03-01

Complex I (CI) of the electron transport chain, a large membrane-embedded NADH dehydrogenase, couples electron transfer to the release of protons into the mitochondrial inner membrane space to promote ATP production through ATP synthase. In addition to being a central conduit for ATP production, CI activity has been linked to neurodegenerative disorders, including Parkinson's disease. CI is built in a stepwise fashion through the actions of several assembly factors. We employed interaction proteomics to interrogate the molecular associations of 15 core subunits and assembly factors previously linked to human CI deficiency, resulting in a network of 101 proteins and 335 interactions (edges). TIMMDC1, a predicted 4-pass membrane protein, reciprocally associated with multiple members of the MCIA CI assembly factor complex and core CI subunits and was localized in the mitochondrial inner membrane, and its depletion resulted in reduced CI activity and cellular respiration. Quantitative proteomics demonstrated a role for TIMMDC1 in assembly of membrane-embedded and soluble arms of the complex. This study defines a new membrane-embedded CI assembly factor and provides a resource for further analysis of CI biology.

Role of Surface Charge Density in Nanoparticle-templated Assembly of Bromovirus Protein Cages

PubMed Central

Daniel, Marie-Christine; Tsvetkova, Irina B.; Quinkert, Zachary T.; Murali, Ayaluru; De, Mrinmoy; Rotello, Vincent M.; Kao, C. Cheng; Dragnea, Bogdan

2010-01-01

Self-assembling icosahedral protein cages have potencially useful physical and chemical characteristics for a variety of nanotechnology applications, ranging from therapeutic or diagnostic vectors to building blocks for hierarchical materials. For application-specific functional control of protein cage assemblies, a deeper understanding of the interaction between the protein cage and its payload is necessary. Protein-cage encapsulated nanoparticles, with their well-defined surface chemistry, allow for systematic control over key parameters of encapsulation such as the surface charge, hydrophobicity, and size. Independent control over these variables allows experimental testing of different assembly mechanism models. Previous studies done with Brome mosaic virus capsids and negatively-charged gold nanoparticles indicated that the result of the self-assembly process depends on the diameter of the particle. However, in these experiments, the surface-ligand density was maintained at saturation levels, while the total charge and the radius of curvature remained coupled variables, making the interpretation of the observed dependence on the core size difficult. The current work furnishes evidence of a critical surface charge density for assembly through an analysis aimed at decoupling the surface charge the core size. PMID:20575505

Native Language Experience Shapes Neural Basis of Addressed and Assembled Phonologies

PubMed Central

Mei, Leilei; Xue, Gui; Lu, Zhong-Lin; He, Qinghua; Wei, Miao; Zhang, Mingxia; Dong, Qi; Chen, Chuansheng

2015-01-01

Previous studies have suggested differential engagement of addressed and assembled phonologies in reading Chinese and alphabetic languages (e.g., English) and the modulatory role of native language in learning to read a second language. However, it is not clear whether native language experience shapes the neural mechanisms of addressed and assembled phonologies. To address this question, we trained native Chinese and native English speakers to read the same artificial language (based on Korean Hangul) either through addressed (i.e., whole-word mapping) or assembled (i.e., grapheme-to-phoneme mapping) phonology. We found that, for both native Chinese and native English speakers, addressed phonology relied on the regions in the ventral pathway, whereas assembled phonology depended on the regions in the dorsal pathway. More importantly, we found that the neural mechanisms of addressed and assembled phonologies were shaped by native language experience. Specifically, two key regions for addressed phonology (i.e., the left middle temporal gyrus and right inferior temporal gyrus) showed greater activation for addressed phonology in native Chinese speakers, while one key region for assembled phonology (i.e., the left supramarginal gyrus) showed more activation for assembled phonology in native English speakers. These results provide direct neuroimaging evidence for the effect of native language experience on the neural mechanisms of phonological access in a new language and support the assimilation-accommodation hypothesis. PMID:25858447

Brain transcriptome sequencing and assembly of three songbird model systems for the study of social behavior

PubMed Central

Mukai, Motoko; Gonser, Rusty A.; Wingfield, John C.; London, Sarah E.; Tuttle, Elaina M.; Clayton, David F.

2014-01-01

Emberizid sparrows (emberizidae) have played a prominent role in the study of avian vocal communication and social behavior. We present here brain transcriptomes for three emberizid model systems, song sparrow Melospiza melodia, white-throated sparrow Zonotrichia albicollis, and Gambel’s white-crowned sparrow Zonotrichia leucophrys gambelii. Each of the assemblies covered fully or in part, over 89% of the previously annotated protein coding genes in the zebra finch Taeniopygia guttata, with 16,846, 15,805, and 16,646 unique BLAST hits in song, white-throated and white-crowned sparrows, respectively. As in previous studies, we find tissue of origin (auditory forebrain versus hypothalamus and whole brain) as an important determinant of overall expression profile. We also demonstrate the successful isolation of RNA and RNA-sequencing from post-mortem samples from building strikes and suggest that such an approach could be useful when traditional sampling opportunities are limited. These transcriptomes will be an important resource for the study of social behavior in birds and for data driven annotation of forthcoming whole genome sequences for these and other bird species. PMID:24883256

Automated assembly of Gallium Arsenide and 50-micron thick silicon solar cell modules

NASA Technical Reports Server (NTRS)

Mesch, H. G.

1984-01-01

The TRW automated solar array assembly equipment was used for the module assembly of 300 GaAs solar cells and 300 50 micron thick silicon solar cells (2 x 4 cm in size). These cells were interconnected with silver plated Invar tabs by means of welding. The GaAs cells were bonded to Kapton graphite aluminum honeycomb graphite substrates and the thin silicon cells were bonded to 0.002 inch thick single layer Kapton substrates. The GaAs solar cell module assembly resulted in a yield of 86% and the thin cell assembly produced a yield of 46% due to intermittent sticking of weld electrodes during the front cell contact welding operation. (Previously assembled thin cell solar modules produced an overall assembly yield of greater than 80%).

Predictive energy landscapes for folding membrane protein assemblies

NASA Astrophysics Data System (ADS)

Truong, Ha H.; Kim, Bobby L.; Schafer, Nicholas P.; Wolynes, Peter G.

2015-12-01

We study the energy landscapes for membrane protein oligomerization using the Associative memory, Water mediated, Structure and Energy Model with an implicit membrane potential (AWSEM-membrane), a coarse-grained molecular dynamics model previously optimized under the assumption that the energy landscapes for folding α-helical membrane protein monomers are funneled once their native topology within the membrane is established. In this study we show that the AWSEM-membrane force field is able to sample near native binding interfaces of several oligomeric systems. By predicting candidate structures using simulated annealing, we further show that degeneracies in predicting structures of membrane protein monomers are generally resolved in the folding of the higher order assemblies as is the case in the assemblies of both nicotinic acetylcholine receptor and V-type Na+-ATPase dimers. The physics of the phenomenon resembles domain swapping, which is consistent with the landscape following the principle of minimal frustration. We revisit also the classic Khorana study of the reconstitution of bacteriorhodopsin from its fragments, which is the close analogue of the early Anfinsen experiment on globular proteins. Here, we show the retinal cofactor likely plays a major role in selecting the final functional assembly.

Retrosynthetic Analysis-Guided Breaking Tile Symmetry for the Assembly of Complex DNA Nanostructures.

PubMed

Wang, Pengfei; Wu, Siyu; Tian, Cheng; Yu, Guimei; Jiang, Wen; Wang, Guansong; Mao, Chengde

2016-10-11

Current tile-based DNA self-assembly produces simple repetitive or highly symmetric structures. In the case of 2D lattices, the unit cell often contains only one basic tile because the tiles often are symmetric (in terms of either the backbone or the sequence). In this work, we have applied retrosynthetic analysis to determine the minimal asymmetric units for complex DNA nanostructures. Such analysis guides us to break the intrinsic structural symmetries of the tiles to achieve high structural complexities. This strategy has led to the construction of several DNA nanostructures that are not accessible from conventional symmetric tile designs. Along with previous studies, herein we have established a set of four fundamental rules regarding tile-based assembly. Such rules could serve as guidelines for the design of DNA nanostructures.

Dynamics of HIV-1 Assembly and Release

PubMed Central

Ivanchenko, Sergey; Godinez, William J.; Lampe, Marko; Kräusslich, Hans-Georg; Eils, Roland; Rohr, Karl; Bräuchle, Christoph; Müller, Barbara; Lamb, Don C.

2009-01-01

Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10−3 s−1, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics. PMID:19893629

Transcriptome assembly and digital gene expression atlas of the rainbow trout

USDA-ARS?s Scientific Manuscript database

Background: Transcriptome analysis is a preferred method for gene discovery, marker development and gene expression profiling in non-model organisms. Previously, we sequenced a transcriptome reference using Sanger-based and 454-pyrosequencing, however, a transcriptome assembly is still incomplete an...

One-piece transparent shell improves design of helmet assembly

NASA Technical Reports Server (NTRS)

Jones, R. L.; Okane, J. H.

1966-01-01

One-piece transparent helmet shell made of polycarbonate is equipped with a helmet protection pad, a visor assembly, a communications skull cap, and an emergency oxygen supply. This design offers improvements over previous designs in weight, visual field, comfort and protection.

Complete Taiwanese Macaque (Macaca cyclopis) Mitochondrial Genome: Reference-Assisted de novo Assembly with Multiple k-mer Strategy.

PubMed

Huang, Yu-Feng; Midha, Mohit; Chen, Tzu-Han; Wang, Yu-Tai; Smith, David Glenn; Pei, Kurtis Jai-Chyi; Chiu, Kuo Ping

2015-01-01

The Taiwanese (Formosan) macaque (Macaca cyclopis) is the only nonhuman primate endemic to Taiwan. This primate species is valuable for evolutionary studies and as subjects in medical research. However, only partial fragments of the mitochondrial genome (mitogenome) of this primate species have been sequenced, not mentioning its nuclear genome. We employed next-generation sequencing to generate 2 x 90 bp paired-end reads, followed by reference-assisted de novo assembly with multiple k-mer strategy to characterize the M. cyclopis mitogenome. We compared the assembled mitogenome with that of other macaque species for phylogenetic analysis. Our results show that, the M. cyclopis mitogenome consists of 16,563 nucleotides encoding for 13 protein-coding genes, 2 ribosomal RNAs and 22 transfer RNAs. Phylogenetic analysis indicates that M. cyclopis is most closely related to M. mulatta lasiota (Chinese rhesus macaque), supporting the notion of Asia-continental origin of M. cyclopis proposed in previous studies based on partial mitochondrial sequences. Our work presents a novel approach for assembling a mitogenome that utilizes the capabilities of de novo genome assembly with assistance of a reference genome. The availability of the complete Taiwanese macaque mitogenome will facilitate the study of primate evolution and the characterization of genetic variations for the potential usage of this species as a non-human primate model for medical research.

Challenging Students' Intuitions—the Influence of a Tangible Model of Virus Assembly on Students' Conceptual Reasoning About the Process of Self-Assembly

NASA Astrophysics Data System (ADS)

Larsson, Caroline; Tibell, Lena A. E.

2015-10-01

A well-ordered biological complex can be formed by the random motion of its components, i.e. self-assemble. This is a concept that incorporates issues that may contradict students' everyday experiences and intuitions. In previous studies, we have shown that a tangible model of virus self-assembly, used in a group exercise, helps students to grasp the process of self-assembly and in particular the facet "random molecular collision". The present study investigates how and why the model and the group exercise facilitate students' learning of this particular facet. The data analysed consist of audio recordings of six group exercises ( n = 35 university students) and individual semi-structured interviews ( n = 5 university students). The analysis is based on constructivist perspectives of learning, a combination of conceptual change theory and learning with external representations. Qualitative analysis indicates that perceived counterintuitive aspects of the process created a cognitive conflict within learners. The tangible model used in the group exercises facilitated a conceptual change in their understanding of the process. In particular, the tangible model appeared to provide cues and possible explanations and functioned as an "eye-opener" and a "thinking tool". Lastly, the results show signs of emotions also being important elements for successful accommodation.

The challenges of lean manufacturing implementation in kitting assembly

NASA Astrophysics Data System (ADS)

Fansuri, A. F. H.; Rose, A. N. M.; Nik Mohamed, N. M. Z.; Ahmad, H.

2017-10-01

Literature studies shows that lean manufacturing goes way back with the original founder Eli Whitney in year 1799. The main purpose of lean manufacturing is to identify and eliminate waste in production. The application of lean manufacturing can be carried out in any industrial processes with regards to the understanding of lean principles, theories and practices. Kitting is one of the important aspects in a successful production. The continuous supply of materials from store to production has to be systematic and able to achieve lean standard for it to be successful. The objective of this paper is to review the implementation of lean manufacturing in kitting assembly. Previous papers show that, the implementation of lean manufacturing in kitting assembly may be beneficial to the organization such as reduce in space occupancy, part shortages, lead time and manpower. Based on previous research, some industries may tend to change between kitting and line stocking which are due to lack of understanding when implementing kitting and causes longer lead time and materials overflow in store. With a proper understanding on what to kit, where to kit, how to kit, why to kit and who kits the material with a standardised process flow may ensure the success of kitting.

Glucose starvation increases V-ATPase assembly and activity in mammalian cells through AMP kinase and phosphatidylinositide 3-kinase/Akt signaling.

PubMed

McGuire, Christina M; Forgac, Michael

2018-06-08

The vacuolar H + -ATPase (V-ATPase) is an ATP-driven proton pump involved in many cellular processes. An important mechanism by which V-ATPase activity is controlled is the reversible assembly of its two domains, namely the peripheral V 1 domain and the integral V 0 domain. Although reversible assembly is conserved across all eukaryotic organisms, the signaling pathways controlling it have not been fully characterized. Here, we identify glucose starvation as a novel regulator of V-ATPase assembly in mammalian cells. During acute glucose starvation, the V-ATPase undergoes a rapid and reversible increase in assembly and activity as measured by lysosomal acidification. Because the V-ATPase has recently been implicated in the activation of AMP kinase (AMPK), a critical cellular energy sensor that is also activated upon glucose starvation, we compared the time course of AMPK activation and V-ATPase assembly upon glucose starvation. We observe that AMPK activation precedes increased V-ATPase activity. Moreover, the starvation-induced increase in V-ATPase activity and assembly are prevented by the AMPK inhibitor dorsomorphin. These results suggest that increased assembly and activity of the V-ATPase upon glucose starvation are dependent upon AMPK. We also find that the PI3K/Akt pathway, which has previously been implicated in controlling V-ATPase assembly in mammalian cells, also plays a role in the starvation-induced increase in V-ATPase assembly and activity. These studies thus identify a novel stimulus of V-ATPase assembly and a novel signaling pathway involved in regulating this process. The possible function of starvation-induced increase in lysosomal V-ATPase activity is discussed. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Simple method for assembly of CRISPR synergistic activation mediator gRNA expression array.

PubMed

Vad-Nielsen, Johan; Nielsen, Anders Lade; Luo, Yonglun

2018-05-20

When studying complex interconnected regulatory networks, effective methods for simultaneously manipulating multiple genes expression are paramount. Previously, we have developed a simple method for generation of an all-in-one CRISPR gRNA expression array. We here present a Golden Gate Assembly-based system of synergistic activation mediator (SAM) compatible CRISPR/dCas9 gRNA expression array for the simultaneous activation of multiple genes. Using this system, we demonstrated the simultaneous activation of the transcription factors, TWIST, SNAIL, SLUG, and ZEB1 a human breast cancer cell line. Copyright © 2018 Elsevier B.V. All rights reserved.

Large-scale assembly bias of dark matter halos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lazeyras, Titouan; Musso, Marcello; Schmidt, Fabian, E-mail: titouan@mpa-garching.mpg.de, E-mail: mmusso@sas.upenn.edu, E-mail: fabians@mpa-garching.mpg.de

We present precise measurements of the assembly bias of dark matter halos, i.e. the dependence of halo bias on other properties than the mass, using curved 'separate universe' N-body simulations which effectively incorporate an infinite-wavelength matter overdensity into the background density. This method measures the LIMD (local-in-matter-density) bias parameters b {sub n} in the large-scale limit. We focus on the dependence of the first two Eulerian biases b {sup E} {sup {sub 1}} and b {sup E} {sup {sub 2}} on four halo properties: the concentration, spin, mass accretion rate, and ellipticity. We quantitatively compare our results with previous worksmore » in which assembly bias was measured on fairly small scales. Despite this difference, our findings are in good agreement with previous results. We also look at the joint dependence of bias on two halo properties in addition to the mass. Finally, using the excursion set peaks model, we attempt to shed new insights on how assembly bias arises in this analytical model.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Metz, Lori A.; Friese, Judah I.; Finn, Erin C.

Critical assemblies provide one method of achieving a fast neutron spectrum that is close to a 235U fission-energy neutron spectrum for nuclear data measurements. Previous work has demonstrated the use of a natural boron carbide capsule for spectral-tailoring in a mixed spectrum reactor as an alternate and complementary method for performing fission-energy neutron experiments. Previous fission products measurements showed that the neutron spectrum achievable with natural boron carbide was not as hard as what can be achieved with critical assemblies. New measurements performed with the Washington State University TRIGA reactor using a boron carbide capsule 96% enriched in 10B formore » irradiations resulted in a neutron spectrum very similar to a critical assembly and a pure 235U fission spectrum. The current work describes an experiment involving a highly-enriched uranium target irradiated under the new 10B4C capsule. Fission product yields were measured following radiochemical separations and are presented here. Reactor dosimetry measurements for characterizing neutron spectra and fluence for the enriched boron carbide capsule and critical assemblies are also discussed.« less

Modeling Effects of RNA on Capsid Assembly Pathways via Coarse-Grained Stochastic Simulation

PubMed Central

Smith, Gregory R.; Xie, Lu; Schwartz, Russell

2016-01-01

The environment of a living cell is vastly different from that of an in vitro reaction system, an issue that presents great challenges to the use of in vitro models, or computer simulations based on them, for understanding biochemistry in vivo. Virus capsids make an excellent model system for such questions because they typically have few distinct components, making them amenable to in vitro and modeling studies, yet their assembly can involve complex networks of possible reactions that cannot be resolved in detail by any current experimental technology. We previously fit kinetic simulation parameters to bulk in vitro assembly data to yield a close match between simulated and real data, and then used the simulations to study features of assembly that cannot be monitored experimentally. The present work seeks to project how assembly in these simulations fit to in vitro data would be altered by computationally adding features of the cellular environment to the system, specifically the presence of nucleic acid about which many capsids assemble. The major challenge of such work is computational: simulating fine-scale assembly pathways on the scale and in the parameter domains of real viruses is far too computationally costly to allow for explicit models of nucleic acid interaction. We bypass that limitation by applying analytical models of nucleic acid effects to adjust kinetic rate parameters learned from in vitro data to see how these adjustments, singly or in combination, might affect fine-scale assembly progress. The resulting simulations exhibit surprising behavioral complexity, with distinct effects often acting synergistically to drive efficient assembly and alter pathways relative to the in vitro model. The work demonstrates how computer simulations can help us understand how assembly might differ between the in vitro and in vivo environments and what features of the cellular environment account for these differences. PMID:27244559

Self-assembly of metal nanowires induced by alternating current electric fields

NASA Astrophysics Data System (ADS)

García-Sánchez, Pablo; Arcenegui, Juan J.; Morgan, Hywel; Ramos, Antonio

2015-01-01

We describe the reversible assembly of an aqueous suspension of metal nanowires into two different 2-dimensional stable configurations. The assembly is induced by an AC electric field of magnitude around 10 kV/m. It is known that single metal nanowires orientate parallel to the electric field for all values of applied frequency, according to two different mechanisms depending on the frequency. These different mechanisms also govern the mutual interaction between nanowires, which leads to directed-assembly into distinctive structures, the shape of which depends on the frequency of the applied field. We show that for frequencies higher than the typical frequency for charging the electrical double layer at the metal-electrolyte interface, dipole-dipole interaction leads to the formation of chains of nanowires. For lower frequencies, the nanowires form wavy bands perpendicular to the electric field direction. This behavior appears to be driven by the electroosmotic flow induced on the metal surface of the nanowires. Remarkably, no similar structures have been reported in previous studies of nanowires.

Modelling of DNA-Mediated of Two- and -Three dimensional Protein-Protein and Protein-Nanoparticle Self-Assembly

NASA Astrophysics Data System (ADS)

Millan, Jaime; McMillan, Janet; Brodin, Jeff; Lee, Byeongdu; Mirkin, Chad; Olvera de La Cruz, Monica

Programmable DNA interactions represent a robust scheme to self-assemble a rich variety of tunable superlattices, where intrinsic and in some cases non-desirable nano-scale building blocks interactions are substituted for DNA hybridization events. Recent advances in synthesis has allowed the extension of this successful scheme to proteins, where DNA distribution can be tuned independently of protein shape by selectively addressing surface residues, giving rise to assembly properties in three dimensional protein-nanoparticle superlattices dependent on DNA distribution. In parallel to this advances, we introduced a scalable coarse-grained model that faithfully reproduces the previously observed co-assemblies from nanoparticles and proteins conjugates. Herein, we implement this numerical model to explain the stability of complex protein-nanoparticle binary superlattices and to elucidate experimentally inaccessible features such as protein orientation. Also, we will discuss systematic studies that highlight the role of DNA distribution and sequence on two-dimensional protein-protein and protein-nanoparticle superlattices.

The nucleolar helicase DDX56 redistributes to West Nile virus assembly sites.

PubMed

Reid, Colleen R; Hobman, Tom C

2017-01-01

Flaviviruses, including the human pathogen, West Nile virus (WNV), are known to co-opt many host factors for their replication and propagation. To this end, we previously reported that the nucleolar DEAD-box RNA helicase, DDX56, is important for production of infectious WNV virions. In this study, we show that WNV infection results in relocalization of DDX56 from nucleoli to virus assembly sites on the endoplasmic reticululm (ER), an observation that is consistent with a role for DDX56 in WNV virion assembly. Super-resolution microscopy revealed that capsid and DDX56 localized to the same subcompartment of the ER, however, unexpectedly, stable interaction between these two proteins was only detected in the nucleus. Together, these data suggest that DDX56 relocalizes to the site of virus assembly during WNV infection and that its interaction with WNV capsid in the cytoplasm may occur transiently during virion morphogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Ministry-Related Burnout and Stress Coping Mechanisms Among Assemblies of God-Ordained Clergy in Minnesota.

PubMed

Visker, Joseph D; Rider, Taylor; Humphers-Ginther, Anastasia

2017-06-01

Clergy members suffering from burnout face both personal and professional complications. This study assessed occupational burnout and stress coping mechanisms among ministers affiliated with the Assemblies of God, a denomination that has received little attention in previous burnout-related studies. A cross-sectional design was employed using two instruments with acceptable psychometric properties. Approximately 65 % of those surveyed were either suffering from burnout or on the verge of burnout. Statistical differences in coping mechanism use were found between those who were and were not experiencing some level of burnout. The results of this study add to the existing understanding of occupational burnout among clergy members and ways in which burnout may be alleviated.

Strategies for optimizing BioNano and Dovetail explored through a second reference quality assembly for the legume model, Medicago truncatula.

PubMed

Moll, Karen M; Zhou, Peng; Ramaraj, Thiruvarangan; Fajardo, Diego; Devitt, Nicholas P; Sadowsky, Michael J; Stupar, Robert M; Tiffin, Peter; Miller, Jason R; Young, Nevin D; Silverstein, Kevin A T; Mudge, Joann

2017-08-04

Third generation sequencing technologies, with sequencing reads in the tens- of kilo-bases, facilitate genome assembly by spanning ambiguous regions and improving continuity. This has been critical for plant genomes, which are difficult to assemble due to high repeat content, gene family expansions, segmental and tandem duplications, and polyploidy. Recently, high-throughput mapping and scaffolding strategies have further improved continuity. Together, these long-range technologies enable quality draft assemblies of complex genomes in a cost-effective and timely manner. Here, we present high quality genome assemblies of the model legume plant, Medicago truncatula (R108) using PacBio, Dovetail Chicago (hereafter, Dovetail) and BioNano technologies. To test these technologies for plant genome assembly, we generated five assemblies using all possible combinations and ordering of these three technologies in the R108 assembly. While the BioNano and Dovetail joins overlapped, they also showed complementary gains in continuity and join numbers. Both technologies spanned repetitive regions that PacBio alone was unable to bridge. Combining technologies, particularly Dovetail followed by BioNano, resulted in notable improvements compared to Dovetail or BioNano alone. A combination of PacBio, Dovetail, and BioNano was used to generate a high quality draft assembly of R108, a M. truncatula accession widely used in studies of functional genomics. As a test for the usefulness of the resulting genome sequence, the new R108 assembly was used to pinpoint breakpoints and characterize flanking sequence of a previously identified translocation between chromosomes 4 and 8, identifying more than 22.7 Mb of novel sequence not present in the earlier A17 reference assembly. Adding Dovetail followed by BioNano data yielded complementary improvements in continuity over the original PacBio assembly. This strategy proved efficient and cost-effective for developing a quality draft assembly compared to traditional reference assemblies.

Environment-Dependent Guest Exchange in Supramolecular Hosts

PubMed Central

2015-01-01

Dynamic exchange of guest molecules, encapsulated in host assemblies, is a phenomenon in supramolecular chemistry that has important implications in several applications. While the mechanism of exchange in micellar assemblies has been previously investigated, the effect of host and guest environment upon the guest-exchange dynamics has received little attention, if any. In this paper, we study the guest-exchange mechanism in pH-sensitive nanogels along with pH-insensitive nanogels as a control. By systematically comparing the behavior of these nanogels, we show that size, concentration, and hydrophobicity can all play a critical role in guest-exchange dynamics. More importantly, these studies reveal that the dominant mechanism of guest exchange can intimately depend on environmental factors. PMID:25244305

Aliphatic peptides show similar self-assembly to amyloid core sequences, challenging the importance of aromatic interactions in amyloidosis.

PubMed

Lakshmanan, Anupama; Cheong, Daniel W; Accardo, Angelo; Di Fabrizio, Enzo; Riekel, Christian; Hauser, Charlotte A E

2013-01-08

The self-assembly of abnormally folded proteins into amyloid fibrils is a hallmark of many debilitating diseases, from Alzheimer's and Parkinson diseases to prion-related disorders and diabetes type II. However, the fundamental mechanism of amyloid aggregation remains poorly understood. Core sequences of four to seven amino acids within natural amyloid proteins that form toxic fibrils have been used to study amyloidogenesis. We recently reported a class of systematically designed ultrasmall peptides that self-assemble in water into cross-β-type fibers. Here we compare the self-assembly of these peptides with natural core sequences. These include core segments from Alzheimer's amyloid-β, human amylin, and calcitonin. We analyzed the self-assembly process using circular dichroism, electron microscopy, X-ray diffraction, rheology, and molecular dynamics simulations. We found that the designed aliphatic peptides exhibited a similar self-assembly mechanism to several natural sequences, with formation of α-helical intermediates being a common feature. Interestingly, the self-assembly of a second core sequence from amyloid-β, containing the diphenylalanine motif, was distinctly different from all other examined sequences. The diphenylalanine-containing sequence formed β-sheet aggregates without going through the α-helical intermediate step, giving a unique fiber-diffraction pattern and simulation structure. Based on these results, we propose a simplified aliphatic model system to study amyloidosis. Our results provide vital insight into the nature of early intermediates formed and suggest that aromatic interactions are not as important in amyloid formation as previously postulated. This information is necessary for developing therapeutic drugs that inhibit and control amyloid formation.

An improved assembly of the loblolly pine mega-genome using long-read single-molecule sequencing.

PubMed

Zimin, Aleksey V; Stevens, Kristian A; Crepeau, Marc W; Puiu, Daniela; Wegrzyn, Jill L; Yorke, James A; Langley, Charles H; Neale, David B; Salzberg, Steven L

2017-01-01

The 22-gigabase genome of loblolly pine (Pinus taeda) is one of the largest ever sequenced. The draft assembly published in 2014 was built entirely from short Illumina reads, with lengths ranging from 100 to 250 base pairs (bp). The assembly was quite fragmented, containing over 11 million contigs whose weighted average (N50) size was 8206 bp. To improve this result, we generated approximately 12-fold coverage in long reads using the Single Molecule Real Time sequencing technology developed at Pacific Biosciences. We assembled the long and short reads together using the MaSuRCA mega-reads assembly algorithm, which produced a substantially better assembly, P. taeda version 2.0. The new assembly has an N50 contig size of 25 361, more than three times as large as achieved in the original assembly, and an N50 scaffold size of 107 821, 61% larger than the previous assembly. © The Author 2017. Published by Oxford University Press.

Self-assembled monolayer and method of making

DOEpatents

Fryxell, Glen E [Kennewick, WA; Zemanian, Thomas S [Richland, WA; Liu, Jun [West Richland, WA; Shin, Yongsoon [Richland, WA

2003-03-11

According to the present invention, the previously known functional material having a self-assembled monolayer on a substrate has a plurality of assembly molecules each with an assembly atom with a plurality of bonding sites (four sites when silicon is the assembly molecule) wherein a bonding fraction (or fraction) of fully bonded assembly atoms (the plurality of bonding sites bonded to an oxygen atom) has a maximum when made by liquid solution deposition, for example a maximum of 40% when silicon is the assembly molecule, and maximum surface density of assembly molecules was 5 silanes per square nanometer. Note that bonding fraction and surface population are independent parameters. The method of the present invention is an improvement to the known method for making a siloxane layer on a substrate, wherein instead of a liquid phase solution chemistry, the improvement is a supercritical phase chemistry. The present invention has the advantages of greater fraction of oxygen bonds, greater surface density of assembly molecules and reduced time for reaction of about 5 minutes to about 24 hours.

Self-assembled monolayer and method of making

DOEpatents

Fryxell, Glen E.; Zemanian, Thomas S.; Liu, Jun; Shin, Yongsoon

2004-05-11

According to the present invention, the previously known functional material having a self-assembled monolayer on a substrate has a plurality of assembly molecules each with an assembly atom with a plurality of bonding sites (four sites when silicon is the assembly molecule) wherein a bonding fraction (or fraction) of fully bonded assembly atoms (the plurality of bonding sites bonded to an oxygen atom) has a maximum when made by liquid solution deposition, for example a maximum of 40% when silicon is the assembly molecule, and maximum surface density of assembly molecules was 5 silanes per square nanometer. Note that bonding fraction and surface population are independent parameters. The method of the present invention is an improvement to the known method for making a siloxane layer on a substrate, wherein instead of a liquid phase solution chemistry, the improvement is a supercritical phase chemistry. The present invention has the advantages of greater fraction of oxygen bonds, greater surface density of assembly molecules and reduced time for reaction of about 5 minutes to about 24 hours.

Self-Assembled Monolayer And Method Of Making

DOEpatents

Fryxell, Glen E.; Zemanian, Thomas S.; Liu, Jun; Shin, Yongsoon

2004-06-22

According to the present invention, the previously known functional material having a self-assembled monolayer on a substrate has a plurality of assembly molecules each with an assembly atom with a plurality of bonding sites (four sites when silicon is the assembly molecule) wherein a bonding fraction (or fraction) of fully bonded assembly atoms (the plurality of bonding sites bonded to an oxygen atom) has a maximum when made by liquid solution deposition, for example a maximum of 40% when silicon is the assembly molecule, and maximum surface density of assembly molecules was 5 silanes per square nanometer. Note that bonding fraction and surface population are independent parameters. The method of the present invention is an improvement to the known method for making a siloxane layer on a substrate, wherein instead of a liquid phase solution chemistry, the improvement is a supercritical phase chemistry. The present invention has the advantages of greater fraction of oxygen bonds, greater surface density of assembly molecules and reduced time for reaction of about 5 minutes to about 24 hours.

Self-Assembled Monolayer And Method Of Making

DOEpatents

Fryxell, Glen E.; Zemanian, Thomas S.; Liu, Jun; Shin, Yongsoon

2005-01-25

According to the present invention, the previously known functional material having a self-assembled monolayer on a substrate has a plurality of assembly molecules each with an assembly atom with a plurality of bonding sites (four sites when silicon is the assembly molecule) wherein a bonding fraction (or fraction) of fully bonded assembly atoms (the plurality of bonding sites bonded to an oxygen atom) has a maximum when made by liquid solution deposition, for example a maximum of 40% when silicon is the assembly molecule, and maximum surface density of assembly molecules was 5 silanes per square nanometer. Note that bonding fraction and surface population are independent parameters. The method of the present invention is an improvement to the known method for making a siloxane layer on a substrate, wherein instead of a liquid phase solution chemistry, the improvement is a supercritical phase chemistry. The present invention has the advantages of greater fraction of oxygen bonds, greater surface density of assembly molecules and reduced time for reaction of about 5 minutes to about 24 hours.

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome.

PubMed

Bickhart, Derek M; Rosen, Benjamin D; Koren, Sergey; Sayre, Brian L; Hastie, Alex R; Chan, Saki; Lee, Joyce; Lam, Ernest T; Liachko, Ivan; Sullivan, Shawn T; Burton, Joshua N; Huson, Heather J; Nystrom, John C; Kelley, Christy M; Hutchison, Jana L; Zhou, Yang; Sun, Jiajie; Crisà, Alessandra; Ponce de León, F Abel; Schwartz, John C; Hammond, John A; Waldbieser, Geoffrey C; Schroeder, Steven G; Liu, George E; Dunham, Maitreya J; Shendure, Jay; Sonstegard, Tad S; Phillippy, Adam M; Van Tassell, Curtis P; Smith, Timothy P L

2017-04-01

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus) based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced what is, to our knowledge, the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ∼400-fold improvement in continuity due to properly assembled gaps, compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex yet produced for an individual of a ruminant species.

Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome

PubMed Central

Bickhart, Derek M.; Rosen, Benjamin D.; Koren, Sergey; Sayre, Brian L.; Hastie, Alex R.; Chan, Saki; Lee, Joyce; Lam, Ernest T.; Liachko, Ivan; Sullivan, Shawn T.; Burton, Joshua N.; Huson, Heather J.; Nystrom, John C.; Kelley, Christy M.; Hutchison, Jana L.; Zhou, Yang; Sun, Jiajie; Crisà, Alessandra; de León, F. Abel Ponce; Schwartz, John C.; Hammond, John A.; Waldbieser, Geoffrey C.; Schroeder, Steven G.; Liu, George E.; Dunham, Maitreya J.; Shendure, Jay; Sonstegard, Tad S.; Phillippy, Adam M.; Van Tassell, Curtis P.; Smith, Timothy P.L.

2018-01-01

The decrease in sequencing cost and increased sophistication of assembly algorithms for short-read platforms has resulted in a sharp increase in the number of species with genome assemblies. However, these assemblies are highly fragmented, with many gaps, ambiguities, and errors, impeding downstream applications. We demonstrate current state of the art for de novo assembly using the domestic goat (Capra hircus), based on long reads for contig formation, short reads for consensus validation, and scaffolding by optical and chromatin interaction mapping. These combined technologies produced the most continuous de novo mammalian assembly to date, with chromosome-length scaffolds and only 649 gaps. Our assembly represents a ~400-fold improvement in continuity due to properly assembled gaps compared to the previously published C. hircus assembly, and better resolves repetitive structures longer than 1 kb, representing the largest repeat family and immune gene complex ever produced for an individual of a ruminant species. PMID:28263316

Erratum to: An improved assembly of the loblolly pine mega-genome using long-read single-molecule sequencing.

PubMed

Zimin, Aleksey V; Stevens, Kristian A; Crepeau, Marc W; Puiu, Daniela; Wegrzyn, Jill L; Yorke, James A; Langley, Charles H; Neale, David B; Salzberg, Steven L

2017-10-01

The 22-gigabase genome of loblolly pine (Pinus taeda) is one of the largest ever sequenced. The draft assembly published in 2014 was built entirely from short Illumina reads, with lengths ranging from 100 to 250 base pairs (bp). The assembly was quite fragmented, containing over 11 million contigs whose weighted average (N50) size was 8206 bp. To improve this result, we generated approximately 12-fold coverage in long reads using the Single Molecule Real Time sequencing technology developed at Pacific Biosciences. We assembled the long and short reads together using the MaSuRCA mega-reads assembly algorithm, which produced a substantially better assembly, P. taeda version 2.0. The new assembly has an N50 contig size of 25 361, more than three times as large as achieved in the original assembly, and an N50 scaffold size of 107 821, 61% larger than the previous assembly. © The Authors 2017. Published by Oxford University Press.

Manual Skill Training of Retarded Children.

ERIC Educational Resources Information Center

Pomerantz, David J.

1975-01-01

In an on-going pilot study, training procedures previously found successful with moderately and severely retarded adolescents and adults have been adapted to teaching trainable retarded children (6-, 8-, and 10-years-old) to assemble a 14-piece coaster brake. Modifications in the carefully detailed task analysis approach have included the need for…

Identification of Genes Related to Learning and Memory in the Brain Transcriptome of the Mollusc, "Hermissenda Crassicornis"

ERIC Educational Resources Information Center

Tamvacakis, Arianna N.; Senatore, Adriano; Katz, Paul S.

2015-01-01

The sea slug "Hermissenda crassicornis" (Mollusca, Gastropoda, Nudibranchia) has been studied extensively in associative learning paradigms. However, lack of genetic information previously hindered molecular-level investigations. Here, the "Hermissenda" brain transcriptome was sequenced and assembled de novo, producing 165,743…

Gal4-VP16 directs ATP-independent chromatin reorganization in a yeast chromatin assembly system.

PubMed

Robinson, Karen M; Schultz, Michael C

2005-03-22

Major insights into the regulation of chromatin organization have stemmed from biochemical studies using Gal4-VP16, a chimeric transcriptional activator in which the DNA binding domain of Gal4p is fused to the activation domain of viral protein VP16. Unexpectedly, given previous intensive efforts to understand how Gal4-VP16 functions in the context of chromatin, we have uncovered a new mode of chromatin reorganization that is dependent on Gal4-VP16. This reorganization is performed by an activity in a crude DEAE (CD) fraction from budding yeast which also supports ATP-dependent assembly of physiologically spaced nucleosome arrays. Biochemical analysis reveals that the activity tightly associates with chromatin and reorganizes nucleosome arrays by a mechanism which is insensitive to ATP depletion after nucleosome assembly. It generates a chromatin organization in which a nucleosome is stably positioned immediately adjacent to Gal4p binding sites in the template DNA. Individual deletion of genes previously implicated in chromatin assembly and remodeling, namely, the histone chaperones NAP1, ASF1, and CAC1 and the SNF2-like DEAD/H ATPases SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20, does not significantly perturb reorganization. Therefore, Gal4-VP16-directed chromatin reorganization in yeast can occur by an ATP-independent mechanism that does not require SAGA, SWI/SNF, Isw1, or Isw2 chromatin remodeling complexes.

Expandable tubulars for use in geologic structures

DOEpatents

Spray, Jeffery A.; Svedeman, Steven; Walter, David; Mckeighan, Peter; Siebanaler, Shane; Dewhurst, Peter; Hobson, Steven; Foss, Doug; Wirz, Holger; Sharpe, Aaron; Apostal, Michael

2014-08-12

An expandable tubular includes a plurality of leaves formed from sheet material that have curved surfaces. The leaves extend around a portion or fully around the diameter of the tubular structure. Some of the adjacent leaves of the tubular are coupled together. The tubular is compressed to a smaller diameter so that it can be inserted through previously deployed tubular assemblies. Once the tubular is properly positioned, it is deployed and coupled or not coupled to a previously deployed tubular assembly. The tubular is useful for all types of wells and boreholes.

j5 DNA assembly design automation.

PubMed

Hillson, Nathan J

2014-01-01

Modern standardized methodologies, described in detail in the previous chapters of this book, have enabled the software-automated design of optimized DNA construction protocols. This chapter describes how to design (combinatorial) scar-less DNA assembly protocols using the web-based software j5. j5 assists biomedical and biotechnological researchers construct DNA by automating the design of optimized protocols for flanking homology sequence as well as type IIS endonuclease-mediated DNA assembly methodologies. Unlike any other software tool available today, j5 designs scar-less combinatorial DNA assembly protocols, performs a cost-benefit analysis to identify which portions of an assembly process would be less expensive to outsource to a DNA synthesis service provider, and designs hierarchical DNA assembly strategies to mitigate anticipated poor assembly junction sequence performance. Software integrated with j5 add significant value to the j5 design process through graphical user-interface enhancement and downstream liquid-handling robotic laboratory automation.

Transcriptome assembly, gene annotation and tissue gene expression atlas of the rainbow trout

USDA-ARS?s Scientific Manuscript database

Efforts to obtain a comprehensive genome sequence for rainbow trout are ongoing and will be complimented by transcriptome information that will enhance genome assembly and annotation. Previously, we reported a transcriptome reference sequence using a 19X coverage of Sanger and 454-pyrosequencing dat...

The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mattiroli, Francesca; Gu, Yajie; Balsbaugh, Jeremy L.

Nucleosome assembly following DNA replication controls epigenome maintenance and genome integrity. Chromatin assembly factor 1 (CAF-1) is the histone chaperone responsible for histone (H3-H4)2 deposition following DNA synthesis. Structural and functional details for this chaperone complex and its interaction with histones are slowly emerging. Using hydrogen-deuterium exchange coupled to mass spectrometry, combined with in vitro and in vivo mutagenesis studies, we identified the regions involved in the direct interaction between the yeast CAF-1 subunits, and mapped the CAF-1 domains responsible for H3-H4 binding. The large subunit, Cac1 organizes the assembly of CAF-1. Strikingly, H3-H4 binding is mediated by a compositemore » interface, shaped by Cac1-bound Cac2 and the Cac1 acidic region. Cac2 is indispensable for productive histone binding, while deletion of Cac3 has only moderate effects on H3-H4 binding and nucleosome assembly. These results define direct structural roles for yeast CAF-1 subunits and uncover a previously unknown critical function of the middle subunit in CAF-1.« less

Genome assembly and transcriptome resource for river buffalo, Bubalus bubalis (2n = 50)

PubMed Central

Iamartino, Daniela; Pruitt, Kim D; Sonstegard, Tad; Smith, Timothy P L; Low, Wai Yee; Biagini, Tommaso; Bomba, Lorenzo; Capomaccio, Stefano; Castiglioni, Bianca; Coletta, Angelo; Corrado, Federica; Ferré, Fabrizio; Iannuzzi, Leopoldo; Lawley, Cynthia; Macciotta, Nicolò; McClure, Matthew; Mancini, Giordano; Matassino, Donato; Mazza, Raffaele; Milanesi, Marco; Moioli, Bianca; Morandi, Nicola; Ramunno, Luigi; Peretti, Vincenzo; Pilla, Fabio; Ramelli, Paola; Schroeder, Steven; Strozzi, Francesco; Thibaud-Nissen, Francoise; Zicarelli, Luigi; Ajmone-Marsan, Paolo; Valentini, Alessio; Chillemi, Giovanni; Zimin, Aleksey

2017-01-01

Abstract Water buffalo is a globally important species for agriculture and local economies. A de novo assembled, well-annotated reference sequence for the water buffalo is an important prerequisite for studying the biology of this species, and is necessary to manage genetic diversity and to use modern breeding and genomic selection techniques. However, no such genome assembly has been previously reported. There are 2 species of domestic water buffalo, the river (2n = 50) and the swamp (2n = 48) buffalo. Here we describe a draft quality reference sequence for the river buffalo created from Illumina GA and Roche 454 short read sequences using the MaSuRCA assembler. The assembled sequence is 2.83 Gb, consisting of 366 983 scaffolds with a scaffold N50 of 1.41 Mb and contig N50 of 21 398 bp. Annotation of the genome was supported by transcriptome data from 30 tissues and identified 21 711 predicted protein coding genes. Searches for complete mammalian BUSCO gene groups found 98.6% of curated single copy orthologs present among predicted genes, which suggests a high level of completeness of the genome. The annotated sequence is available from NCBI at accession GCA_000471725.1. PMID:29048578

The effect of Au amount on size uniformity of self-assembled Au nanoparticles

NASA Astrophysics Data System (ADS)

Chen, S.-H.; Wang, D.-C.; Chen, G.-Y.; Chen, K.-Y.

2008-03-01

The self-assembled fabrication of nanostructure, a dreaming approach in the area of fabrication engineering, is the ultimate goal of this research. A finding was proved through previous research that the size of the self-assembled gold nanoparticles could be controlled with the mole ratio between AuCl4- and thiol. In this study, the moles of Au were fixed, only the moles of thiol were adjusted. Five different mole ratios of Au/S with their effect on size uniformity were investigated. The mole ratios were 1:1/16, 1:1/8, 1:1, 1:8, 1:16, respectively. The size distributions of the gold nanoparticles were analyzed by Mac-View analysis software. HR-TEM was used to derive images of self-assembled gold nanoparticles. The result reached was also the higher the mole ratio between AuCl4- and thiol the bigger the self-assembled gold nanoparticles. Under the condition of moles of Au fixed, the most homogeneous nanoparticles in size distribution derived with the mole ratio of 1:1/8 between AuCl4- and thiol. The obtained nanoparticles could be used, for example, in uniform surface nanofabrication, leading to the fabrication of ordered array of quantum dots.

Structural Study of the RIPoptosome Core Reveals a Helical Assembly for Kinase Recruitment

PubMed Central

2015-01-01

Receptor interaction protein kinase 1 (RIP1) is a molecular cell-fate switch. RIP1, together with Fas-associated protein with death domain (FADD) and caspase-8, forms the RIPoptosome that activates apoptosis. RIP1 also associates with RIP3 to form the necrosome that triggers necroptosis. The RIPoptosome assembles through interactions between the death domains (DDs) of RIP1 and FADD and between death effector domains (DEDs) of FADD and caspase-8. In this study, we analyzed the overall structure of the RIP1 DD/FADD DD complex, the core of the RIPoptosome, by negative-stain electron microscopy and modeling. The results show that RIP1 DD and FADD DD form a stable complex in vitro similar to the previously described Fas DD/FADD DD complex, suggesting that the RIPoptosome and the Fas death-inducing signaling complex share a common assembly mechanism. Both complexes adopt a helical conformation that requires type I, II, and III interactions between the death domains. PMID:25119434

Bacteriophage Tail-Tube Assembly Studied by Proton-Detected 4D Solid-State NMR

DOE PAGES

Zinke, Maximilian; Fricke, Pascal; Samson, Camille; ...

2017-07-07

Obtaining unambiguous resonance assignments remains a major bottleneck in solid-state NMR studies of protein structure and dynamics. Particularly for supramolecular assemblies with large subunits (>150 residues), the analysis of crowded spectral data presents a challenge, even if three-dimensional (3D) spectra are used. Here, we present a proton-detected 4D solid-state NMR assignment procedure that is tailored for large assemblies. The key to recording 4D spectra with three indirect carbon or nitrogen dimensions with their inherently large chemical shift dispersion lies in the use of sparse non-uniform sampling (as low as 2 %). As a proof of principle, we acquired 4D (H)COCANH,more » (H)CACONH, and (H)CBCANH spectra of the 20 kDa bacteriophage tail-tube protein gp17.1 in a total time of two and a half weeks. These spectra were sufficient to obtain complete resonance assignments in a straightforward manner without use of previous solution NMR data.« less

Bacteriophage Tail-Tube Assembly Studied by Proton-Detected 4D Solid-State NMR

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zinke, Maximilian; Fricke, Pascal; Samson, Camille

Obtaining unambiguous resonance assignments remains a major bottleneck in solid-state NMR studies of protein structure and dynamics. Particularly for supramolecular assemblies with large subunits (>150 residues), the analysis of crowded spectral data presents a challenge, even if three-dimensional (3D) spectra are used. Here, we present a proton-detected 4D solid-state NMR assignment procedure that is tailored for large assemblies. The key to recording 4D spectra with three indirect carbon or nitrogen dimensions with their inherently large chemical shift dispersion lies in the use of sparse non-uniform sampling (as low as 2 %). As a proof of principle, we acquired 4D (H)COCANH,more » (H)CACONH, and (H)CBCANH spectra of the 20 kDa bacteriophage tail-tube protein gp17.1 in a total time of two and a half weeks. These spectra were sufficient to obtain complete resonance assignments in a straightforward manner without use of previous solution NMR data.« less

Numerical simulation of colloidal self-assembly of super-hydrophobic arachnid cerotegument structures.

PubMed

Filippov, Alexander É; Wolff, Jonas O; Seiter, Michael; Gorb, Stanislav N

2017-10-07

Certain arachnids exhibit complex coatings of their exoskeleton, consisting of globular structures with complex surface features. This, so-called, cerotegument is formed by a multi-component colloidal secretion that self-assembles and cures on the body surface, and leads to high water repellency. Previous ultrastructural studies revealed the involvement of different glandular cells that contribute different components to the secretion mixture, but the overall process of self-assembly into the complex regular structures observed remained highly unclear. Here we study this process from a theoretical point of view, starting from the so-called Tammes-problem. We show that slight changes of simple parameters lead to a variety of morphologies that are highly similar to the ones observed in the species specific cerotegument structures of whip-spiders. These results are not only important for our understanding of the formation of globular hierarchical structures in nature, but also for the fabrication of novel surface coatings by colloidal lithography. Copyright © 2017 Elsevier Ltd. All rights reserved.

The relationship between physical workload and quality within line-based assembly.

PubMed

Ivarsson, Anna; Eek, Frida

2016-07-01

Reducing costs and improvement of product quality are considered important to ensure productivity within a company. Quality deviations during production processes and ergonomics have previously shown to be associated. This study explored the relationship between physical workload and real (found during production processes) and potential (need of extra time and assistance to complete tasks) quality deviations in a line-based assembly plant. The physical workload on and the work rotation between 52 workstations were assessed. As the outcome, real and potential quality deviations were studied during 10 weeks. Results show that workstations with higher physical workload had significantly more real deviations compared to lower workload stations. Static work posture had significantly more potential deviations. Rotation between high and low workload was related to fewer quality deviations compared to rotation between only high workload stations. In conclusion, physical ergonomics seems to be related to real and potential quality deviation within line-based assembly. Practitioner Summary: To ensure good productivity in manufacturing industries, it is important to reduce costs and improve product quality. This study shows that high physical workload is associated with quality deviations and need of extra time and assistance to complete tasks within line-based assembly, which can be financially expensive for a company.

Scale dependence of the mechanics of active gels with increasing motor concentration.

PubMed

Sonn-Segev, Adar; Bernheim-Groswasser, Anne; Roichman, Yael

2017-10-18

Actin is a protein that plays an essential role in maintaining the mechanical integrity of cells. In response to strong external stresses, it can assemble into large bundles, but it grows into a fine branched network to induce cell motion. In some cases, the self-organization of actin fibers and networks involves the action of bipolar filaments of the molecular motor myosin. Such self-organization processes mediated by large myosin bipolar filaments have been studied extensively in vitro. Here we create active gels, composed of single actin filaments and small myosin bipolar filaments. The active steady state in these gels persists long enough to enable the characterization of their mechanical properties using one and two point microrheology. We study the effect of myosin concentration on the mechanical properties of this model system for active matter, for two different motor assembly sizes. In contrast to previous studies of networks with large motor assemblies, we find that the fluctuations of tracer particles embedded in the network decrease in amplitude as motor concentration increases. Nonetheless, we show that myosin motors stiffen the actin networks, in accordance with bulk rheology measurements of networks containing larger motor assemblies. This implies that such stiffening is of universal nature and may be relevant to a wider range of cytoskeleton-based structures.

Nonmedially assembled F-actin cables incorporate into the actomyosin ring in fission yeast

PubMed Central

Huang, Junqi; Huang, Yinyi; Yu, Haochen; Subramanian, Dhivya; Padmanabhan, Anup; Thadani, Rahul; Tao, Yaqiong; Tang, Xie; Wedlich-Soldner, Roland

2012-01-01

In many eukaryotes, cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring. Despite the central role of this ring in cytokinesis, the mechanism of F-actin assembly and accumulation in the ring is not fully understood. In this paper, we investigate the mechanism of F-actin assembly during cytokinesis in Schizosaccharomyces pombe using lifeact as a probe to monitor actin dynamics. Previous work has shown that F-actin in the actomyosin ring is assembled de novo at the division site. Surprisingly, we find that a significant fraction of F-actin in the ring was recruited from formin-Cdc12p nucleated long actin cables that were generated at multiple nonmedial locations and incorporated into the ring by a combination of myosin II and myosin V activities. Our results, together with findings in animal cells, suggest that de novo F-actin assembly at the division site and directed transport of F-actin cables assembled elsewhere can contribute to ring assembly. PMID:23185032

QSRA: a quality-value guided de novo short read assembler.

PubMed

Bryant, Douglas W; Wong, Weng-Keen; Mockler, Todd C

2009-02-24

New rapid high-throughput sequencing technologies have sparked the creation of a new class of assembler. Since all high-throughput sequencing platforms incorporate errors in their output, short-read assemblers must be designed to account for this error while utilizing all available data. We have designed and implemented an assembler, Quality-value guided Short Read Assembler, created to take advantage of quality-value scores as a further method of dealing with error. Compared to previous published algorithms, our assembler shows significant improvements not only in speed but also in output quality. QSRA generally produced the highest genomic coverage, while being faster than VCAKE. QSRA is extremely competitive in its longest contig and N50/N80 contig lengths, producing results of similar quality to those of EDENA and VELVET. QSRA provides a step closer to the goal of de novo assembly of complex genomes, improving upon the original VCAKE algorithm by not only drastically reducing runtimes but also increasing the viability of the assembly algorithm through further error handling capabilities.

Carbon nanotubes as templates for polymerized lipid assemblies

NASA Astrophysics Data System (ADS)

Thauvin, Cédric; Rickling, Stéphane; Schultz, Patrick; Célia, Hervé; Meunier, Stéphane; Mioskowski, Charles

2008-12-01

Amphiphilic molecules-molecules that have both hydrophobic and hydrophilic properties-can self-assemble in water to form diverse structures such as micelles, vesicles and tubes, and these nanostructures can be used for delivering drugs, stabilizing membrane proteins or as nanoreactors. We have previously shown that lipids can self-organize on the surface of single-walled carbon nanotubes into regular ring-shaped assemblies. Here we show that these lipid assemblies can be polymerized and isolated from the nanotube template by application of an electric field. We also demonstrate that these assemblies are monodispersed, water-soluble, and can dissolve various hydrophobic rylene dyes, fullerenes and membrane proteins. The stability of these constructs and their diverse applications will be useful in the fields of cosmetics, medicine and material sciences.

Optimal use of human and machine resources for Space Station assembly operations

NASA Technical Reports Server (NTRS)

Parrish, Joseph C.

1988-01-01

This paper investigates the issues involved in determining the best mix of human and machine resources for assembly of the Space Station. It presents the current Station assembly sequence, along with descriptions of the available assembly resources. A number of methodologies for optimizing the human/machine tradeoff problem have been developed, but the Space Station assembly offers some unique issues that have not yet been addressed. These include a strong constraint on available EVA time for early flights and a phased deployment of assembly resources over time. A methodology for incorporating the previously developed decision methods to the special case of the Space Station is presented. This methodology emphasizes an application of multiple qualitative and quantitative techniques, including simulation and decision analysis, for producing an objective, robust solution to the tradeoff problem.

Better ILP models for haplotype assembly.

PubMed

Etemadi, Maryam; Bagherian, Mehri; Chen, Zhi-Zhong; Wang, Lusheng

2018-02-19

The haplotype assembly problem for diploid is to find a pair of haplotypes from a given set of aligned Single Nucleotide Polymorphism (SNP) fragments (reads). It has many applications in association studies, drug design, and genetic research. Since this problem is computationally hard, both heuristic and exact algorithms have been designed for it. Although exact algorithms are much slower, they are still of great interest because they usually output significantly better solutions than heuristic algorithms in terms of popular measures such as the Minimum Error Correction (MEC) score, the number of switch errors, and the QAN50 score. Exact algorithms are also valuable because they can be used to witness how good a heuristic algorithm is. The best known exact algorithm is based on integer linear programming (ILP) and it is known that ILP can also be used to improve the output quality of every heuristic algorithm with a little decline in speed. Therefore, faster ILP models for the problem are highly demanded. As in previous studies, we consider not only the general case of the problem but also its all-heterozygous case where we assume that if a column of the input read matrix contains at least one 0 and one 1, then it corresponds to a heterozygous SNP site. For both cases, we design new ILP models for the haplotype assembly problem which aim at minimizing the MEC score. The new models are theoretically better because they contain significantly fewer constraints. More importantly, our experimental results show that for both simulated and real datasets, the new model for the all-heterozygous (respectively, general) case can usually be solved via CPLEX (an ILP solver) at least 5 times (respectively, twice) faster than the previous bests. Indeed, the running time can sometimes be 41 times better. This paper proposes a new ILP model for the haplotype assembly problem and its all-heterozygous case, respectively. Experiments with both real and simulated datasets show that the new models can be solved within much shorter time by CPLEX than the previous bests. We believe that the models can be used to improve heuristic algorithms as well.

Self-assembled nitrogen-doped fullerenes and their catalysis for fuel cell and rechargeable metal-air battery applications.

PubMed

Noh, Seung Hyo; Kwon, Choah; Hwang, Jeemin; Ohsaka, Takeo; Kim, Beom-Jun; Kim, Tae-Young; Yoon, Young-Gi; Chen, Zhongwei; Seo, Min Ho; Han, Byungchan

2017-06-08

In this study, we report self-assembled nitrogen-doped fullerenes (N-fullerene) as non-precious catalysts, which are active for the oxygen reduction reaction (ORR) and oxygen evolution reaction (OER), and thus applicable for energy conversion and storage devices such as fuel cells and metal-air battery systems. We screen the best N-fullerene catalyst at the nitrogen doping level of 10 at%, not at the previously known doping level of 5 or 20 at% for graphene. We identify that the compressive surface strain induced by doped nitrogen plays a key role in the fine-tuning of catalytic activity.

Failure Engineering Study and Accelerated Stress Test Results for the Mars Global Surveyor Spacecraft's Power Shunt Assemblies

NASA Technical Reports Server (NTRS)

Gibbel, Mark; Larson, Tim

1999-01-01

Due to a post launch failure of a part a new plan for the Mars Global Surveyor was developed. This new plan involved the addition of many deep thermal cycles to the Power Shunt Assemblies (PSA's). This new plan exceeds the previous acceptance cold level, and fatigue life on packaging design. This presentation reviews the experiments that were used to test the capabilities of the PSA to function in the new situation. It also reviews the analyses preformed to verify the most likely failure mechanism, and the likelihood that these failures would impact the new mission requirements.

The Meeting of the Americas

NASA Astrophysics Data System (ADS)

Bates, John; Fucugauchi, Jaime Urrutia

2008-05-01

The AGU Joint Assembly, previously known as the Spring Meeting, has formally adopted the designation ``The Meeting of the Americas.'' This designation reflects the objective of the Joint Assembly to promote cooperative efforts among Earth and science societies in the Americas. To achieve this objective, future Joint Assembly locations will rotate among different cities in the United States, Canada, and Latin America; the Program Committee includes representatives from throughout the Americas; and the meetings will be convened jointly with scientific societies throughout the Americas.

The Evolution, Design and Implementation of the Minds in Motion Curriculum

ERIC Educational Resources Information Center

Cottone, Elizabeth; Chen, Wei-Bing; Brock, Laura

2013-01-01

Building on the empirical work of the previous two studies, this paper describes the development of the Minds In Motion curriculum (MIM), as well as the setting and circumstances of a randomized controlled trial conducted to evaluate this intervention. Throughout this paper the authors emphasize the benefits and challenges of assembling an…

Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (Fragaria vesca) with chromosome-scale contiguity.

PubMed

Edger, Patrick P; VanBuren, Robert; Colle, Marivi; Poorten, Thomas J; Wai, Ching Man; Niederhuth, Chad E; Alger, Elizabeth I; Ou, Shujun; Acharya, Charlotte B; Wang, Jie; Callow, Pete; McKain, Michael R; Shi, Jinghua; Collier, Chad; Xiong, Zhiyong; Mower, Jeffrey P; Slovin, Janet P; Hytönen, Timo; Jiang, Ning; Childs, Kevin L; Knapp, Steven J

2018-02-01

Although draft genomes are available for most agronomically important plant species, the majority are incomplete, highly fragmented, and often riddled with assembly and scaffolding errors. These assembly issues hinder advances in tool development for functional genomics and systems biology. Here we utilized a robust, cost-effective approach to produce high-quality reference genomes. We report a near-complete genome of diploid woodland strawberry (Fragaria vesca) using single-molecule real-time sequencing from Pacific Biosciences (PacBio). This assembly has a contig N50 length of ∼7.9 million base pairs (Mb), representing a ∼300-fold improvement of the previous version. The vast majority (>99.8%) of the assembly was anchored to 7 pseudomolecules using 2 sets of optical maps from Bionano Genomics. We obtained ∼24.96 Mb of sequence not present in the previous version of the F. vesca genome and produced an improved annotation that includes 1496 new genes. Comparative syntenic analyses uncovered numerous, large-scale scaffolding errors present in each chromosome in the previously published version of the F. vesca genome. Our results highlight the need to improve existing short-read based reference genomes. Furthermore, we demonstrate how genome quality impacts commonly used analyses for addressing both fundamental and applied biological questions. © The Authors 2017. Published by Oxford University Press.

Direct-Write Printing on Three-Dimensional Geometries for Miniaturized Detector and Electronic Assemblies

NASA Technical Reports Server (NTRS)

Paquette, Beth; Samuels, Margaret; Chen, Peng

2017-01-01

Direct-write printing techniques will enable new detector assemblies that were not previously possible with traditional assembly processes. Detector concepts were manufactured using this technology to validate repeatability. Additional detector applications and printed wires on a 3-dimensional magnetometer bobbin will be designed for print. This effort focuses on evaluating performance for direct-write manufacturing techniques on 3-dimensional surfaces. Direct-write manufacturing has the potential to reduce mass and volume for fabrication and assembly of advanced detector concepts by reducing trace widths down to 10 microns, printing on complex geometries, allowing new electronic concept production, and reduced production times of complex those electronics.

A Study on the Rectification Property of Self-Assembled Viologen Single Molecules Using a Scanning Tunneling Microscopy.

PubMed

Lee, Nam-Suk; Shin, Hoon-Kyu; Kwon, Young-Soo

2015-02-01

An ultrahigh vacuum scanning tunneling microscopy (UHV-STM) and a scanning tunneling spectroscopy (STS) are used measure the rectification property of self-assembled viologen single molecules (VC8SH, VC10SH, HSC8VC8SH, and HSC10VC10SH) in the previous study. Using STM we observe viologen single molecules in the self-assembled octanethiol (OT) SAM matrix. In the OT matrix a mixed phase that includes a c(4 x 2) superlattice of high-density standing up-phase is observed. We indicate high peak current-like rectifications at + 1.68 V(VC8SH), + 1.56 V(VC10SH), + 1.14 V(HSC8VC8SH), and + 1.04 V(HSC10VC10SH) based on the experiment implemented in this study. In addition, transition voltages (Vtrans) from direct tunneling to the Fowler-Nordheim tunneling are presented at 1.08 V(VC8SH), 0.97 V(VC10SH), 0.99 V(HSC8VC8SH), and 0.89 V(HSC1VC1SH).

Characterization and kinetics of surface functionalization and binding of biologically and chemically significant molecules

NASA Astrophysics Data System (ADS)

Steiner, Rachel

The purpose of this project is to investigate intermolecular interactions of organic molecular assemblies. By understanding the structure and physical interactions in these assemblies, we gain insights into practical applications for nanoscale systems built upon these surface structures. It is possible for organic chemists to create many forms of modified organic molecules, functionalizing them with specific reactive end groups. Through surface functionalization, enabling covalent or highly associative binding, it is possible to create ordered molecular assemblies of these molecules. Scientists can study the nature of this structure and the intermolecular interactions through spectroscopic, optical, and scattering experiments. To understand the self-assembly process in molecular systems, we preliminarily created monolayer films on silica substrates with a variety of organic molecules. In particular, we functionalized silica substrates with hydroxyl groups and covalently bound acid chloride functionalized aromatic compounds, with and without an underlying adhesion layer of 3-aminopropyltriethoxysilane. We characterized the monolayer assemblies with ellipsometry, UV-vis absorption spectroscopy, FTIR spectroscopy, and fluorescence/photoemission spectroscopy, obtaining a quantitative measure of the molecular surface coverage. In order to understand the nature of these molecular assemblies, we also pursued an in-depth kinetic study to control and optimize the monolayer formation process. Through use of UV-vis spectroscopy, we determined that the monolayer formation can best be modeled with diffusion-limited Langmuir kinetics. Specifically, we concluded that for anthracene acid chloride in dichloromethane the average diffusion coefficient was 1.6x10-7 cm2/sec. Additionally, we find we are able to achieve surface coverages of approximately 2x1014 molecules/cm2. Having established the ability to create ordered molecular assemblies, through surface functionalization, enabling covalent or highly associative binding, we continued to explore the field of molecular assemblies by studying the binding and structure of molecules to carbon nanostructures. Previous studies have shown that alkyl side chains and aromatic compounds, such as pyrene, will bind non-covalently to the sidewalls of carbon nanotubes through pi-pi interactions. We explored functionalization of carbon nanotubes and graphene by using microscopy to examine the adsorption of biomolecules onto nanotube sidewalls and graphene.

Kinetics of Surface-Driven Self-Assembly and Fatigue-Induced Disassembly of a Virus-Based Nanocoating.

PubMed

Valbuena, Alejandro; Mateu, Mauricio G

2017-02-28

Self-assembling protein layers provide a "bottom-up" approach for precisely organizing functional elements at the nanoscale over a large solid surface area. The design of protein sheets with architecture and physical properties suitable for nanotechnological applications may be greatly facilitated by a thorough understanding of the principles that underlie their self-assembly and disassembly. In a previous study, the hexagonal lattice formed by the capsid protein (CA) of human immunodeficiency virus (HIV) was self-assembled as a monomolecular layer directly onto a solid substrate, and its mechanical properties and dynamics at equilibrium were analyzed by atomic force microscopy. Here, we use atomic force microscopy to analyze the kinetics of self-assembly of the planar CA lattice on a substrate and of its disassembly, either spontaneous or induced by materials fatigue. Both self-assembly and disassembly of the CA layer are cooperative reactions that proceed until a phase equilibrium is reached. Self-assembly requires a critical protein concentration and is initiated by formation of nucleation points on the substrate, followed by lattice growth and eventual merging of CA patches into a continuous monolayer. Disassembly of the CA layer showed hysteresis and appears to proceed only after large enough defects (nucleation points) are formed in the lattice, whose number is largely increased by inducing materials fatigue that depends on mechanical load and its frequency. Implications of the kinetic results obtained for a better understanding of self-assembly and disassembly of the HIV capsid and protein-based two-dimensional nanomaterials and the design of anti-HIV drugs targeting (dis)assembly and biocompatible nanocoatings are discussed. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Robustness analysis of non-ordinary Petri nets for flexible assembly systems

NASA Astrophysics Data System (ADS)

Hsieh, Fu-Shiung

2010-05-01

Non-ordinary controlled Petri nets (NCPNs) have the advantages to model flexible assembly systems in which multiple identical resources may be required to perform an operation. However, existing studies on NCPNs are still limited. For example, the robustness properties of NCPNs have not been studied. This motivates us to develop an analysis method for NCPNs. Robustness analysis concerns the ability for a system to maintain operation in the presence of uncertainties. It provides an alternative way to analyse a perturbed system without reanalysis. In our previous research, we have analysed the robustness properties of several subclasses of ordinary controlled Petri nets. To study the robustness properties of NCPNs, we augment NCPNs with an uncertainty model, which specifies an upper bound on the uncertainties for each reachable marking. The resulting PN models are called non-ordinary controlled Petri nets with uncertainties (NCPNU). Based on NCPNU, the problem is to characterise the maximal tolerable uncertainties for each reachable marking. The computational complexities to characterise maximal tolerable uncertainties for each reachable marking grow exponentially with the size of the nets. Instead of considering general NCPNU, we limit our scope to a subclass of PN models called non-ordinary controlled flexible assembly Petri net with uncertainties (NCFAPNU) for assembly systems and study its robustness. We will extend the robustness analysis to NCFAPNU. We identify two types of uncertainties under which the liveness of NCFAPNU can be maintained.

Preparation of a novel composite nanofiber gel-encapsulated human placental extract through layer-by-layer self-assembly

PubMed Central

LIU, GUOHUI; CHEN, XI; ZHOU, WU; YANG, SHUHUA; YE, SHUNAN; CAO, FAQI; LIU, YI; XIONG, YUAN

2016-01-01

Aqueous human placenta extract (HPE) has been previously used to treat chronic soft tissue ulcer; however, the optimal dosage of HPE has yet to be elucidated. The present study investigated a novel nanofiber gel composed through layer-by-layer (LbL) self-assembly, in which HPE was encapsulated. IKVAV, RGD, RAD16 and FGL-PA were screened and combined to produce an optimal vehicle nanofiber gel through LbL assembly. Subsequently, the aqueous HPE was encapsulated into this nanofiber at the appropriate concentration, and the morphology, particle size, drug loading efficacy, encapsulation rate, release efficiency and structure validation were detected. The encapsulation efficiency of all three HPE samples was >90%, the nanofiber gel exhibited a slow releasing profile, and the structure of HPE encapsulated in the nanofiber gel was unvaried. In conclusion, this type of novel composite nanocapsules may offer a promising delivery system for HPE. PMID:27073463

Fractal dimension of microbead assemblies used for protein detection.

PubMed

Hecht, Ariel; Commiskey, Patrick; Lazaridis, Filippos; Argyrakis, Panos; Kopelman, Raoul

2014-11-10

We use fractal analysis to calculate the protein concentration in a rotating magnetic assembly of microbeads of size 1 μm, which has optimized parameters of sedimentation, binding sites and magnetic volume. We utilize the original Forrest-Witten method, but due to the relatively small number of bead particles, which is of the order of 500, we use a large number of origins and also a large number of algorithm iterations. We find a value of the fractal dimension in the range 1.70-1.90, as a function of the thrombin concentration, which plays the role of binding the microbeads together. This is in good agreement with previous results from magnetorotation studies. The calculation of the fractal dimension using multiple points of reference can be used for any assembly with a relatively small number of particles. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Conformational changes of the N-terminal part of Mason-Pfizer monkey virus p12 protein during multimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Knejzlik, Zdenek; Ulbrich, Pavel; Strohalm, Martin

2009-10-10

The Mason-Pfizer monkey virus is a prototype Betaretrovirus with the defining characteristic that it assembles spherical immature particles from Gag-related polyprotein precursors within the cytoplasm of the infected cell. It was shown previously that the N-terminal part of the Gag p12 domain (wt-Np12) is required for efficient assembly. However, the precise role for p12 in mediating Gag-Gag interaction is still poorly understood. In this study we employed detailed circular dichroism spectroscopy, electron microscopy and ultracentrifugation analyses of recombinant wt-Np12 prepared by in vitro transcription and translation. The wt-Np12 domain fragment forms fibrillar structures in a concentration-dependent manner. Assembly into fibersmore » is linked to a conformational transition from unfolded or another non-periodical state to alpha-helix during multimerization.« less

Self-assembly of marine exudate particles and their impact on the CCN properties of nascent marine aerosol

NASA Astrophysics Data System (ADS)

Schill, S.; Zimmermann, K.; Ryder, O. S.; Campbell, N.; Collins, D. B.; Gianneschi, N.; Bertram, T. H.

2013-12-01

Spontaneous self-assembly of marine exudate particles has previously been observed in filtered seawater samples. The chemicophysical properties of these particles may alter the chemical composition and CCN properties of nascent marine aerosol, yet to date simultaneous measurement of seawater exudate particle formation rates and number distributions, with aerosol particle formation rates and CCN activity are lacking. Here, we use a novel Marine Aerosol Reference Tank (MART) system to experimentally mimic a phytoplankton bloom via sequential addition of biological surrogates, including sterol, galactose, lipopolysaccharide, BSA protein, and dipalmitoylphosphatidylcholine. Nascent sea-spray aerosol are generated in the MART system via a continuous plunging waterfall. Exudate particle assembly in the water is monitored via dynamic light scattering (DLS) and transmission electron microscopy (TEM) to obtain both the assembly kinetics of the particles as well as particle number distributions Simultaneous characterization of both particle production rates and super-saturated particle hygroscopicity are also discussed. This study permits analysis of the controlling role of the molecular composition of dissolved organic carbon in setting the production rates of colloidal material in the surface oceans.

Nup133 Is Required for Proper Nuclear Pore Basket Assembly and Dynamics in Embryonic Stem Cells.

PubMed

Souquet, Benoit; Freed, Ellen; Berto, Alessandro; Andric, Vedrana; Audugé, Nicolas; Reina-San-Martin, Bernardo; Lacy, Elizabeth; Doye, Valérie

2018-05-22

Nup133 belongs to the Y-complex, a key component of the nuclear pore complex (NPC) scaffold. Studies on a null mutation in mice previously revealed that Nup133 is essential for embryonic development but not for mouse embryonic stem cell (mESC) proliferation. Using single-pore detection and average NE-fluorescence intensity, we find that Nup133 is dispensable for interphase and postmitotic NPC scaffold assembly in pluripotent mESCs. However, loss of Nup133 specifically perturbs the formation of the nuclear basket as manifested by the absence of Tpr in about half of the NPCs combined with altered dynamics of Nup153. We further demonstrate that its central domain mediates Nup133's role in assembling Tpr and Nup153 into a properly configured nuclear basket. Our findings thus revisit the role of the Y-complex in pore biogenesis and provide insights into the interplay between NPC scaffold architecture, nuclear basket assembly, and the generation of heterogeneity among NPCs. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Design for Manufacture and Assembly for Product Development (Case study : Emergency Lamp)

NASA Astrophysics Data System (ADS)

Ngatilah, Y.; Pulansari, F.; Ernawati, Dira; Pujiastuti, C.; Parwati, C. I.; Prasetyo, B.

2018-01-01

Community needs that are not primary but important in everyday life are lights for lighting. State electricity company (PLN) is experiencing limitations in supplying electricity for this puIDRose. Therefore emergency lights (emergency lights) are already marketed in the community, which limited function only illuminate a very limited space. Therefore we developed the design of energy saving lamps using “Light Emitting Diode” (LED) which can illuminate the whole house as well as functioning as mobile phone charger (HP). The method used is Design for Manufacture and Assembly (DFMA), with the result of design development The percentage increase in assembly efficiency (E) is 0.01071 - 0.00645 = 0.00426 or = 39.76%. The decrease in material costs is IDR 234,000 - IDR 214,000 = IDR 20,000 or = 8.54% .Development design is received because of more assembly efficiency than the initial design. Power usage on previous products with series and designs of the original product can last only 4-5 hours non-stop, while the development of the design can survive 9-10 hours 2x more energy efficient.

Not5-dependent co-translational assembly of Ada2 and Spt20 is essential for functional integrity of SAGA

PubMed Central

Kassem, Sari; Villanyi, Zoltan

2017-01-01

Abstract Acetylation of histones regulates gene expression in eukaryotes. In the yeast Saccharomyces cerevisiae it depends mainly upon the ADA and SAGA histone acetyltransferase complexes for which Gcn5 is the catalytic subunit. Previous screens have determined that global acetylation is reduced in cells lacking subunits of the Ccr4–Not complex, a global regulator of eukaryotic gene expression. In this study we have characterized the functional connection between the Ccr4–Not complex and SAGA. We show that SAGA mRNAs encoding a core set of SAGA subunits are tethered together for co-translational assembly of the encoded proteins. Ccr4–Not subunits bind SAGA mRNAs and promote the co-translational assembly of these subunits. This is needed for integrity of SAGA. In addition, we determine that a glycolytic enzyme, the glyceraldehyde-3-phosphate dehydrogenase Tdh3, a prototypical moonlighting protein, is tethered at this site of Ccr4–Not-dependent co-translational SAGA assembly and functions as a chaperone. PMID:28180299

Two-Step Self-Assembly of Liposome-Multidomain Peptide Nanofiber Hydrogel for Time-Controlled Release

PubMed Central

2015-01-01

Progress in self-assembly and supramolecular chemistry has been directed toward obtaining macromolecular assemblies with higher degrees of complexity, simulating the highly structured environment in natural systems. One approach to this type of complexity are multistep, multicomponent, self-assembling systems that allow approaches comparable to traditional multistep synthetic organic chemistry; however, only a few examples of this approach have appeared in the literature. Our previous work demonstrated nanofibrous mimics of the extracellular matrix. Here we demonstrate the ability to create a unique hydrogel, developed by stepwise self-assembly of multidomain peptide fibers and liposomes. The two-component system allows for controlled release of bioactive factors at multiple time points. The individual components of the self-assembled gel and the composite hydrogel were characterized by TEM, SEM, and rheometry, demonstrating that peptide nanofibers and lipid vesicles both retain their structural integrity in the composite gel. The rheological robustness of the hydrogel is shown to be largely unaffected by the presence of liposomes. Release studies from the composite gels loaded with different growth factors EGF, MCP-1, and PlGF-1 showed delay and prolongation of release by liposomes entrapped in the hydrogel compared to more rapid release from the hydrogel alone. This bimodal release system may have utility in systems where timed cascades of biological signals may be valuable, such as in tissue regeneration. PMID:25308335

Assembly of hydrogel units for 3D microenvironment in a poly(dimethylsiloxane) channel

NASA Astrophysics Data System (ADS)

Cho, Chang Hyun; Kwon, Seyong; Park, Je-Kyun

2017-12-01

Construction of three-dimensional (3D) microenvironment become an important issue in recent biological studies due to their biological relevance compared to conventional two-dimensional (2D) microenvironment. Various fabrication techniques have been employed to construct a 3D microenvironment, however, it is difficult to fully satisfy the biological and mechanical properties required for the 3D cell culture system, such as heterogeneous tissue structures generated from the functional differences or diseases. We propose here an assembly method for facile construction of 3D microenvironment in a poly(dimethylsiloxane) (PDMS) channel using hydrogel units. The high-aspect-ratio of hydrogel units was achieved by fabricating these units using a 2D mold. With this approach, 3D heterogeneous hydrogel units were produced and assembled in a PDMS channel by structural hookup. In vivo-like 3D heterogeneous microenvironment in a precisely controllable fluidic system was also demonstrated using a controlled assembly of different types of hydrogel units, which was difficult to obtain from previous methods. By regulating the flow condition, the mechanical stability of the assembled hydrogel units was verified by the flow-induced deformation of hydrogel units. In addition, in vivo-like cell culture environment was demonstrated using an assembly of cell-coated hydrogel units in the fluidic channel. Based on these features, our method expects to provide a beneficial tool for the 3D cell culture module and biomimetic engineering.

Measuring phenotypic variability and plasticity in influenza A virus using multispectral viral strains

NASA Astrophysics Data System (ADS)

Vahey, Michael

Despite relevance to human health, the mechanisms of enveloped virus assembly remain largely mysterious. This is particularly true of influenza A virus (IAV), which (unlike viral capsids with stereotyped shape and composition) forms heterogeneous particles whose assembly cannot be described in terms of equilibrium thermodynamics. Although the ability to assemble into particles with diverse size and composition could have important implications for infectivity, understanding how virion-to-virion differences arise and how they ultimately influence virus replication has proven challenging due to the lack of available tools for studying the assembly process. To address this challenge and establish a dynamic picture of how IAV assembles, we have developed virus strains that harbor small, non-disruptive fluorescent tags on each of the virus's five major structural proteins. Using these multispectral strains, we are able to quantify the protein composition and dynamics of virions as they assemble in live infected cells - measurements that have been previously inaccessible, and which reveal subpopulations of virus that favor either the binding or destruction of host receptors. The occupancy of these different subpopulations is malleable, shifting in response to environmental stimuli, including antiviral drugs that block receptor-destruction. In complex environments like the human respiratory tract, this phenotypic diversity could act as an evolutionary hedge. We acknowledge the Burroughs Wellcome Fund and NIH NIGMS for supporting this work.

Packing and self-assembly of truncated triangular bipyramids

NASA Astrophysics Data System (ADS)

Haji-Akbari, Amir; Chen, Elizabeth R.; Engel, Michael; Glotzer, Sharon C.

2013-07-01

Motivated by breakthroughs in the synthesis of faceted nano- and colloidal particles, as well as theoretical and computational studies of their packings, we investigate a family of truncated triangular bipyramids. We report dense periodic packings with small unit cells that were obtained via numerical and analytical optimization. The maximal packing fraction ϕmax changes continuously with the truncation parameter t. Eight distinct packings are identified based on discontinuities in the first and second derivatives of ϕmax(t). These packings differ in the number of particles in the fundamental domain (unit cell) and the type of contacts between the particles. In particular, we report two packings with four particles in the unit cell for which both ϕmax(t) and ϕmax'(t) are continuous and the discontinuity occurs in the second derivative only. In the self-assembly simulations that we perform for larger boxes with 2048 particles, only one out of eight packings is found to assemble. In addition, the degenerate quasicrystal reported previously for triangular bipyramids without truncation [Haji-Akbari , Phys. Rev. Lett.PRLTAO0031-900710.1103/PhysRevLett.107.215702 107, 215702 (2011)] assembles for truncations as high as 0.45. The self-assembly propensities for the structures formed in the thermodynamic limit are explained using the isoperimetric quotient of the particles and the coordination number in the disordered fluid and in the assembled structure.

Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly.

PubMed

Dilley, Kari A; Nikolaitchik, Olga A; Galli, Andrea; Burdick, Ryan C; Levine, Louis; Li, Kelvin; Rein, Alan; Pathak, Vinay K; Hu, Wei-Shau

2017-08-15

Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly. IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly. Understanding the mechanism used by HIV-1 to ensure genome packaging provides significant insights into viral assembly and replication. Copyright © 2017 American Society for Microbiology.

aTRAM 2.0: An Improved, Flexible Locus Assembler for NGS Data

PubMed Central

Allen, Julie M; LaFrance, Raphael; Folk, Ryan A; Johnson, Kevin P; Guralnick, Robert P

2018-01-01

Massive strides have been made in technologies for collecting genome-scale data. However, tools for efficiently and flexibly assembling raw outputs into downstream analytical workflows are still nascent. aTRAM 1.0 was designed to assemble any locus from genome sequencing data but was neither optimized for efficiency nor able to serve as a single toolkit for all assembly needs. We have completely re-implemented aTRAM and redesigned its structure for faster read retrieval while adding a number of key features to improve flexibility and functionality. The software can now (1) assemble single- or paired-end data, (2) utilize both read directions in the database, (3) use an additional de novo assembly module, and (4) leverage new built-in pipelines to automate common workflows in phylogenomics. Owing to reimplementation of databasing strategies, we demonstrate that aTRAM 2.0 is much faster across all applications compared to the previous version. PMID:29881251

Draft de novo transcriptome assembly and proteome characterization of the electric lobe of Tetronarce californica: a molecular tool for the study of cholinergic neurotransmission in the electric organ.

PubMed

Stavrianakou, Maria; Perez, Ricardo; Wu, Cheng; Sachs, Matthew S; Aramayo, Rodolfo; Harlow, Mark

2017-08-14

The electric organ of Tetronarce californica (an electric ray formerly known as Torpedo californica) is a classic preparation for biochemical studies of cholinergic neurotransmission. To broaden the usefulness of this preparation, we have performed a transcriptome assembly of the presynaptic component of the electric organ (the electric lobe). We combined our assembled transcriptome with a previous transcriptome of the postsynaptic electric organ, to define a MetaProteome containing pre- and post-synaptic components of the electric organ. Sequencing yielded 102 million paired-end 100 bp reads. De novo Trinity assembly was performed at Kmer 25 (default) and Kmers 27, 29, and 31. Trinity, generated around 103,000 transcripts, and 78,000 genes per assembly. Assemblies were evaluated based on the number of bases/transcripts assembled, RSEM-EVAL scores and informational content and completeness. We found that different assemblies scored differently according to the evaluation criteria used, and that while each individual assembly contained unique information, much of the assembly information was shared by all assemblies. To generate the presynaptic transcriptome (electric lobe), while capturing all information, assemblies were first clustered and then combined with postsynaptic transcripts (electric organ) downloaded from NCBI. The completness of the resulting clustered predicted MetaProteome was rigorously evaluated by comparing its information against the predicted proteomes from Homo sapiens, Callorhinchus milli, and the Transporter Classification Database (TCDB). In summary, we obtained a MetaProteome containing 92%, 88.5%, and 66% of the expected set of ultra-conserved sequences (i.e., BUSCOs), expected to be found for Eukaryotes, Metazoa, and Vertebrata, respectively. We cross-annotated the conserved set of proteins shared between the T. californica MetaProteome and the proteomes of H. sapiens and C. milli, using the H. sapiens genome as a reference. This information was used to predict the position in human pathways of the conserved members of the T. californica MetaProteome. We found proteins not detected before in T. californica, corresponding to processes involved in synaptic vesicle biology. Finally, we identified 42 transporter proteins in TCDB that were detected by the T. californica MetaProteome (electric fish) and not selected by a control proteome consisting of the combined proteomes of 12 widely diverse non-electric fishes by Reverse-Blast-Hit Blast. Combined, the information provided here is not only a unique tool for the study of cholinergic neurotransmission, but it is also a starting point for understanding the evolution of early vertebrates.

Field-induced assembly of colloidal ellipsoids into well-defined microtubules

PubMed Central

Crassous, Jérôme J.; Mihut, Adriana M.; Wernersson, Erik; Pfleiderer, Patrick; Vermant, Jan; Linse, Per; Schurtenberger, Peter

2014-01-01

Current theoretical attempts to understand the reversible formation of stable microtubules and virus shells are generally based on shape-specific building blocks or monomers, where the local curvature of the resulting structure is explicitly built-in via the monomer geometry. Here we demonstrate that even simple ellipsoidal colloids can reversibly self-assemble into regular tubular structures when subjected to an alternating electric field. Supported by model calculations, we discuss the combined effects of anisotropic shape and field-induced dipolar interactions on the reversible formation of self-assembled structures. Our observations show that the formation of tubular structures through self-assembly requires much less geometrical and interaction specificity than previously thought, and advance our current understanding of the minimal requirements for self-assembly into regular virus-like structures. PMID:25409686

NEDDylation promotes stress granule assembly.

PubMed

Jayabalan, Aravinth Kumar; Sanchez, Anthony; Park, Ra Young; Yoon, Sang Pil; Kang, Gum-Yong; Baek, Je-Hyun; Anderson, Paul; Kee, Younghoon; Ohn, Takbum

2016-07-06

Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly.

Assembly of transcriptionally inactive chromatin in vitro.

PubMed

Shanahan, M M; Kmiec, E B

1989-07-01

We have successfully uncoupled the previously interlocked activities of chromatin assembly and in vitro transcription promoted by the Xenopus oocyte S-150 cell-free extract. Our isolated fraction catalyzes extensive chromatin assembly measured both by changes in DNA topology and Micrococcal nuclease digestions. The assembly of chromatin is slowed by the exogenous addition of ATP. In the absence of exogenously added ATP, the fraction forms a chromatin template that is transcriptionally inert. Addition of small amounts of the HeLa cell extract (S-100) converts these templates into transcriptionally active ones without disrupting the chromatin structure. Our protocol defines a method for the isolation of a fraction from the Xenopus cell free extract that catalyzes the assembly of transcriptionally inactive chromatin. We characterize this reaction and establish conditions for the transcriptional activation of these inactive minichromosomes.

Genome assembly from synthetic long read clouds

PubMed Central

Kuleshov, Volodymyr; Snyder, Michael P.; Batzoglou, Serafim

2016-01-01

Motivation: Despite rapid progress in sequencing technology, assembling de novo the genomes of new species as well as reconstructing complex metagenomes remains major technological challenges. New synthetic long read (SLR) technologies promise significant advances towards these goals; however, their applicability is limited by high sequencing requirements and the inability of current assembly paradigms to cope with combinations of short and long reads. Results: Here, we introduce Architect, a new de novo scaffolder aimed at SLR technologies. Unlike previous assembly strategies, Architect does not require a costly subassembly step; instead it assembles genomes directly from the SLR’s underlying short reads, which we refer to as read clouds. This enables a 4- to 20-fold reduction in sequencing requirements and a 5-fold increase in assembly contiguity on both genomic and metagenomic datasets relative to state-of-the-art assembly strategies aimed directly at fully subassembled long reads. Availability and Implementation: Our source code is freely available at https://github.com/kuleshov/architect. Contact: kuleshov@stanford.edu PMID:27307620

The Escherichia coli P and Type 1 Pilus Assembly Chaperones PapD and FimC Are Monomeric in Solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sarowar, Samema; Hu, Olivia J.; Werneburg, Glenn T.

ABSTRACT The chaperone/usher pathway is used by Gram-negative bacteria to assemble adhesive surface structures known as pili or fimbriae. Uropathogenic strains ofEscherichia coliuse this pathway to assemble P and type 1 pili, which facilitate colonization of the kidney and bladder, respectively. Pilus assembly requires a periplasmic chaperone and outer membrane protein termed the usher. The chaperone allows folding of pilus subunits and escorts the subunits to the usher for polymerization into pili and secretion to the cell surface. Based on previous structures of mutant versions of the P pilus chaperone PapD, it was suggested that the chaperone dimerizes in themore » periplasm as a self-capping mechanism. Such dimerization is counterintuitive because the chaperone G1 strand, important for chaperone-subunit interaction, is buried at the dimer interface. Here, we show that the wild-type PapD chaperone also forms a dimer in the crystal lattice; however, the dimer interface is different from the previously solved structures. In contrast to the crystal structures, we found that both PapD and the type 1 pilus chaperone, FimC, are monomeric in solution. Our findings indicate that pilus chaperones do not sequester their G1 β-strand by forming a dimer. Instead, the chaperones may expose their G1 strand for facile interaction with pilus subunits. We also found that the type 1 pilus adhesin, FimH, is flexible in solution while in complex with its chaperone, whereas the P pilus adhesin, PapGII, is rigid. Our study clarifies a crucial step in pilus biogenesis and reveals pilus-specific differences that may relate to biological function. IMPORTANCEPili are critical virulence factors for many bacterial pathogens. UropathogenicE. colirelies on P and type 1 pili assembled by the chaperone/usher pathway to adhere to the urinary tract and establish infection. Studying pilus assembly is important for understanding mechanisms of protein secretion, as well as for identifying points for therapeutic intervention. Pilus biogenesis is a multistep process. This work investigates the oligomeric state of the pilus chaperone in the periplasm, which is important for understanding early assembly events. Our work unambiguously demonstrates that both PapD and FimC chaperones are monomeric in solution. We further demonstrate that the solution behavior of the FimH and PapGII adhesins differ, which may be related to functional differences between the two pilus systems.« less

Effects of short read quality and quantity on a de novo vertebrate transcriptome assembly.

PubMed

Garcia, T I; Shen, Y; Catchen, J; Amores, A; Schartl, M; Postlethwait, J; Walter, R B

2012-01-01

For many researchers, next generation sequencing data holds the key to answering a category of questions previously unassailable. One of the important and challenging steps in achieving these goals is accurately assembling the massive quantity of short sequencing reads into full nucleic acid sequences. For research groups working with non-model or wild systems, short read assembly can pose a significant challenge due to the lack of pre-existing EST or genome reference libraries. While many publications describe the overall process of sequencing and assembly, few address the topic of how many and what types of reads are best for assembly. The goal of this project was use real world data to explore the effects of read quantity and short read quality scores on the resulting de novo assemblies. Using several samples of short reads of various sizes and qualities we produced many assemblies in an automated manner. We observe how the properties of read length, read quality, and read quantity affect the resulting assemblies and provide some general recommendations based on our real-world data set. Published by Elsevier Inc.

Synthesis and characterization of designed BMHP1-derived self-assembling peptides for tissue engineering applications

NASA Astrophysics Data System (ADS)

Silva, Diego; Natalello, Antonino; Sanii, Babak; Vasita, Rajesh; Saracino, Gloria; Zuckermann, Ronald N.; Doglia, Silvia Maria; Gelain, Fabrizio

2012-12-01

The importance of self-assembling peptides (SAPs) in regenerative medicine is becoming increasingly recognized. The propensity of SAPs to form nanostructured fibers is governed by multiple forces including hydrogen bonds, hydrophobic interactions and π-π aromatic interactions among side chains of the amino acids. Single residue modifications in SAP sequences can significantly affect these forces. BMHP1-derived SAPs is a class of biotinylated oligopeptides, which self-assemble in β-structured fibers to form a self-healing hydrogel. In the current study, selected modifications in previously described BMHP1-derived SAPs were designed in order to investigate the influence of modified residues on self-assembly kinetics and scaffold formation properties. The Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis demonstrated the secondary structure (β-sheet) formation in all modified SAP sequences, whereas atomic force microscopy (AFM) analysis further confirmed the presence of nanofibers. Furthermore, the fiber shape and dimension analysis by AFM showed flattened and twisted fiber morphology ranging from ~8 nm to ~70 nm. The mechanical properties of the pre-assembled and post assembled solution were investigated by rheometry. The shear-thinning behavior and rapid re-healing properties of the pre-assembled solutions make them a preferable choice for injectable scaffolds. The wide range of stiffnesses (G') -from ~1000 to ~27 000 Pa - exhibited by the post-assembled scaffolds demonstrated their potential for a variety of tissue engineering applications. The extra cellular matrix (ECM) mimicking (physically and chemically) properties of SAP scaffolds enhanced cell adhesion and proliferation. The capability of the scaffold to facilitate murine neural stem cell (mNSC) proliferation was evaluated in vitro: the increased mNSCs adhesion and proliferation demonstrated the potential of newly synthesized SAPs for regenerative medicine approaches.The importance of self-assembling peptides (SAPs) in regenerative medicine is becoming increasingly recognized. The propensity of SAPs to form nanostructured fibers is governed by multiple forces including hydrogen bonds, hydrophobic interactions and π-π aromatic interactions among side chains of the amino acids. Single residue modifications in SAP sequences can significantly affect these forces. BMHP1-derived SAPs is a class of biotinylated oligopeptides, which self-assemble in β-structured fibers to form a self-healing hydrogel. In the current study, selected modifications in previously described BMHP1-derived SAPs were designed in order to investigate the influence of modified residues on self-assembly kinetics and scaffold formation properties. The Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis demonstrated the secondary structure (β-sheet) formation in all modified SAP sequences, whereas atomic force microscopy (AFM) analysis further confirmed the presence of nanofibers. Furthermore, the fiber shape and dimension analysis by AFM showed flattened and twisted fiber morphology ranging from ~8 nm to ~70 nm. The mechanical properties of the pre-assembled and post assembled solution were investigated by rheometry. The shear-thinning behavior and rapid re-healing properties of the pre-assembled solutions make them a preferable choice for injectable scaffolds. The wide range of stiffnesses (G') -from ~1000 to ~27 000 Pa - exhibited by the post-assembled scaffolds demonstrated their potential for a variety of tissue engineering applications. The extra cellular matrix (ECM) mimicking (physically and chemically) properties of SAP scaffolds enhanced cell adhesion and proliferation. The capability of the scaffold to facilitate murine neural stem cell (mNSC) proliferation was evaluated in vitro: the increased mNSCs adhesion and proliferation demonstrated the potential of newly synthesized SAPs for regenerative medicine approaches. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr32656f

Team assembly mechanisms determine collaboration network structure and team performance.

PubMed

Guimerà, Roger; Uzzi, Brian; Spiro, Jarrett; Amaral, Luís A Nunes

2005-04-29

Agents in creative enterprises are embedded in networks that inspire, support, and evaluate their work. Here, we investigate how the mechanisms by which creative teams self-assemble determine the structure of these collaboration networks. We propose a model for the self-assembly of creative teams that has its basis in three parameters: team size, the fraction of newcomers in new productions, and the tendency of incumbents to repeat previous collaborations. The model suggests that the emergence of a large connected community of practitioners can be described as a phase transition. We find that team assembly mechanisms determine both the structure of the collaboration network and team performance for teams derived from both artistic and scientific fields.

Evolution of an RNP assembly system: A minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster

PubMed Central

Kroiss, Matthias; Schultz, Jörg; Wiesner, Julia; Chari, Ashwin; Sickmann, Albert; Fischer, Utz

2008-01-01

In vertebrates, assembly of spliceosomal uridine-rich small nuclear ribonucleoproteins (UsnRNPs) is mediated by the SMN complex, a macromolecular entity composed of the proteins SMN and Gemins 2–8. Here we have studied the evolution of this machinery using complete genome assemblies of multiple model organisms. The SMN complex has gained complexity in evolution by a blockwise addition of Gemins onto an ancestral core complex composed of SMN and Gemin2. In contrast to this overall evolutionary trend to more complexity in metazoans, orthologs of most Gemins are missing in dipterans. In accordance with these bioinformatic data a previously undescribed biochemical purification strategy elucidated that the dipteran Drosophila melanogaster contains an SMN complex of remarkable simplicity. Surprisingly, this minimal complex not only mediates the assembly reaction in a manner very similar to its vertebrate counterpart, but also prevents misassembly onto nontarget RNAs. Our data suggest that only a minority of Gemins are required for the assembly reaction per se, whereas others may serve additional functions in the context of UsnRNP biogenesis. The evolution of the SMN complex is an interesting example of how the simplification of a biochemical process contributes to genome compaction. PMID:18621711

Genome assembly and transcriptome resource for river buffalo, Bubalus bubalis (2n = 50).

PubMed

Williams, John L; Iamartino, Daniela; Pruitt, Kim D; Sonstegard, Tad; Smith, Timothy P L; Low, Wai Yee; Biagini, Tommaso; Bomba, Lorenzo; Capomaccio, Stefano; Castiglioni, Bianca; Coletta, Angelo; Corrado, Federica; Ferré, Fabrizio; Iannuzzi, Leopoldo; Lawley, Cynthia; Macciotta, Nicolò; McClure, Matthew; Mancini, Giordano; Matassino, Donato; Mazza, Raffaele; Milanesi, Marco; Moioli, Bianca; Morandi, Nicola; Ramunno, Luigi; Peretti, Vincenzo; Pilla, Fabio; Ramelli, Paola; Schroeder, Steven; Strozzi, Francesco; Thibaud-Nissen, Francoise; Zicarelli, Luigi; Ajmone-Marsan, Paolo; Valentini, Alessio; Chillemi, Giovanni; Zimin, Aleksey

2017-10-01

Water buffalo is a globally important species for agriculture and local economies. A de novo assembled, well-annotated reference sequence for the water buffalo is an important prerequisite for studying the biology of this species, and is necessary to manage genetic diversity and to use modern breeding and genomic selection techniques. However, no such genome assembly has been previously reported. There are 2 species of domestic water buffalo, the river (2 n = 50) and the swamp (2 n = 48) buffalo. Here we describe a draft quality reference sequence for the river buffalo created from Illumina GA and Roche 454 short read sequences using the MaSuRCA assembler. The assembled sequence is 2.83 Gb, consisting of 366 983 scaffolds with a scaffold N50 of 1.41 Mb and contig N50 of 21 398 bp. Annotation of the genome was supported by transcriptome data from 30 tissues and identified 21 711 predicted protein coding genes. Searches for complete mammalian BUSCO gene groups found 98.6% of curated single copy orthologs present among predicted genes, which suggests a high level of completeness of the genome. The annotated sequence is available from NCBI at accession GCA_000471725.1. © The Author 2017. Published by Oxford University Press.

Nanoscale Platelet Formation by Monounsaturated and Saturated Sophorolipids under Basic pH Conditions.

PubMed

Cuvier, Anne-Sophie; Babonneau, Florence; Berton, Jan; Stevens, Christian V; Fadda, Giulia C; Péhau-Arnaudet, Gérard; Le Griel, Patrick; Prévost, Sylvain; Perez, Javier; Baccile, Niki

2015-12-21

The self-assembly behavior of the yeast-derived bolaamphiphile sophorolipid (SL) is generally studied under acidic/neutral pH conditions, at which micellar and fibrillar aggregates are commonly found, according to the (un)saturation of the aliphatic chain: the cis form, which corresponds to the oleic acid form of SL, spontaneously forms micelles, whereas the saturated form, which corresponds to the stearic acid form of SL, preferentially forms chiral fibers. By using small-angle light and X-ray scattering (SLS, SAXS) combined with high-sensitivity transmission electron microscopy imaging under cryogenic conditions (cryo-TEM), the nature of the self-assembled structures formed by these two compounds above pH 10, which is the pH at which they are negatively charged due to the presence of a carboxylate group, has been explored. Under these conditions, these compounds self-assemble into nanoscale platelets, despite the different molecular structures. This work shows that the electrostatic repulsion forces generated by COO(-) mainly drive the self-assembly process at basic pH, in contrast with that found at pH below neutrality, at which self-assembly is driven by van der Waals forces and hydrogen bonding, and thus, is in agreement with previous findings on carbohydrate-based gemini surfactants. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

B. subtilis as a Model for Studying the Assembly of Fe-S Clusters in Gram-Positive Bacteria.

PubMed

Dos Santos, Patricia C

2017-01-01

Complexes of iron and sulfur (Fe-S clusters) are widely distributed in nature and participate in essential biochemical reactions. The biological formation of Fe-S clusters involves dedicated pathways responsible for the mobilization of sulfur, the assembly of Fe-S clusters, and the transfer of these clusters to target proteins. Genomic analysis of Bacillus subtilis and other Gram-positive bacteria indicated the presence of only one Fe-S cluster biosynthesis pathway, which is distinct in number of components and organization from previously studied systems. B. subtilis has been used as a model system for the characterization of cysteine desulfurases responsible for sulfur mobilization reactions in the biogenesis of Fe-S clusters and other sulfur-containing cofactors. Cysteine desulfurases catalyze the cleavage of the C-S bond from the amino acid cysteine and subsequent transfer of sulfur to acceptor molecules. These reactions can be monitored by the rate of alanine formation, the first product in the reaction, and sulfide formation, a byproduct of reactions performed under reducing conditions. The assembly of Fe-S clusters on protein scaffolds and the transfer of these clusters to target acceptors are determined through a combination of spectroscopic methods probing the rate of cluster assembly and transfer. This chapter provides a description of reactions promoting the assembly of Fe-S clusters in bacteria as well as methods used to study functions of each biosynthetic component and identify mechanistic differences employed by these enzymes across different pathways. © 2017 Elsevier Inc. All rights reserved.

Stochastic assembly in a subtropical forest chronosequence: evidence from contrasting changes of species, phylogenetic and functional dissimilarity over succession.

PubMed

Mi, Xiangcheng; Swenson, Nathan G; Jia, Qi; Rao, Mide; Feng, Gang; Ren, Haibao; Bebber, Daniel P; Ma, Keping

2016-09-07

Deterministic and stochastic processes jointly determine the community dynamics of forest succession. However, it has been widely held in previous studies that deterministic processes dominate forest succession. Furthermore, inference of mechanisms for community assembly may be misleading if based on a single axis of diversity alone. In this study, we evaluated the relative roles of deterministic and stochastic processes along a disturbance gradient by integrating species, functional, and phylogenetic beta diversity in a subtropical forest chronosequence in Southeastern China. We found a general pattern of increasing species turnover, but little-to-no change in phylogenetic and functional turnover over succession at two spatial scales. Meanwhile, the phylogenetic and functional beta diversity were not significantly different from random expectation. This result suggested a dominance of stochastic assembly, contrary to the general expectation that deterministic processes dominate forest succession. On the other hand, we found significant interactions of environment and disturbance and limited evidence for significant deviations of phylogenetic or functional turnover from random expectations for different size classes. This result provided weak evidence of deterministic processes over succession. Stochastic assembly of forest succession suggests that post-disturbance restoration may be largely unpredictable and difficult to control in subtropical forests.

Detection of large-scale concentric gravity waves from a Chinese airglow imager network

NASA Astrophysics Data System (ADS)

Lai, Chang; Yue, Jia; Xu, Jiyao; Yuan, Wei; Li, Qinzeng; Liu, Xiao

2018-06-01

Concentric gravity waves (CGWs) contain a broad spectrum of horizontal wavelengths and periods due to their instantaneous localized sources (e.g., deep convection, volcanic eruptions, or earthquake, etc.). However, it is difficult to observe large-scale gravity waves of >100 km wavelength from the ground for the limited field of view of a single camera and local bad weather. Previously, complete large-scale CGW imagery could only be captured by satellite observations. In the present study, we developed a novel method that uses assembling separate images and applying low-pass filtering to obtain temporal and spatial information about complete large-scale CGWs from a network of all-sky airglow imagers. Coordinated observations from five all-sky airglow imagers in Northern China were assembled and processed to study large-scale CGWs over a wide area (1800 km × 1 400 km), focusing on the same two CGW events as Xu et al. (2015). Our algorithms yielded images of large-scale CGWs by filtering out the small-scale CGWs. The wavelengths, wave speeds, and periods of CGWs were measured from a sequence of consecutive assembled images. Overall, the assembling and low-pass filtering algorithms can expand the airglow imager network to its full capacity regarding the detection of large-scale gravity waves.

Self-assembled three-dimensional chiral colloidal architecture

NASA Astrophysics Data System (ADS)

Ben Zion, Matan Yah; He, Xiaojin; Maass, Corinna C.; Sha, Ruojie; Seeman, Nadrian C.; Chaikin, Paul M.

2017-11-01

Although stereochemistry has been a central focus of the molecular sciences since Pasteur, its province has previously been restricted to the nanometric scale. We have programmed the self-assembly of micron-sized colloidal clusters with structural information stemming from a nanometric arrangement. This was done by combining DNA nanotechnology with colloidal science. Using the functional flexibility of DNA origami in conjunction with the structural rigidity of colloidal particles, we demonstrate the parallel self-assembly of three-dimensional microconstructs, evincing highly specific geometry that includes control over position, dihedral angles, and cluster chirality.

Automated ensemble assembly and validation of microbial genomes.

PubMed

Koren, Sergey; Treangen, Todd J; Hill, Christopher M; Pop, Mihai; Phillippy, Adam M

2014-05-03

The continued democratization of DNA sequencing has sparked a new wave of development of genome assembly and assembly validation methods. As individual research labs, rather than centralized centers, begin to sequence the majority of new genomes, it is important to establish best practices for genome assembly. However, recent evaluations such as GAGE and the Assemblathon have concluded that there is no single best approach to genome assembly. Instead, it is preferable to generate multiple assemblies and validate them to determine which is most useful for the desired analysis; this is a labor-intensive process that is often impossible or unfeasible. To encourage best practices supported by the community, we present iMetAMOS, an automated ensemble assembly pipeline; iMetAMOS encapsulates the process of running, validating, and selecting a single assembly from multiple assemblies. iMetAMOS packages several leading open-source tools into a single binary that automates parameter selection and execution of multiple assemblers, scores the resulting assemblies based on multiple validation metrics, and annotates the assemblies for genes and contaminants. We demonstrate the utility of the ensemble process on 225 previously unassembled Mycobacterium tuberculosis genomes as well as a Rhodobacter sphaeroides benchmark dataset. On these real data, iMetAMOS reliably produces validated assemblies and identifies potential contamination without user intervention. In addition, intelligent parameter selection produces assemblies of R. sphaeroides comparable to or exceeding the quality of those from the GAGE-B evaluation, affecting the relative ranking of some assemblers. Ensemble assembly with iMetAMOS provides users with multiple, validated assemblies for each genome. Although computationally limited to small or mid-sized genomes, this approach is the most effective and reproducible means for generating high-quality assemblies and enables users to select an assembly best tailored to their specific needs.

Hierarchical self-assembly of hexagonal single-crystal nanosheets into 3D layered superlattices with high conductivity

NASA Astrophysics Data System (ADS)

Tao, Yulun; Shen, Yuhua; Yang, Liangbao; Han, Bin; Huang, Fangzhi; Li, Shikuo; Chu, Zhuwang; Xie, Anjian

2012-05-01

While the number of man-made nano superstructures realized by self-assembly is growing in recent years, assemblies of conductive polymer nanocrystals, especially for superlattices, are still a significant challenge, not only because of the simplicity of the shape of the nanocrystal building blocks and their interactions, but also because of the poor control over these parameters in the fabrication of more elaborate nanocrystals. Here, we firstly report a facile and general route to a new generation of 3D layered superlattices of polyaniline doped with CSA (PANI-CSA) and show how PANI crystallize and self-assemble, in a suitable single solution environment. In cyclohexane, 1D amorphous nanofibers transformed to 1D nanorods as building blocks, and then to 2D single-crystal nanosheets with a hexagonal phase, and lastly to 3D ordered layered superlattices with the narrowest polydispersity value (Mw/Mn = 1.47). Remarkably, all the instructions for the hierarchical self-assembly are encoded in the layered shape in other non-polar solvents (hexane, octane) and their conductivity in the π-π stacking direction is improved to about 50 S cm-1, which is even higher than that of the highest previously reported value (16 S cm-1). The method used in this study is greatly expected to be readily scalable to produce superlattices of conductive polymers with high quality and low cost.While the number of man-made nano superstructures realized by self-assembly is growing in recent years, assemblies of conductive polymer nanocrystals, especially for superlattices, are still a significant challenge, not only because of the simplicity of the shape of the nanocrystal building blocks and their interactions, but also because of the poor control over these parameters in the fabrication of more elaborate nanocrystals. Here, we firstly report a facile and general route to a new generation of 3D layered superlattices of polyaniline doped with CSA (PANI-CSA) and show how PANI crystallize and self-assemble, in a suitable single solution environment. In cyclohexane, 1D amorphous nanofibers transformed to 1D nanorods as building blocks, and then to 2D single-crystal nanosheets with a hexagonal phase, and lastly to 3D ordered layered superlattices with the narrowest polydispersity value (Mw/Mn = 1.47). Remarkably, all the instructions for the hierarchical self-assembly are encoded in the layered shape in other non-polar solvents (hexane, octane) and their conductivity in the π-π stacking direction is improved to about 50 S cm-1, which is even higher than that of the highest previously reported value (16 S cm-1). The method used in this study is greatly expected to be readily scalable to produce superlattices of conductive polymers with high quality and low cost. Electronic supplementary information (ESI) available: SEM, and TEM images. See DOI: 10.1039/c2nr30743j

Decay heat uncertainty for BWR used fuel due to modeling and nuclear data uncertainties

DOE PAGES

Ilas, Germina; Liljenfeldt, Henrik

2017-05-19

Characterization of the energy released from radionuclide decay in nuclear fuel discharged from reactors is essential for the design, safety, and licensing analyses of used nuclear fuel storage, transportation, and repository systems. There are a limited number of decay heat measurements available for commercial used fuel applications. Because decay heat measurements can be expensive or impractical for covering the multitude of existing fuel designs, operating conditions, and specific application purposes, decay heat estimation relies heavily on computer code prediction. Uncertainty evaluation for calculated decay heat is an important aspect when assessing code prediction and a key factor supporting decision makingmore » for used fuel applications. While previous studies have largely focused on uncertainties in code predictions due to nuclear data uncertainties, this study discusses uncertainties in calculated decay heat due to uncertainties in assembly modeling parameters as well as in nuclear data. Capabilities in the SCALE nuclear analysis code system were used to quantify the effect on calculated decay heat of uncertainties in nuclear data and selected manufacturing and operation parameters for a typical boiling water reactor (BWR) fuel assembly. Furthermore, the BWR fuel assembly used as the reference case for this study was selected from a set of assemblies for which high-quality decay heat measurements are available, to assess the significance of the results through comparison with calculated and measured decay heat data.« less

Decay heat uncertainty for BWR used fuel due to modeling and nuclear data uncertainties

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ilas, Germina; Liljenfeldt, Henrik

Characterization of the energy released from radionuclide decay in nuclear fuel discharged from reactors is essential for the design, safety, and licensing analyses of used nuclear fuel storage, transportation, and repository systems. There are a limited number of decay heat measurements available for commercial used fuel applications. Because decay heat measurements can be expensive or impractical for covering the multitude of existing fuel designs, operating conditions, and specific application purposes, decay heat estimation relies heavily on computer code prediction. Uncertainty evaluation for calculated decay heat is an important aspect when assessing code prediction and a key factor supporting decision makingmore » for used fuel applications. While previous studies have largely focused on uncertainties in code predictions due to nuclear data uncertainties, this study discusses uncertainties in calculated decay heat due to uncertainties in assembly modeling parameters as well as in nuclear data. Capabilities in the SCALE nuclear analysis code system were used to quantify the effect on calculated decay heat of uncertainties in nuclear data and selected manufacturing and operation parameters for a typical boiling water reactor (BWR) fuel assembly. Furthermore, the BWR fuel assembly used as the reference case for this study was selected from a set of assemblies for which high-quality decay heat measurements are available, to assess the significance of the results through comparison with calculated and measured decay heat data.« less

Polymer-mediated nanorod self-assembly predicted by dissipative particle dynamics simulations.

PubMed

Khani, Shaghayegh; Jamali, Safa; Boromand, Arman; Hore, Michael J A; Maia, Joao

2015-09-14

Self-assembly of nanoparticles in polymer matrices is an interesting and growing subject in the field of nanoscience and technology. We report herein on modelling studies of the self-assembly and phase behavior of nanorods in a homopolymer matrix, with the specific goal of evaluating the role of deterministic entropic and enthalpic factors that control the aggregation/dispersion in such systems. Grafting polymer brushes from the nanorods is one approach to control/impact their self-assembly capabilities within a polymer matrix. From an energetic point of view, miscible interactions between the brush and the matrix are required for achieving a better dispersibility; however, grafting density and brush length are the two important parameters in dictating the morphology. Unlike in previous computational studies, the present Dissipative Particle Dynamics (DPD) simulation framework is able to both predict dispersion or aggregation of nanorods and determine the self-assembled structure, allowing for the determination of a phase diagram, which takes all of these factors into account. Three types of morphologies are predicted: dispersion, aggregation and partial aggregation. Moreover, favorable enthalpic interactions between the brush and the matrix are found to be essential for expanding the window for achieving a well-dispersed morphology. A three-dimensional phase diagram is mapped on which all the afore-mentioned parameters are taken into account. Additionally, in the case of immiscibility between brushes and the matrix, simulations predict the formation of some new and tunable structures.

Management of surgical instruments with radio frequency identification tags.

PubMed

Kusuda, Kaori; Yamashita, Kazuhiko; Ohnishi, Akiko; Tanaka, Kiyohito; Komino, Masaru; Honda, Hiroshi; Tanaka, Shinichi; Okubo, Takashi; Tripette, Julien; Ohta, Yuji

2016-01-01

To prevent malpractices, medical staff has adopted inventory time-outs and/or checklists. Accurate inventory and maintenance of surgical instruments decreases the risk of operating room miscounting and malfunction. In our previous study, an individual management of surgical instruments was accomplished using Radio Frequency Identification (RFID) tags. The purpose of this paper is to evaluate a new management method of RFID-tagged instruments. The management system of RFID-tagged surgical instruments was used for 27 months in clinical areas. In total, 13 study participants assembled surgical trays in the central sterile supply department. While using the management system, trays were assembled 94 times. During this period, no assembly errors occurred. An instrument malfunction had occurred after the 19th, 56th, and 73 th uses, no malfunction caused by the RFID tags, and usage history had been recorded. Additionally, the time it took to assemble surgical trays was recorded, and the long-term usability of the management system was evaluated. The system could record the number of uses and the defective history of each surgical instrument. In addition, the history of the frequency of instruments being transferred from one tray to another was recorded. The results suggest that our system can be used to manage instruments safely. Additionally, the management system was acquired of the learning effect and the usability on daily maintenance. This finding suggests that the management system examined here ensures surgical instrument and tray assembly quality.

Analysis of de novo sequencing and transcriptome assembly and lignocellulolytic enzymes gene expression of Coriolopsis gallica HTC.

PubMed

Chen, Yuehong; Cao, Qinghua; Tao, Xiang; Shao, Huanhuan; Zhang, Kun; Zhang, Yizheng; Tan, Xuemei

2017-03-01

White-rot basidiomycete Coriolopsis gallica HTC is one of the main biodegraders of poplar. In our previous study, we have shown the strong capacity of C. gallica HTC to degrade lignocellulose. In this study, equal amounts of total RNA fromC. Gallica HTC cultures grown in different conditions were pooled together. Illumina paired-end RNA sequencing was performed, and 13.2 million 90-bp paired-end reads were generated. We chose the Merged Assembly of Oases data-set for the following blast searches and gene ontology analyses. The reads were assembled de novo into 28,034 transcripts (≥ 100 bp) using combined assembly strategy MAO. The transcripts were annotated using Blast2GO. In all, 18,810 transcripts (≥100 bp) achieved BLASTX hits, of which, 7048 transcripts had GO term and 2074 had ECs. The expression level of 11 lignocellulolytic enzyme genes from the assembled C. gallica HTC transcriptome were detected by real-time quantitative polymerase chain reaction. The results showed that expression levels of these genes were affected by carbon source and nitrogen source at the level of transcription. The current abundant transcriptome data allowed the identification of many new transcripts in C. gallica HTC. Data provided here represent the most comprehensive and integrated genomic resources for cloning and identifying genes of interest from C. gallica HTC. Characterization of C. gallica HTC transcriptome provides an effective tool to understand mechanisms underlying cellular and molecular functions of C. gallica HTC.

Unknown Aspects of Self-Assembly of PbS Microscale Superstructures

PubMed Central

Querejeta-Fernández, Ana; Hernández-Garrido, Juan C.; Yang, Hengxi; Zhou, Yunlong; Varela, Aurea; Parras, Marina; Calvino-Gámez, José J.; González-Calbet, Jose M.; Green, Peter F.; Kotov, Nicholas A.

2012-01-01

A lot of interesting and sophisticated examples of nanoparticle (NP) self-assembly (SA) are known. From both fundamental and technological standpoints this field requires advancements in three principle directions: a) understanding the mechanism and driving forces of three-dimensional (3D) SA with both nano- and micro-levels of organization; b) understanding of disassembly/deconstruction processes; and c) finding synthetic methods of assembly into continuous superstructures without insulating barriers. From this perspective, we investigated the formation of well-known star-like PbS superstructures and found a number of previously unknown or overlooked aspects that can advance the knowledge of NP self-assembly in these three directions. The primary one is that the formation of large seemingly monocrystalline PbS superstructures with multiple levels of octahedral symmetry can be explained only by SA of small octahedral NPs. We found five distinct periods in the formation PbS hyperbranched stars: 1) nucleation of early PbS NPs with an average diameter of 31 nm; 2) assembly into 100–500 nm octahedral mesocrystals; 3) assembly into 1000–2500 nm hyperbranched stars; 4) assembly and ionic recrystallization into six-arm rods accompanied by disappearance of fine nanoscale structure; 5) deconstruction into rods and cubooctahedral NPs. The switches in assembly patterns between the periods occur due to variable dominance of pattern–determining forces that include vander Waals and electrostatic (charge-charge, dipole-dipole, and polarization) interactions. The superstructure deconstruction is triggered by chemical changes in the deep eutectic solvent (DES) used as the media. PbS superstructures can be excellent models for fundamental studies of nanoscale organization and SA manufacturing of (opto)electronics and energy harvesting devices which require organization of PbS components at multiple scales. PMID:22515512

Unknown aspects of self-assembly of PbS microscale superstructures.

PubMed

Querejeta-Fernández, Ana; Hernández-Garrido, Juan C; Yang, Hengxi; Zhou, Yunlong; Varela, Aurea; Parras, Marina; Calvino-Gámez, José J; González-Calbet, Jose M; Green, Peter F; Kotov, Nicholas A

2012-05-22

A lot of interesting and sophisticated examples of nanoparticle (NP) self-assembly (SA) are known. From both fundamental and technological standpoints, this field requires advancements in three principle directions: (a) understanding the mechanism and driving forces of three-dimensional (3D) SA with both nano- and microlevels of organization; (b) understanding disassembly/deconstruction processes; and (c) finding synthetic methods of assembly into continuous superstructures without insulating barriers. From this perspective, we investigated the formation of well-known star-like PbS superstructures and found a number of previously unknown or overlooked aspects that can advance the knowledge of NP self-assembly in these three directions. The primary one is that the formation of large seemingly monocrystalline PbS superstructures with multiple levels of octahedral symmetry can be explained only by SA of small octahedral NPs. We found five distinct periods in the formation PbS hyperbranched stars: (1) nucleation of early PbS NPs with an average diameter of 31 nm; (2) assembly into 100-500 nm octahedral mesocrystals; (3) assembly into 1000-2500 nm hyperbranched stars; (4) assembly and ionic recrystallization into six-arm rods accompanied by disappearance of fine nanoscale structure; (5) deconstruction into rods and cuboctahedral NPs. The switches in assembly patterns between the periods occur due to variable dominance of pattern-determining forces that include van der Waals and electrostatic (charge-charge, dipole-dipole, and polarization) interactions. The superstructure deconstruction is triggered by chemical changes in the deep eutectic solvent (DES) used as the media. PbS superstructures can be excellent models for fundamental studies of nanoscale organization and SA manufacturing of (opto)electronics and energy-harvesting devices which require organization of PbS components at multiple scales.

Fluorescent J-aggregates of core-substituted perylene bisimides: studies on structure-property relationship, nucleation-elongation mechanism, and sergeants-and-soldiers principle.

PubMed

Kaiser, Theo E; Stepanenko, Vladimir; Würthner, Frank

2009-05-20

A series of highly soluble and fluorescent, at core tetraaryloxy-substituted and in imide positions hydrogen atom containing perylene bisimide (PBI) dyes 1a-e with varying peripheral side chains have been synthesized and thoroughly characterized. The self-assembly of these PBIs has been studied in detail by UV/vis, linear dichroism (LD) and circular dichroism (CD) spectroscopy, and scanning probe microscopy (AFM, STM). These studies revealed that the present PBIs self-assemble into extended double string cables, which consist of two hydrogen-bonded supramolecular polymeric chains of densely packed and strongly excitonically coupled PBI chromophores, providing highly fluorescent J-aggregates. The aggregation strength ("melting" temperature) and the fluorescence properties of these J-aggregates are dependent on the number and chain length of the peripheral alkoxy substituents, thus revealing a structure-property relationship. In contrast to previously reported assemblies of PBIs, for which the aggregation process is described by the isodesmic (or equal K) model, a cooperative nucleation-elongation mechanism applies for the aggregation of the present assemblies as revealed by concentration-dependent UV/vis absorption studies with the chiral PBI 1e, providing equilibrium constants for dimerization (= nucleation) of K(2) = 13 +/- 11 L mol(-1) and for elongation of K = 2.3 +/- 0.1 x 10(6) L mol(-1) in methylcyclohexane (MCH). LD spectroscopic measurements have been performed to analyze the orientation of the monomers within the aggregates. The nonlinearity of chiral amplification in PBI aggregates directed by sergeants-and-soldiers principle has been elucidated by coaggregation experiments of different PBI dyes using CD spectroscopy. The dimensions as well as the molecular arrangement of the monomeric units in assemblies have been explored by atomic force microscopy (AFM) and scanning tunneling microscopy (STM).

NEDDylation promotes stress granule assembly

PubMed Central

Jayabalan, Aravinth Kumar; Sanchez, Anthony; Park, Ra Young; Yoon, Sang Pil; Kang, Gum-Yong; Baek, Je-Hyun; Anderson, Paul; Kee, Younghoon; Ohn, Takbum

2016-01-01

Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly. PMID:27381497

Self-assembly of pi-conjugated peptides in aqueous environments leading to energy-transporting bioelectronic nanostructures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tavor, John

The realization of new supramolecular pi-conjugated organic structures inspired and driven by peptide-based self-assembly will offer a new approach to interface with the biotic environment in a way that will help to meet many DOE-recognized grand challenges. Previously, we developed pi-conjugated peptides that undergo supramolecular self-assembly into one-dimensional (1-D) organic electronic nanomaterials under benign aqueous conditions. The intermolecular interactions among the pi-conjugated organic segments within these nanomaterials lead to defined perturbations of their optoelectronic properties and yield nanoscale conduits that support energy transport within individual nanostructures and throughout bulk macroscopic collections of nanomaterials. Our objectives for future research are tomore » construct and study biomimetic electronic materials for energy-related technology optimized for harsher non-biological environments where peptide-driven self-assembly enhances pi-stacking within nanostructured biomaterials, as detailed in the following specific tasks: (1) synthesis and detailed optoelectronic characterization of new pi-electron units to embed within homogeneous self assembling peptides, (2) molecular and data-driven modeling of the nanomaterial aggregates and their higher-order assemblies, and (3) development of new hierarchical assembly paradigms to organize multiple electronic subunits within the nanomaterials leading to heterogeneous electronic properties (i.e. gradients and localized electric fields). These intertwined research tasks will lead to the continued development and fundamental mechanistic understanding of a powerful bioinspired materials set capable of making connections between nanoscale electronic materials and macroscopic bulk interfaces, be they those of a cell, a protein or a device.« less

Designer Self-Assembling Peptide Nanofiber Scaffolds Containing Link Protein N-Terminal Peptide Induce Chondrogenesis of Rabbit Bone Marrow Stem Cells

PubMed Central

Wang, Baichuan; Sun, Caixia; Shao, Zengwu; Yang, Shuhua; Che, Biao; Wu, Qiang; Liu, Jianxiang

2014-01-01

Designer self-assembling peptide nanofiber hydrogel scaffolds have been considered as promising biomaterials for tissue engineering because of their excellent biocompatibility and biofunctionality. Our previous studies have shown that a novel designer functionalized self-assembling peptide nanofiber hydrogel scaffold (RLN/RADA16, LN-NS) containing N-terminal peptide sequence of link protein (link N) can promote nucleus pulposus cells (NPCs) adhesion and three-dimensional (3D) migration and stimulate biosynthesis of type II collagen and aggrecan by NPCs in vitro. The present study has extended these investigations to determine the effects of this functionalized LN-NS on bone marrow stem cells (BMSCs), a potential cell source for NP regeneration. Although the functionalized LN-NS cannot promote BMSCs proliferation, it significantly promotes BMSCs adhesion compared with that of the pure RADA16 hydrogel scaffold. Moreover, the functionalized LN-NS remarkably stimulates biosynthesis and deposition of type II collagen and aggrecan. These data demonstrate that the functionalized peptide nanofiber hydrogel scaffold containing link N peptide as a potential matrix substrate will be very useful in the NP tissue regeneration. PMID:25243141

Identification of a novel interspecific hybrid yeast from a metagenomic spontaneously inoculated beer sample using Hi-C.

PubMed

Smukowski Heil, Caiti; Burton, Joshua N; Liachko, Ivan; Friedrich, Anne; Hanson, Noah A; Morris, Cody L; Schacherer, Joseph; Shendure, Jay; Thomas, James H; Dunham, Maitreya J

2018-01-01

Interspecific hybridization is a common mechanism enabling genetic diversification and adaptation; however, the detection of hybrid species has been quite difficult. The identification of microbial hybrids is made even more complicated, as most environmental microbes are resistant to culturing and must be studied in their native mixed communities. We have previously adapted the chromosome conformation capture method Hi-C to the assembly of genomes from mixed populations. Here, we show the method's application in assembling genomes directly from an uncultured, mixed population from a spontaneously inoculated beer sample. Our assembly method has enabled us to de-convolute four bacterial and four yeast genomes from this sample, including a putative yeast hybrid. Downstream isolation and analysis of this hybrid confirmed its genome to consist of Pichia membranifaciens and that of another related, but undescribed, yeast. Our work shows that Hi-C-based metagenomic methods can overcome the limitation of traditional sequencing methods in studying complex mixtures of genomes. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Assembly/disassembly of a complex icosahedral virus to incorporate heterologous nucleic acids

NASA Astrophysics Data System (ADS)

Pascual, Elena; Mata, Carlos P.; Carrascosa, José L.; Castón, José R.

2017-12-01

Hollow protein containers are widespread in nature, and include virus capsids as well as eukaryotic and bacterial complexes. Protein cages are studied extensively for applications in nanotechnology, nanomedicine and materials science. Their inner and outer surfaces can be modified chemically or genetically, and the internal cavity can be used to template, store and/or arrange molecular cargos. Virus capsids and virus-like particles (VLP, noninfectious particles) provide versatile platforms for nanoscale bioengineering. Study of capsid protein self-assembly into monodispersed particles, and of VLP structure and biophysics is necessary not only to understand natural processes, but also to infer how these platforms can be redesigned to furnish novel functional VLP. Here we address the assembly dynamics of infectious bursal disease virus (IBDV), a complex icosahedral virus. IBDV has a ~70 nm-diameter T  =  13 capsid with VP2 trimers as the only structural subunits. During capsid assembly, VP2 is synthesized as a precursor (pVP2) whose C terminus is cleaved. The pVP2 C terminus has an amphipathic helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, necessary for control of assembly, 466/456-residue pVP2 intermediates bearing this helix assemble into VLP only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for genetic insertion of proteins (cargo space ~78 000 nm3). We established an in vitro assembly/disassembly system of HT-VP2-466-based VLP for heterologous nucleic acid packaging and/or encapsulation of drugs and other molecules. HT-VP2-466 (empty) capsids were disassembled and reassembled by dialysis against low-salt/basic pH and high-salt/acid pH buffers, respectively, thus illustrating the reversibility in vitro of IBDV capsid assembly. HT-VP2-466 VLP also packed heterologous DNA by non-specific confinement during assembly. These and previous results establish the bases for biotechnological applications based on the IBDV capsid and its ability to incorporate exogenous proteins and nucleic acids.

Cost-effective sequencing of full-length cDNA clones powered by a de novo-reference hybrid assembly.

PubMed

Kuroshu, Reginaldo M; Watanabe, Junichi; Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka; Kasahara, Masahiro

2010-05-07

Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence approximately 800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only approximately US$3 per clone, demonstrating a significant advantage over previous approaches.

Gene space and transcriptome assemblies of leafy spurge (Euphorbia esula) identify promoter sequences, repetitive elements, high-quality markers, and a full-length chloroplast genome

USDA-ARS?s Scientific Manuscript database

Leafy spurge is an invasive perennial weed infesting range and recreational lands of North America. Previous research and omics projects with leafy spurge have helped develop it as a model for studying numerous aspects of perennial plant development and response to abiotic stress. However, the lack ...

Input dependent cell assembly dynamics in a model of the striatal medium spiny neuron network.

PubMed

Ponzi, Adam; Wickens, Jeff

2012-01-01

The striatal medium spiny neuron (MSN) network is sparsely connected with fairly weak GABAergic collaterals receiving an excitatory glutamatergic cortical projection. Peri-stimulus time histograms (PSTH) of MSN population response investigated in various experimental studies display strong firing rate modulations distributed throughout behavioral task epochs. In previous work we have shown by numerical simulation that sparse random networks of inhibitory spiking neurons with characteristics appropriate for UP state MSNs form cell assemblies which fire together coherently in sequences on long behaviorally relevant timescales when the network receives a fixed pattern of constant input excitation. Here we first extend that model to the case where cortical excitation is composed of many independent noisy Poisson processes and demonstrate that cell assembly dynamics is still observed when the input is sufficiently weak. However if cortical excitation strength is increased more regularly firing and completely quiescent cells are found, which depend on the cortical stimulation. Subsequently we further extend previous work to consider what happens when the excitatory input varies as it would when the animal is engaged in behavior. We investigate how sudden switches in excitation interact with network generated patterned activity. We show that sequences of cell assembly activations can be locked to the excitatory input sequence and outline the range of parameters where this behavior is shown. Model cell population PSTH display both stimulus and temporal specificity, with large population firing rate modulations locked to elapsed time from task events. Thus the random network can generate a large diversity of temporally evolving stimulus dependent responses even though the input is fixed between switches. We suggest the MSN network is well suited to the generation of such slow coherent task dependent response which could be utilized by the animal in behavior.

Input Dependent Cell Assembly Dynamics in a Model of the Striatal Medium Spiny Neuron Network

PubMed Central

Ponzi, Adam; Wickens, Jeff

2012-01-01

The striatal medium spiny neuron (MSN) network is sparsely connected with fairly weak GABAergic collaterals receiving an excitatory glutamatergic cortical projection. Peri-stimulus time histograms (PSTH) of MSN population response investigated in various experimental studies display strong firing rate modulations distributed throughout behavioral task epochs. In previous work we have shown by numerical simulation that sparse random networks of inhibitory spiking neurons with characteristics appropriate for UP state MSNs form cell assemblies which fire together coherently in sequences on long behaviorally relevant timescales when the network receives a fixed pattern of constant input excitation. Here we first extend that model to the case where cortical excitation is composed of many independent noisy Poisson processes and demonstrate that cell assembly dynamics is still observed when the input is sufficiently weak. However if cortical excitation strength is increased more regularly firing and completely quiescent cells are found, which depend on the cortical stimulation. Subsequently we further extend previous work to consider what happens when the excitatory input varies as it would when the animal is engaged in behavior. We investigate how sudden switches in excitation interact with network generated patterned activity. We show that sequences of cell assembly activations can be locked to the excitatory input sequence and outline the range of parameters where this behavior is shown. Model cell population PSTH display both stimulus and temporal specificity, with large population firing rate modulations locked to elapsed time from task events. Thus the random network can generate a large diversity of temporally evolving stimulus dependent responses even though the input is fixed between switches. We suggest the MSN network is well suited to the generation of such slow coherent task dependent response which could be utilized by the animal in behavior. PMID:22438838

Membrane electrode gasket assembly (MEGA) technology for polymer electrolyte fuel cells

NASA Astrophysics Data System (ADS)

Pozio, A.; Giorgi, L.; De Francesco, M.; Silva, R. F.; Lo Presti, R.; Danzi, A.

A new technology for the production of a membrane electrode gasket assembly (MEGA) for polymer electrolyte fuel cells (PEFCs) is defined. The MEGA system was prepared by sealing a previously prepared membrane electrode assembly (MEA) in a moulded gasket. For this aim, a proprietary silicone based liquid mixture was injected directly into the MEA borders. Gaskets obtained in different shapes and hardness grades are stable in a wide temperature range. The MEGA technology shows several advantages with respect to traditional PEFCs stack assembling systems: effective membrane saving, reduced fabrication time, possibility of quality control and failed elements substitution. This technology was successfully tested at the ENEA laboratories and the results were acquired in laboratory scale, but industrial production appears to be simple and cheap.

Metal Ion-Assembled Micro-Collagen Heterotrimers

PubMed Central

LeBruin, Lyndelle Toni; Banerjee, Sunandan; O'Rourke, Bruce Delany; Case, Martin Ashley

2011-01-01

Collagen mimetic peptides (CMPs) provide critical insight into the assembly, stability and structure of the triple helical collagen protein. The majority of natural fibrous collagens are aab or abc heterotrimers, yet few examples of heterotrimeric CMPs have been reported. Previously CMP heterotrimers have only been accessible by total syntheses or by introducing complementary interstrand electrostatic or steric interactions. Here we describe an abc CMP heterotrimer in which each contributing CMP consists of only three amino acids: glycine, proline and 4-hydroxyproline. Assembly of the heterotrimeric triple helix is directed by a combination of metal-ion coordination to set the relative register of the CMPs, and minimization of valence frustration to direct heterotrimerization. Assembly of the four-component mixture is facile and extremely rapid, and equilibration to the abc heterotrimer occurs within a few hours at modestly elevated temperatures. The melting temperatures of the metal-assembled collagen trimers are higher by some 30 °C than the apopeptide assemblies. Two iterations of the design are described, and the outcomes suggest possibilities for designing self-assembling abc and abb heterotrimers. PMID:21590759

Localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center

PubMed Central

2017-01-01

Adenovirus (AdV) morphogenesis is a complex process, many aspects of which remain unclear. In particular, it is not settled where in the nucleus assembly and packaging occur, and whether these processes occur in a sequential or a concerted manner. Here we use immunofluorescence and immunoelectron microscopy (immunoEM) to trace packaging factors and structural proteins at late times post infection by either wildtype virus or a delayed packaging mutant. We show that representatives of all assembly factors are present in the previously recognized peripheral replicative zone, which therefore is the AdV assembly factory. Assembly intermediates and abortive products observed in this region favor a concurrent assembly and packaging model comprising two pathways, one for capsid proteins and another one for core components. Only when both pathways are coupled by correct interaction between packaging proteins and the genome is the viral particle produced. Decoupling generates accumulation of empty capsids and unpackaged cores. PMID:28448571

FANTOM5 CAGE profiles of human and mouse reprocessed for GRCh38 and GRCm38 genome assemblies.

PubMed

Abugessaisa, Imad; Noguchi, Shuhei; Hasegawa, Akira; Harshbarger, Jayson; Kondo, Atsushi; Lizio, Marina; Severin, Jessica; Carninci, Piero; Kawaji, Hideya; Kasukawa, Takeya

2017-08-29

The FANTOM5 consortium described the promoter-level expression atlas of human and mouse by using CAGE (Cap Analysis of Gene Expression) with single molecule sequencing. In the original publications, GRCh37/hg19 and NCBI37/mm9 assemblies were used as the reference genomes of human and mouse respectively; later, the Genome Reference Consortium released newer genome assemblies GRCh38/hg38 and GRCm38/mm10. To increase the utility of the atlas in forthcoming researches, we reprocessed the data to make them available on the recent genome assemblies. The data include observed frequencies of transcription starting sites (TSSs) based on the realignment of CAGE reads, and TSS peaks that are converted from those based on the previous reference. Annotations of the peak names were also updated based on the latest public databases. The reprocessed results enable us to examine frequencies of transcription initiations on the recent genome assemblies and to refer promoters with updated information across the genome assemblies consistently.

Assembly-directed antivirals differentially bind quasiequivalent pockets to modify hepatitis B virus capsid tertiary and quaternary structure.

PubMed

Katen, Sarah P; Tan, Zhenning; Chirapu, Srinivas Reddy; Finn, M G; Zlotnick, Adam

2013-08-06

Hepatitis B virus (HBV) is a major cause of liver disease. Assembly of the HBV capsid is a critical step in virus production and an attractive target for new antiviral therapies. We determined the structure of HBV capsid in complex with AT-130, a member of the phenylpropenamide family of assembly effectors. AT-130 causes tertiary and quaternary structural changes but does not disrupt capsid structure. AT-130 binds a hydrophobic pocket that also accommodates the previously characterized heteroaryldihydropyrimidine compounds but favors a unique quasiequivalent location on the capsid surface. Thus, this pocket is a promiscuous drug-binding site and a likely target for different assembly effectors with a broad range of mechanisms of activity. That AT-130 successfully decreases virus production by increasing capsid assembly rate without disrupting capsid structure delineates a paradigm in antiviral design, that disrupting reaction timing is a viable strategy for assembly effectors of HBV and other viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Interset: A natural language interface for teleoperated robotic assembly of the EASE space structure

NASA Technical Reports Server (NTRS)

Boorsma, Daniel K.

1989-01-01

A teleoperated robot was used to assemble the Experimental Assembly of Structures in Extra-vehicular activity (EASE) space structure under neutral buoyancy conditions, simulating a telerobot performing structural assembly in the zero gravity of space. This previous work used a manually controlled teleoperator as a test bed for system performance evaluations. From these results several Artificial Intelligence options were proposed. One of these was further developed into a real time assembly planner. The interface for this system is effective in assembling EASE structures using windowed graphics and a set of networked menus. As the problem space becomes more complex and hence the set of control options increases, a natural language interface may prove to be beneficial to supplement the menu based control strategy. This strategy can be beneficial in situations such as: describing the local environment, maintaining a data base of task event histories, modifying a plan or a heuristic dynamically, summarizing a task in English, or operating in a novel situation.

Flow-oriented dynamic assembly algorithm in TCP over OBS networks

NASA Astrophysics Data System (ADS)

Peng, Shuping; Li, Zhengbin; He, Yongqi; Xu, Anshi

2008-11-01

OBS is envisioned as a promising infrastructure for the next generation optical network, and TCP is likely to be the dominant transport protocol in the next generation network. Therefore, it is necessary to evaluate the performance of TCP over OBS networks. The assembly at the ingress edge nodes will impact the network performance. There have been several Fixed Assembly Period (FAP) algorithms proposed. However, the assembly period in FAP is fixed, and it can not be adjusted according to the network condition. Moreover, in FAP, the packets from different TCP sources are assembled into one burst. In that case, if such a burst is dropped, the TCP windows of the corresponding sources will shrink and the throughput will be reduced. In this paper, we introduced a flow-oriented Dynamic Assembly Period (DAP) algorithm for TCP over OBS networks. Through comparing the previous and current burst lengths, DAP can track the variation of TCP window, and update the assembly period dynamically for the next assembly. The performance of DAP is evaluated over a single TCP connection and multiple connections, respectively. The simulation results show that DAP performs better than FAP at almost the whole range of burst dropping probability.

MILP model for integrated balancing and sequencing mixed-model two-sided assembly line with variable launching interval and assignment restrictions

NASA Astrophysics Data System (ADS)

Azmi, N. I. L. Mohd; Ahmad, R.; Zainuddin, Z. M.

2017-09-01

This research explores the Mixed-Model Two-Sided Assembly Line (MMTSAL). There are two interrelated problems in MMTSAL which are line balancing and model sequencing. In previous studies, many researchers considered these problems separately and only few studied them simultaneously for one-sided line. However in this study, these two problems are solved simultaneously to obtain more efficient solution. The Mixed Integer Linear Programming (MILP) model with objectives of minimizing total utility work and idle time is generated by considering variable launching interval and assignment restriction constraint. The problem is analysed using small-size test cases to validate the integrated model. Throughout this paper, numerical experiment was conducted by using General Algebraic Modelling System (GAMS) with the solver CPLEX. Experimental results indicate that integrating the problems of model sequencing and line balancing help to minimise the proposed objectives function.

Observation of quantum criticality with ultracold atoms in optical lattices

NASA Astrophysics Data System (ADS)

Zhang, Xibo

As biological problems are becoming more complex and data growing at a rate much faster than that of computer hardware, new and faster algorithms are required. This dissertation investigates computational problems arising in two of the fields: comparative genomics and epigenomics, and employs a variety of computational techniques to address the problems. One fundamental question in the studies of chromosome evolution is whether the rearrangement breakpoints are happening at random positions or along certain hotspots. We investigate the breakpoint reuse phenomenon, and show the analyses that support the more recently proposed fragile breakage model as opposed to the conventional random breakage models for chromosome evolution. The identification of syntenic regions between chromosomes forms the basis for studies of genome architectures, comparative genomics, and evolutionary genomics. The previous synteny block reconstruction algorithms could not be scaled to a large number of mammalian genomes being sequenced; neither did they address the issue of generating non-overlapping synteny blocks suitable for analyzing rearrangements and evolutionary history of large-scale duplications prevalent in plant genomes. We present a new unified synteny block generation algorithm based on A-Bruijn graph framework that overcomes these shortcomings. In the epigenome sequencing, a sample may contain a mixture of epigenomes and there is a need to resolve the distinct methylation patterns from the mixture. Many sequencing applications, such as haplotype inference for diploid or polyploid genomes, and metagenomic sequencing, share the similar objective: to infer a set of distinct assemblies from reads that are sequenced from a heterogeneous sample and subsequently aligned to a reference genome. We model the problem from both a combinatorial and a statistical angles. First, we describe a theoretical framework. A linear-time algorithm is then given to resolve a minimum number of assemblies that are consistent with all reads, substantially improving on previous algorithms. An efficient algorithm is also described to determine a set of assemblies that is consistent with a maximum subset of the reads, a previously untreated problem. We then prove that allowing nested reads or permitting mismatches between reads and their assemblies renders these problems NP-hard. Second, we describe a mixture model-based approach, and applied the model for the detection of allele-specific methylations.

Understanding self-assembly of charged-neutral block copolymer (BCP) and surfactant complexes using molecular dynamics (MD) simulation

NASA Astrophysics Data System (ADS)

Goswami, Monojoy; Sumpter, Bobby; Kilbey, Michael

Here we report the formation of phase separated BCP-surfactant complexes resulting from the electrostatic self-assembly of charge-neutral block copolymers with oppositely charged surfactants. Complexation behaviors of oppositely charged polyelectrolytes has gained considerable attention in the field of soft condensed matter physics due to their potential application as functional nanomaterials for batteries, wastewater treatment and drug delivery systems. Numerous experiments have examined the self-assembled structures resulting from complexation of charge-neutral BCP and surfactants, however, there is a lack of comprehensive understanding at the fundamental level. To help bridge this gap, we use, MD simulations to study self-assembly and dynamics of the BCP-surfactant complex at the molecular level. Our results show an overcharging effect in BCPs with hydrophobic neutral blocks and a formation of core-shell colloidal structure. Hydrophilic neutral blocks, on the other hand, show stable, hairy colloidal structures with neutral blocks forming a loosely-bound, fuzzy outer layer. Our results qualitatively agree with previous SANS and SAXS experiments. This work was supported by the U.S. Department of Energy (DOE), Office of Basic Energy Sciences, Materials Science and Engineering Division.

Self-assembly programming of DNA polyominoes.

PubMed

Ong, Hui San; Syafiq-Rahim, Mohd; Kasim, Noor Hayaty Abu; Firdaus-Raih, Mohd; Ramlan, Effirul Ikhwan

2016-10-20

Fabrication of functional DNA nanostructures operating at a cellular level has been accomplished through molecular programming techniques such as DNA origami and single-stranded tiles (SST). During implementation, restrictive and constraint dependent designs are enforced to ensure conformity is attainable. We propose a concept of DNA polyominoes that promotes flexibility in molecular programming. The fabrication of complex structures is achieved through self-assembly of distinct heterogeneous shapes (i.e., self-organised optimisation among competing DNA basic shapes) with total flexibility during the design and assembly phases. In this study, the plausibility of the approach is validated using the formation of multiple 3×4 DNA network fabricated from five basic DNA shapes with distinct configurations (monomino, tromino and tetrominoes). Computational tools to aid the design of compatible DNA shapes and the structure assembly assessment are presented. The formations of the desired structures were validated using Atomic Force Microscopy (AFM) imagery. Five 3×4 DNA networks were successfully constructed using combinatorics of these five distinct DNA heterogeneous shapes. Our findings revealed that the construction of DNA supra-structures could be achieved using a more natural-like orchestration as compared to the rigid and restrictive conventional approaches adopted previously. Copyright © 2016 Elsevier B.V. All rights reserved.

Efficient process for previous metal recovery from cell membrane electrode assemblies

DOEpatents

Shore, Lawrence; Matlin, Ramail; Heinz, Robert

2010-05-04

A method is provided for recovering a catalytic element from a fuel cell membrane electrode assembly. The method includes grinding the membrane electrode assembly into a powder, extracting the catalytic element by forming a slurry comprising the powder and an acid leachate adapted to dissolve the catalytic element into a soluble salt, and separating the slurry into a depleted powder and a supernatant containing the catalytic element salt. The depleted powder is washed to remove any catalytic element salt retained within pores in the depleted powder and the catalytic element is purified from the salt.

Comparing highly ordered monolayers of nanoparticles fabricated using electrophoretic deposition: Cobalt ferrite nanoparticles versus iron oxide nanoparticles

DOE PAGES

Dickerson, James H.; Krejci, Alex J.; Garcia, Adriana -Mendoza; ...

2015-08-01

Ordered assemblies of nanoparticles remain challenging to fabricate, yet could open the door to many potential applications of nanomaterials. Here, we demonstrate that locally ordered arrays of nanoparticles, using electrophoretic deposition, can be extended to produce long-range order among the constituents. Voronoi tessellations along with multiple statistical analyses show dramatic increases in order compared with previously reported assemblies formed through electric field-assisted assembly. As a result, based on subsequent physical measurements of the nanoparticles and the deposition system, the underlying mechanisms that generate increased order are inferred.

De novo assembly of a haplotype-resolved human genome.

PubMed

Cao, Hongzhi; Wu, Honglong; Luo, Ruibang; Huang, Shujia; Sun, Yuhui; Tong, Xin; Xie, Yinlong; Liu, Binghang; Yang, Hailong; Zheng, Hancheng; Li, Jian; Li, Bo; Wang, Yu; Yang, Fang; Sun, Peng; Liu, Siyang; Gao, Peng; Huang, Haodong; Sun, Jing; Chen, Dan; He, Guangzhu; Huang, Weihua; Huang, Zheng; Li, Yue; Tellier, Laurent C A M; Liu, Xiao; Feng, Qiang; Xu, Xun; Zhang, Xiuqing; Bolund, Lars; Krogh, Anders; Kristiansen, Karsten; Drmanac, Radoje; Drmanac, Snezana; Nielsen, Rasmus; Li, Songgang; Wang, Jian; Yang, Huanming; Li, Yingrui; Wong, Gane Ka-Shu; Wang, Jun

2015-06-01

The human genome is diploid, and knowledge of the variants on each chromosome is important for the interpretation of genomic information. Here we report the assembly of a haplotype-resolved diploid genome without using a reference genome. Our pipeline relies on fosmid pooling together with whole-genome shotgun strategies, based solely on next-generation sequencing and hierarchical assembly methods. We applied our sequencing method to the genome of an Asian individual and generated a 5.15-Gb assembled genome with a haplotype N50 of 484 kb. Our analysis identified previously undetected indels and 7.49 Mb of novel coding sequences that could not be aligned to the human reference genome, which include at least six predicted genes. This haplotype-resolved genome represents the most complete de novo human genome assembly to date. Application of our approach to identify individual haplotype differences should aid in translating genotypes to phenotypes for the development of personalized medicine.

Self-Assembly of Trimer Colloids: Effect of Shape and Interaction Range†

PubMed Central

Hatch, Harold W.; Yang, Seung-Yeob; Mittal, Jeetain; Shen, Vincent K.

2016-01-01

Trimers with one attractive bead and two repulsive beads, similar to recently synthesized trimer patchy colloids, were simulated with flat-histogram Monte Carlo methods to obtain the stable self-assembled structures for different shapes and interaction potentials. Extended corresponding states principle was successfully applied to self-assembling systems in order to approximately collapse the results for models with the same shape, but different interaction range. This helps us directly compare simulation results with previous experiment, and good agreement was found between the two. In addition, a variety of self-assembled structures were observed by varying the trimer geometry, including spherical clusters, elongated clusters, monolayers, and spherical shells. In conclusion, our results help to compare simulations and experiments, via extended corresponding states, and we predict the formation of self-assembled structures for trimer shapes that have not been experimentally synthesized. PMID:27087490

Engineering multifunctional capsules through the assembly of metal-phenolic networks.

PubMed

Guo, Junling; Ping, Yuan; Ejima, Hirotaka; Alt, Karen; Meissner, Mirko; Richardson, Joseph J; Yan, Yan; Peter, Karlheinz; von Elverfeldt, Dominik; Hagemeyer, Christoph E; Caruso, Frank

2014-05-26

Metal-organic coordination materials are of widespread interest because of the coupled benefits of inorganic and organic building blocks. These materials can be assembled into hollow capsules with a range of properties, which include selective permeability, enhanced mechanical/thermal stability, and stimuli-responsiveness. Previous studies have primarily focused on the assembly aspects of metal-coordination capsules; however, the engineering of metal-specific functionality for capsule design has not been explored. A library of functional metal-phenolic network (MPN) capsules prepared from a phenolic ligand (tannic acid) and a range of metals is reported. The properties of the MPN capsules are determined by the coordinated metals, allowing for control over film thickness, disassembly characteristics, and fluorescence behavior. Furthermore, the functional properties of the MPN capsules were tailored for drug delivery, positron emission tomography (PET), magnetic resonance imaging (MRI), and catalysis. The ability to incorporate multiple metals into MPN capsules demonstrates that a diverse range of functional materials can be generated. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Assembly of 913 microbial genomes from metagenomic sequencing of the cow rumen.

PubMed

Stewart, Robert D; Auffret, Marc D; Warr, Amanda; Wiser, Andrew H; Press, Maximilian O; Langford, Kyle W; Liachko, Ivan; Snelling, Timothy J; Dewhurst, Richard J; Walker, Alan W; Roehe, Rainer; Watson, Mick

2018-02-28

The cow rumen is adapted for the breakdown of plant material into energy and nutrients, a task largely performed by enzymes encoded by the rumen microbiome. Here we present 913 draft bacterial and archaeal genomes assembled from over 800 Gb of rumen metagenomic sequence data derived from 43 Scottish cattle, using both metagenomic binning and Hi-C-based proximity-guided assembly. Most of these genomes represent previously unsequenced strains and species. The draft genomes contain over 69,000 proteins predicted to be involved in carbohydrate metabolism, over 90% of which do not have a good match in public databases. Inclusion of the 913 genomes presented here improves metagenomic read classification by sevenfold against our own data, and by fivefold against other publicly available rumen datasets. Thus, our dataset substantially improves the coverage of rumen microbial genomes in the public databases and represents a valuable resource for biomass-degrading enzyme discovery and studies of the rumen microbiome.

Assessment of lead health hazards in a body shop of an automobile assembly plant.

PubMed

Lilis, R; Valciukas, J A; Kon, S; Sarkosi, L; Campbell, C; Selikoff, I J

1982-01-01

This study of an occupationally lead-exposed group in the automobile assembly industry (body shop employees) has provided information with regard to current and recent lead-exposure levels characteristic for these operations. Comparison with findings in other, previously studied occupational groups (secondary smelter workers) indicated that current and recent lead exposure was significantly lower in the autobody shop employees. Prevalence of symptoms was found to be higher than in a control, non-lead-exposed group of maintenance workers in the same plant, but the differences reached the level of statistical significance only sporadically. Long-term effects on renal function, as reflected in blood urea nitrogen (BUN) and creatinine levels, were documented to be significant in this group of autobody shop employees. When compared with two secondary lead smelter populations previously studied, correlations between indicators of renal function, BUN and creatinine, and duration of lead exposure were consistently higher in the secondary lead smelter populations than in the group of autobody shop employees, although duration of lead exposure was greater in the latter. These findings are consistent with a dose-response relationship with regard to renal function impairment due to lead absorption. An increased prevalence of hypertension, especially diastolic, was also found, probably the consequence of renal effect.

Whole Genome Sequence Analysis of Pig Respiratory Bacterial Pathogens with Elevated Minimum Inhibitory Concentrations for Macrolides.

PubMed

Dayao, Denise Ann Estarez; Seddon, Jennifer M; Gibson, Justine S; Blackall, Patrick J; Turni, Conny

2016-10-01

Macrolides are often used to treat and control bacterial pathogens causing respiratory disease in pigs. This study analyzed the whole genome sequences of one clinical isolate of Actinobacillus pleuropneumoniae, Haemophilus parasuis, Pasteurella multocida, and Bordetella bronchiseptica, all isolated from Australian pigs to identify the mechanism underlying the elevated minimum inhibitory concentrations (MICs) for erythromycin, tilmicosin, or tulathromycin. The H. parasuis assembled genome had a nucleotide transition at position 2059 (A to G) in the six copies of the 23S rRNA gene. This mutation has previously been associated with macrolide resistance but this is the first reported mechanism associated with elevated macrolide MICs in H. parasuis. There was no known macrolide resistance mechanism identified in the other three bacterial genomes. However, strA and sul2, aminoglycoside and sulfonamide resistance genes, respectively, were detected in one contiguous sequence (contig 1) of A. pleuropneumoniae assembled genome. This contig was identical to plasmids previously identified in Pasteurellaceae. This study has provided one possible explanation of elevated MICs to macrolides in H. parasuis. Further studies are necessary to clarify the mechanism causing the unexplained macrolide resistance in other Australian pig respiratory pathogens including the role of efflux systems, which were detected in all analyzed genomes.

Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging.

PubMed

Barajas, Brook C; Tanaka, Motoko; Robinson, Bridget A; Phuong, Daryl J; Chutiraka, Kasana; Reed, Jonathan C; Lingappa, Jaisri R

2018-04-01

During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA.

Identifying the assembly intermediate in which Gag first associates with unspliced HIV-1 RNA suggests a novel model for HIV-1 RNA packaging

PubMed Central

Barajas, Brook C.; Tanaka, Motoko; Robinson, Bridget A.; Phuong, Daryl J.; Reed, Jonathan C.

2018-01-01

During immature capsid assembly, HIV-1 genome packaging is initiated when Gag first associates with unspliced HIV-1 RNA by a poorly understood process. Previously, we defined a pathway of sequential intracellular HIV-1 capsid assembly intermediates; here we sought to identify the intermediate in which HIV-1 Gag first associates with unspliced HIV-1 RNA. In provirus-expressing cells, unspliced HIV-1 RNA was not found in the soluble fraction of the cytosol, but instead was largely in complexes ≥30S. We did not detect unspliced HIV-1 RNA associated with Gag in the first assembly intermediate, which consists of soluble Gag. Instead, the earliest assembly intermediate in which we detected Gag associated with unspliced HIV-1 RNA was the second assembly intermediate (~80S intermediate), which is derived from a host RNA granule containing two cellular facilitators of assembly, ABCE1 and the RNA granule protein DDX6. At steady-state, this RNA-granule-derived ~80S complex was the smallest assembly intermediate that contained Gag associated with unspliced viral RNA, regardless of whether lysates contained intact or disrupted ribosomes, or expressed WT or assembly-defective Gag. A similar complex was identified in HIV-1-infected T cells. RNA-granule-derived assembly intermediates were detected in situ as sites of Gag colocalization with ABCE1 and DDX6; moreover these granules were far more numerous and smaller than well-studied RNA granules termed P bodies. Finally, we identified two steps that lead to association of assembling Gag with unspliced HIV-1 RNA. Independent of viral-RNA-binding, Gag associates with a broad class of RNA granules that largely lacks unspliced viral RNA (step 1). If a viral-RNA-binding domain is present, Gag further localizes to a subset of these granules that contains unspliced viral RNA (step 2). Thus, our data raise the possibility that HIV-1 packaging is initiated not by soluble Gag, but by Gag targeted to a subset of host RNA granules containing unspliced HIV-1 RNA. PMID:29664940

Effects of radial compression on a novel simulated intervertebral disc-like assembly using bone marrow-derived mesenchymal stem cell cell-sheets for annulus fibrosus regeneration.

PubMed

See, Eugene Yong-Shun; Toh, Siew Lok; Goh, James Cho-Hong

2011-10-01

The aim of this study was to develop a tissue engineering approach in regenerating the annulus fibrosus (AF) as part of an overall strategy to produce a tissue-engineered intervertebral disc (IVD) replacement. To determine whether a rehabilitative simulation regime on bone marrow–derived mesenchymal stem cell cell-sheet is able to aid the regeneration of the AF. No previous study has used bone marrow–derived mesenchymal stem cell cell-sheets simulated by a rehabilitative regime to regenerate the AF. The approach was to use bone marrow–derived stem cells to form cell-sheets and incorporating them onto silk scaffolds to simulate the native lamellae of the AF. The in vitro experimental model used to study the efficacy of such a system was made up of the tissue engineering AF construct wrapped around a silicone disc to form a simulated IVD-like assembly. The assembly was cultured within a custom-designed bioreactor that provided a compressive mechanical stimulation onto the silicone disc. The silicone nucleus pulposus would bulge radially and compress the simulated AF to mimic the physiological conditions. The simulated IVD-like assembly was compressed using a rehabilitative regime that lasted for 4 weeks at 0.25 Hz, for 15 minutes each day. With the rehabilitative regime, the cell-sheets remained viable but showed a decrease in cell numbers and viability. Gene expression analysis showed significant upregulation of IVD-related genes and there was an increased ratio of collagen type II to collagen type I found within the extracellular matrix. The results suggested that a rehabilitative regime caused extensive remodeling to take place within the simulated IVD-like assembly, producing extracellular matrix similar to that found in the inner AF.

RNA-Seq Analysis of Cocos nucifera: Transcriptome Sequencing and De Novo Assembly for Subsequent Functional Genomics Approaches

PubMed Central

Xia, Wei; Mason, Annaliese S.; Xia, Zhihui; Qiao, Fei; Zhao, Songlin; Tang, Haoru

2013-01-01

Background Cocos nucifera (coconut), a member of the Arecaceae family, is an economically important woody palm grown in tropical regions. Despite its agronomic importance, previous germplasm assessment studies have relied solely on morphological and agronomical traits. Molecular biology techniques have been scarcely used in assessment of genetic resources and for improvement of important agronomic and quality traits in Cocos nucifera, mostly due to the absence of available sequence information. Methodology/Principal Findings To provide basic information for molecular breeding and further molecular biological analysis in Cocos nucifera, we applied RNA-seq technology and de novo assembly to gain a global overview of the Cocos nucifera transcriptome from mixed tissue samples. Using Illumina sequencing, we obtained 54.9 million short reads and conducted de novo assembly to obtain 57,304 unigenes with an average length of 752 base pairs. Sequence comparison between assembled unigenes and released cDNA sequences of Cocos nucifera and Elaeis guineensis indicated that the assembled sequences were of high quality. Approximately 99.9% of unigenes were novel compared to the released coconut EST sequences. Using BLASTX, 68.2% of unigenes were successfully annotated based on the Genbank non-redundant (Nr) protein database. The annotated unigenes were then further classified using the Gene Ontology (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Conclusions/Significance Our study provides a large quantity of novel genetic information for Cocos nucifera. This information will act as a valuable resource for further molecular genetic studies and breeding in coconut, as well as for isolation and characterization of functional genes involved in different biochemical pathways in this important tropical crop species. PMID:23555859

RNA-Seq analysis of Cocos nucifera: transcriptome sequencing and de novo assembly for subsequent functional genomics approaches.

PubMed

Fan, Haikuo; Xiao, Yong; Yang, Yaodong; Xia, Wei; Mason, Annaliese S; Xia, Zhihui; Qiao, Fei; Zhao, Songlin; Tang, Haoru

2013-01-01

Cocos nucifera (coconut), a member of the Arecaceae family, is an economically important woody palm grown in tropical regions. Despite its agronomic importance, previous germplasm assessment studies have relied solely on morphological and agronomical traits. Molecular biology techniques have been scarcely used in assessment of genetic resources and for improvement of important agronomic and quality traits in Cocos nucifera, mostly due to the absence of available sequence information. To provide basic information for molecular breeding and further molecular biological analysis in Cocos nucifera, we applied RNA-seq technology and de novo assembly to gain a global overview of the Cocos nucifera transcriptome from mixed tissue samples. Using Illumina sequencing, we obtained 54.9 million short reads and conducted de novo assembly to obtain 57,304 unigenes with an average length of 752 base pairs. Sequence comparison between assembled unigenes and released cDNA sequences of Cocos nucifera and Elaeis guineensis indicated that the assembled sequences were of high quality. Approximately 99.9% of unigenes were novel compared to the released coconut EST sequences. Using BLASTX, 68.2% of unigenes were successfully annotated based on the Genbank non-redundant (Nr) protein database. The annotated unigenes were then further classified using the Gene Ontology (GO), Clusters of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Our study provides a large quantity of novel genetic information for Cocos nucifera. This information will act as a valuable resource for further molecular genetic studies and breeding in coconut, as well as for isolation and characterization of functional genes involved in different biochemical pathways in this important tropical crop species.

Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2

PubMed Central

Aijaz, Saima; Sanchez-Heras, Elena; Balda, Maria S; Matter, Karl

2007-01-01

Background Tight junctions are required for epithelial barrier formation and participate in the regulation of signalling mechanisms that control proliferation and differentiation. ZO-1 is a tight junction-associated adaptor protein that regulates gene expression, junction assembly and epithelial morphogenesis. We have previously demonstrated that the heat shock protein Apg-2 binds ZO-1 and thereby regulates its role in cell proliferation. Here, we addressed the question whether Apg-2 is also important for junction formation and epithelial morphogenesis. Results We demonstrate that depletion of Apg-2 by RNAi in MDCK cells did not prevent formation of functional tight junctions. Similar to ZO-1, however, reduced expression of Apg-2 retarded de novo junction assembly if analysed in a Ca-switch model. Formation of functional junctions, as monitored by measuring transepithelial electrical resistance, and recruitment of tight and adherens junction markers were retarded. If cultured in three dimensional extracellular matrix gels, Apg-2 depleted cells, as previously shown for ZO-1 depleted cells, did not form hollow polarised cysts but poorly organised, irregular structures. Conclusion Our data indicate that Apg-2 regulates junction assembly and is required for normal epithelial morphogenesis in a three-dimensional culture system, suggesting that Apg-2 is an important regulator of epithelial differentiation. As the observed phenotypes are similar to those previously described for ZO-1 depleted cells and depletion of Apg-2 retards junctional recruitment of ZO-1, regulation of ZO-1 is likely to be an important functional role for Apg-2 during epithelial differentiation. PMID:18028534

Regulation of tight junction assembly and epithelial morphogenesis by the heat shock protein Apg-2.

PubMed

Aijaz, Saima; Sanchez-Heras, Elena; Balda, Maria S; Matter, Karl

2007-11-20

Tight junctions are required for epithelial barrier formation and participate in the regulation of signalling mechanisms that control proliferation and differentiation. ZO-1 is a tight junction-associated adaptor protein that regulates gene expression, junction assembly and epithelial morphogenesis. We have previously demonstrated that the heat shock protein Apg-2 binds ZO-1 and thereby regulates its role in cell proliferation. Here, we addressed the question whether Apg-2 is also important for junction formation and epithelial morphogenesis. We demonstrate that depletion of Apg-2 by RNAi in MDCK cells did not prevent formation of functional tight junctions. Similar to ZO-1, however, reduced expression of Apg-2 retarded de novo junction assembly if analysed in a Ca-switch model. Formation of functional junctions, as monitored by measuring transepithelial electrical resistance, and recruitment of tight and adherens junction markers were retarded. If cultured in three dimensional extracellular matrix gels, Apg-2 depleted cells, as previously shown for ZO-1 depleted cells, did not form hollow polarised cysts but poorly organised, irregular structures. Our data indicate that Apg-2 regulates junction assembly and is required for normal epithelial morphogenesis in a three-dimensional culture system, suggesting that Apg-2 is an important regulator of epithelial differentiation. As the observed phenotypes are similar to those previously described for ZO-1 depleted cells and depletion of Apg-2 retards junctional recruitment of ZO-1, regulation of ZO-1 is likely to be an important functional role for Apg-2 during epithelial differentiation.

Failure Engineering Study and Accelerated Stress Test Results for the Mars Global Surveyor Spacecraft's Power Shunt Assemblies

NASA Technical Reports Server (NTRS)

Gibbel, Mark; Larson, Timothy

2000-01-01

An Engineering-of-Failure approach to designing and executing an accelerated product qualification test was performed to support a risk assessment of a "work-around" necessitated by an on-orbit failure of another piece of hardware on the Mars Global Surveyor spacecraft. The proposed work-around involved exceeding the previous qualification experience both in terms of extreme cold exposure level and in terms of demonstrated low cycle fatigue life for the power shunt assemblies. An analysis was performed to identify potential failure sites, modes and associated failure mechanisms consistent with the new use conditions. A test was then designed and executed which accelerated the failure mechanisms identified by analysis. Verification of the resulting failure mechanism concluded the effort.

Preparation of Self-Assembled Chitin Nanofiber-Natural Rubber Composite Sheets and Porous Materials

PubMed Central

Kawano, Akito; Yamamoto, Kazuya

2017-01-01

We previously reported the preparation of a self-assembled chitin nanofiber (CNF) film via regeneration from an ion gel with an ionic liquid, followed by sonication and filtration. Based on the finding that CNFs were redispersed in a mixture of the film with ammonia aqueous solution (aq.), in this study, CNF-natural rubber (NR) composite sheets were fabricated by mixing redispersed CNF with NR latex stabilized by ammonia, followed by drying under reduced pressure. Tensile testing of the sheets indicated the reinforcing effect of CNFs. Further, CNF-NR composite porous materials were fabricated by evaporating ammonia from the CNF-NR dispersion, followed by lyophilization. The mechanism for the formation of porous structures was evaluated. PMID:28671578

A method for the further assembly of targeted unigenes in a transcriptome after assembly by Trinity

PubMed Central

Xiao, Xinlong; Ma, Jinbiao; Sun, Yufang; Yao, Yinan

2015-01-01

RNA-sequencing has been widely used to obtain high throughput transcriptome sequences in various species, but the assembly of a full set of complete transcripts is still a significant challenge. Judging by the number of expected transcripts and assembled unigenes in a transcriptome library, we believe that some unigenes could be reassembled. In this study, using the nitrate transporter (NRT) gene family and phosphate transporter (PHT) gene family in Salicornia europaea as examples, we introduced an approach to further assemble unigenes found in transcriptome libraries which had been previously generated by Trinity. To find the unigenes of a particular transcript that contained gaps, we respectively selected 16 NRT candidate unigene pairs and 12 PHT candidate unigene pairs for which the two unigenes had the same annotations, the same expression patterns among various RNA-seq samples, and different positions of the proteins coded as mapped to a reference protein. To fill a gap between the two unigenes, PCR was performed using primers that mapped to the two unigenes and the PCR products were sequenced, which demonstrated that 5 unigene pairs of NRT and 3 unigene pairs of PHT could be reassembled when the gaps were filled using the corresponding PCR product sequences. This fast and simple method will reduce the redundancy of targeted unigenes and allow acquisition of complete coding sequences (CDS). PMID:26528307

Depletion forces drive polymer-like self-assembly in vibrofluidized granular materials†

PubMed Central

Nossal, Ralph

2011-01-01

Ranging from nano- to granular-scales, control of particle assembly can be achieved by limiting the available free space, for example by increasing the concentration of particles (“crowding”) or through their restriction to 2D environments. It is unclear, however, if self-assembly principles governing thermally-equilibrated molecules can also apply to mechanically-excited macroscopic particles in non-equilibrium steady-state. Here we show that low densities of vibrofluidized steel rods, when crowded by high densities of spheres and confined to quasi-2D planes, can self-assemble into linear polymer-like structures. Our 2D Monte Carlo simulations show similar finite sized aggregates in thermally equilibrated binary mixtures. Using theory and simulations, we demonstrate how depletion interactions create oriented “binding” forces between rigid rods to form these “living polymers.” Unlike rod-sphere mixtures in 3D that can demonstrate well-defined equilibrium phases, our mixtures confined to 2D lack these transitions because lower dimensionality favors the formation of linear aggregates, thus suppressing a true phase transition. The qualitative and quantitative agreement between equilibrium and granular patterning for these mixtures suggests that entropy maximization is the determining driving force for bundling. Furthermore, this study uncovers a previously unknown patterning behavior at both the granular and nanoscales, and may provide insights into the role of crowding at interfaces in molecular assembly. PMID:22039392

Improving amphibian genomic resources: a multitissue reference transcriptome of an iconic invader.

PubMed

Richardson, Mark F; Sequeira, Fernando; Selechnik, Daniel; Carneiro, Miguel; Vallinoto, Marcelo; Reid, Jack G; West, Andrea J; Crossland, Michael R; Shine, Richard; Rollins, Lee A

2018-01-01

Cane toads (Rhinella marina) are an iconic invasive species introduced to 4 continents and well utilized for studies of rapid evolution in introduced environments. Despite the long introduction history of this species, its profound ecological impacts, and its utility for demonstrating evolutionary principles, genetic information is sparse. Here we produce a de novo transcriptome spanning multiple tissues and life stages to enable investigation of the genetic basis of previously identified rapid phenotypic change over the introduced range. Using approximately 1.9 billion reads from developing tadpoles and 6 adult tissue-specific cDNA libraries, as well as a transcriptome assembly pipeline encompassing 100 separate de novo assemblies, we constructed 62 202 transcripts, of which we functionally annotated ∼50%. Our transcriptome assembly exhibits 90% full-length completeness of the Benchmarking Universal Single-Copy Orthologs data set. Robust assembly metrics and comparisons with several available anuran transcriptomes and genomes indicate that our cane toad assembly is one of the most complete anuran genomic resources available. This comprehensive anuran transcriptome will provide a valuable resource for investigation of genes under selection during invasion in cane toads, but will also greatly expand our general knowledge of anuran genomes, which are underrepresented in the literature. The data set is publically available in NCBI and GigaDB to serve as a resource for other researchers. © The Authors 2017. Published by Oxford University Press.

Hierarchical self-assembly of hexagonal single-crystal nanosheets into 3D layered superlattices with high conductivity.

PubMed

Tao, Yulun; Shen, Yuhua; Yang, Liangbao; Han, Bin; Huang, Fangzhi; Li, Shikuo; Chu, Zhuwang; Xie, Anjian

2012-06-21

While the number of man-made nano superstructures realized by self-assembly is growing in recent years, assemblies of conductive polymer nanocrystals, especially for superlattices, are still a significant challenge, not only because of the simplicity of the shape of the nanocrystal building blocks and their interactions, but also because of the poor control over these parameters in the fabrication of more elaborate nanocrystals. Here, we firstly report a facile and general route to a new generation of 3D layered superlattices of polyaniline doped with CSA (PANI-CSA) and show how PANI crystallize and self-assemble, in a suitable single solution environment. In cyclohexane, 1D amorphous nanofibers transformed to 1D nanorods as building blocks, and then to 2D single-crystal nanosheets with a hexagonal phase, and lastly to 3D ordered layered superlattices with the narrowest polydispersity value (M(w)/M(n) = 1.47). Remarkably, all the instructions for the hierarchical self-assembly are encoded in the layered shape in other non-polar solvents (hexane, octane) and their conductivity in the π-π stacking direction is improved to about 50 S cm(-1), which is even higher than that of the highest previously reported value (16 S cm(-1)). The method used in this study is greatly expected to be readily scalable to produce superlattices of conductive polymers with high quality and low cost.

α-Synuclein Fibrils Exhibit Gain of Toxic Function, Promoting Tau Aggregation and Inhibiting Microtubule Assembly*

PubMed Central

Oikawa, Takayuki; Nonaka, Takashi; Terada, Makoto; Tamaoka, Akira; Hisanaga, Shin-ichi; Hasegawa, Masato

2016-01-01

α-Synuclein is the major component of Lewy bodies and Lewy neurites in Parkinson disease and dementia with Lewy bodies and of glial cytoplasmic inclusions in multiple system atrophy. It has been suggested that α-synuclein fibrils or intermediate protofibrils in the process of fibril formation may have a toxic effect on neuronal cells. In this study, we investigated the ability of soluble monomeric α-synuclein to promote microtubule assembly and the effects of conformational changes of α-synuclein on Tau-promoted microtubule assembly. In marked contrast to previous findings, monomeric α-synuclein had no effect on microtubule polymerization. However, both α-synuclein fibrils and protofibrils inhibited Tau-promoted microtubule assembly. The inhibitory effect of α-synuclein fibrils was greater than that of the protofibrils. Dot blot overlay assay and spin-down techniques revealed that α-synuclein fibrils bind to Tau and inhibit microtubule assembly by depleting the Tau available for microtubule polymerization. Using various deletion mutants of α-synuclein and Tau, the acidic C-terminal region of α-synuclein and the basic central region of Tau were identified as regions involved in the binding. Furthermore, introduction of α-synuclein fibrils into cultured cells overexpressing Tau protein induced Tau aggregation. These results raise the possibility that α-synuclein fibrils interact with Tau, inhibit its function to stabilize microtubules, and also promote Tau aggregation, leading to dysfunction of neuronal cells. PMID:27226637

Improving amphibian genomic resources: a multitissue reference transcriptome of an iconic invader

PubMed Central

Reid, Jack G; Crossland, Michael R

2018-01-01

Abstract Background Cane toads (Rhinella marina) are an iconic invasive species introduced to 4 continents and well utilized for studies of rapid evolution in introduced environments. Despite the long introduction history of this species, its profound ecological impacts, and its utility for demonstrating evolutionary principles, genetic information is sparse. Here we produce a de novo transcriptome spanning multiple tissues and life stages to enable investigation of the genetic basis of previously identified rapid phenotypic change over the introduced range. Findings Using approximately 1.9 billion reads from developing tadpoles and 6 adult tissue-specific cDNA libraries, as well as a transcriptome assembly pipeline encompassing 100 separate de novo assemblies, we constructed 62 202 transcripts, of which we functionally annotated ∼50%. Our transcriptome assembly exhibits 90% full-length completeness of the Benchmarking Universal Single-Copy Orthologs data set. Robust assembly metrics and comparisons with several available anuran transcriptomes and genomes indicate that our cane toad assembly is one of the most complete anuran genomic resources available. Conclusions This comprehensive anuran transcriptome will provide a valuable resource for investigation of genes under selection during invasion in cane toads, but will also greatly expand our general knowledge of anuran genomes, which are underrepresented in the literature. The data set is publically available in NCBI and GigaDB to serve as a resource for other researchers. PMID:29186423

Superresolution Imaging Captures Carbohydrate Utilization Dynamics in Human Gut Symbionts

PubMed Central

Karunatilaka, Krishanthi S.; Cameron, Elizabeth A.; Martens, Eric C.; Koropatkin, Nicole M.

2014-01-01

ABSTRACT Gut microbes play a key role in human health and nutrition by catabolizing a wide variety of glycans via enzymatic activities that are not encoded in the human genome. The ability to recognize and process carbohydrates strongly influences the structure of the gut microbial community. While the effects of diet on the microbiota are well documented, little is known about the molecular processes driving metabolism. To provide mechanistic insight into carbohydrate catabolism in gut symbionts, we studied starch processing in real time in the model Bacteroides thetaiotaomicron starch utilization system (Sus) by single-molecule fluorescence. Although previous studies have explored Sus protein structure and function, the transient interactions, assembly, and collaboration of these outer membrane proteins have not yet been elucidated in live cells. Our live-cell superresolution imaging reveals that the polymeric starch substrate dynamically recruits Sus proteins, serving as an external scaffold for bacterial membrane assembly of the Sus complex, which may promote efficient capturing and degradation of starch. Furthermore, by simultaneously localizing multiple Sus outer membrane proteins on the B. thetaiotaomicron cell surface, we have characterized the dynamics and stoichiometry of starch-induced Sus complex assembly on the molecular scale. Finally, based on Sus protein knockout strains, we have discerned the mechanism of starch-induced Sus complex assembly in live anaerobic cells with nanometer-scale resolution. Our insights into the starch-induced outer membrane protein assembly central to this conserved nutrient uptake mechanism pave the way for the development of dietary or pharmaceutical therapies to control Bacteroidetes in the intestinal tract to enhance human health and treat disease. PMID:25389179

Supramolecular self-assemblies of beta-cyclodextrins with aromatic tethers: factors governing the helical columnar versus linear channel superstructures.

PubMed

Liu, Yu; Fan, Zhi; Zhang, Heng-Yi; Yang, Ying-Wei; Ding, Fei; Liu, Shuang-Xi; Wu, Xue; Wada, Takehiko; Inoue, Yoshihisa

2003-10-31

A series of 6-O-(p-substituted phenyl)-modified beta-cyclodextrin derivatives, i.e., 6-O-(4-bromophenyl)-beta-CD (1), 6-O-(4-nitrophenyl)-beta-CD (2), 6-O-(4-formylphenyl)-beta-CD (3), 6-phenylselenyl-6-deoxy-beta-CD (4), and 6-O-(4-hydroxybenzoyl)-beta-CD (5), were synthesized, and their inclusion complexation behavior in aqueous solution and self-assembling behavior in the solid state were comparatively studied by NMR spectroscopy, microcalorimetry, crystallography, and scanning tunneling microscopy. Interestingly, (seleno)ethers 1-4 and ester 5 displayed distinctly different self-assembling behavior in the solid state, affording a successively threading head-to-tail polymeric helical structure for the (seleno)ethers or a mutually penetrating tail-to-tail dimeric columnar channel structure for the ester. Combining the present and previous structures reported for the relevant beta-CD derivatives, we further deduce that the pivot heteroatom, through which the aromatic substituent is tethered to beta-CD, plays a critical role in determining the helix structure, endowing the 2-fold and 4-fold axes to the N/O- and S/Se-pivoted beta-CD aggregates, respectively. This means that one can control the self-assembling orientation, alignment, and helicity in the solid state by finely tuning the pivot atom and the tether length. Further NMR and calorimetric studies on the self-assembling behavior in aqueous solution revealed that the dimerization step is the key to the formation of linear polymeric supramolecular architecture, which is driven by favorable entropic contributions.

Cost-Effective Sequencing of Full-Length cDNA Clones Powered by a De Novo-Reference Hybrid Assembly

PubMed Central

Sugano, Sumio; Morishita, Shinichi; Suzuki, Yutaka

2010-01-01

Background Sequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded. Methodology We developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence ∼800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species. Conclusions The entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only ∼US$3 per clone, demonstrating a significant advantage over previous approaches. PMID:20479877

Upgraded HFIR Fuel Element Welding System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sease, John D

2010-02-01

The welding of aluminum-clad fuel plates into aluminum alloy 6061 side plate tubing is a unique design feature of the High Flux Isotope Reactor (HFIR) fuel assemblies as 101 full-penetration circumferential gas metal arc welds (GMAW) are required in the fabrication of each assembly. In a HFIR fuel assembly, 540 aluminum-clad fuel plates are assembled into two nested annular fuel elements 610 mm (24-inches) long. The welding process for the HFIR fuel elements was developed in the early 1960 s and about 450 HFIR fuel assemblies have been successfully welded using the GMAW process qualified in the 1960 s. Inmore » recent years because of the degradation of the electronic and mechanical components in the old HFIR welding system, reportable defects in plate attachment or adapter welds have been present in almost all completed fuel assemblies. In October 2008, a contract was awarded to AMET, Inc., of Rexburg, Idaho, to replace the old welding equipment with standard commercially available welding components to the maximum extent possible while maintaining the qualified HFIR welding process. The upgraded HFIR welding system represents a major improvement in the welding system used in welding HFIR fuel elements for the previous 40 years. In this upgrade, the new inner GMAW torch is a significant advancement over the original inner GMAW torch previously used. The innovative breakthrough in the new inner welding torch design is the way the direction of the cast in the 0.762 mm (0.030-inch) diameter aluminum weld wire is changed so that the weld wire emerging from the contact tip is straight in the plane perpendicular to the welding direction without creating any significant drag resistance in the feeding of the weld wire.« less

A Functional Link between RNA Replication and Virion Assembly in the Potyvirus Plum Pox Virus.

PubMed

Gallo, Araiz; Valli, Adrian; Calvo, María; García, Juan Antonio

2018-05-01

Accurate assembly of viral particles in the potyvirus Plum pox virus (PPV) has been shown to depend on the contribution of the multifunctional viral protein HCPro. In this study, we show that other viral factors, in addition to the capsid protein (CP) and HCPro, are necessary for the formation of stable PPV virions. The CP produced in Nicotiana benthamiana leaves from a subviral RNA termed LONG, which expresses a truncated polyprotein that lacks P1 and HCPro, together with HCPro supplied in trans , was assembled into virus-like particles and remained stable after in vitro incubation. In contrast, deletions in multiple regions of the LONG coding sequence prevented the CP stabilization mediated by HCPro. In particular, we demonstrated that the first 178 amino acids of P3, but not a specific nucleotide sequence coding for them, are required for CP stability and proper assembly of PPV particles. Using a sequential coagroinfiltration assay, we observed that the subviral LONG RNA replicates and locally spreads in N. benthamiana leaves expressing an RNA silencing suppressor. The analysis of the effect of both point and deletion mutations affecting RNA replication in LONG and full-length PPV demonstrated that this process is essential for the assembly of stable viral particles. Interestingly, in spite of this requirement, the CP produced by a nonreplicating viral RNA can be stably assembled into virions as long as it is coexpressed with a replication-proficient RNA. Altogether, these results highlight the importance of coupling encapsidation to other viral processes to secure a successful infection. IMPORTANCE Viruses of the family Potyviridae are among the most dangerous threats for basically every important crop, and such socioeconomical relevance has made them a subject of many research studies. In spite of this, very little is currently known about proteins and processes controlling viral genome encapsidation by the coat protein. In the case of Plum pox virus (genus Potyvirus ), for instance, we have previously shown that the multitasking viral factor HCPro plays a role in the production of stable virions. Here, by using this potyvirus as a model, we move further to show that additional factors are also necessary for the efficient production of potyviral particles. More importantly, a comprehensive screening for such factors led us to the identification of a functional link between virus replication and packaging, unraveling a previously unknown connection of these two key events of the potyviral infection cycle. Copyright © 2018 American Society for Microbiology.

In Touch with Industry. ICAF Industry Studies 1999

DTIC Science & Technology

1999-01-01

Plant , Hamtramck, MI General Motors Corporation, Warren, MI General Motors Technical Center, Warren, MI General Motors Truck Assembly Plant , Pontiac...indicate that they cannot modernize plant equipment because workers lack the necessary skills to operate more advanced equipment. The National...construct new plants . General Motors is using M&S in new car designs to replace the clumsy clay models previously used. Now, when GM is making design

Similarities and Differences in Teachers' and Researchers' Conceptions of Communicative Language Teaching: Does the Use of an Educational Model Cast a Better Light?

ERIC Educational Resources Information Center

Mangubhai, Francis; Marland, Perc; Dashwood, Ann; Son, Jeong-Bae

2005-01-01

This study seeks to document teachers' conceptions of communicative language teaching (CLT) and to compare their conceptions with a composite view of CLT assembled, in part, from researchers' accounts of the distinctive features of CLT. The research was prompted by a review of the relevant research literature showing that, though previous studies…

ADVANCEMENTS IN TIME-SPECTRA ANALYSIS METHODS FOR LEAD SLOWING-DOWN SPECTROSCOPY

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, Leon E.; Anderson, Kevin K.; Gesh, Christopher J.

2010-08-11

Direct measurement of Pu in spent nuclear fuel remains a key challenge for safeguarding nuclear fuel cycles of today and tomorrow. Lead slowing-down spectroscopy (LSDS) is an active nondestructive assay method that has the potential to provide independent, direct measurement of Pu and U isotopic mass with an uncertainty lower than the approximately 10 percent typical of today’s confirmatory assay methods. Pacific Northwest National Laboratory’s (PNNL) previous work to assess the viability of LSDS for the assay of pressurized water reactor (PWR) assemblies indicated that the method could provide direct assay of Pu-239 and U-235 (and possibly Pu-240 and Pu-241)more » with uncertainties less than a few percent, assuming suitably efficient instrumentation, an intense pulsed neutron source, and improvements in the time-spectra analysis methods used to extract isotopic information from a complex LSDS signal. This previous simulation-based evaluation used relatively simple PWR fuel assembly definitions (e.g. constant burnup across the assembly) and a constant initial enrichment and cooling time. The time-spectra analysis method was founded on a preliminary analytical model of self-shielding intended to correct for assay-signal nonlinearities introduced by attenuation of the interrogating neutron flux within the assembly.« less

Evidence for adverse reproductive outcomes among women microelectronic assembly workers.

PubMed Central

Huel, G; Mergler, D; Bowler, R

1990-01-01

Microelectronics assembly entails complex processes where several potentially fetotoxic chemical compounds are used extensively. This study was undertaken to assess the potential adverse reproductive outcomes among former women workers in a microelectronics assembly plant in New Mexico with respect to a comparable population from the same geographical region and to examine the relation between these outcomes and employment history in this plant. After matching a pool of 143 former microelectronic female workers and 105 referents, 90 former microelectronic female worker-referent pairs were constituted (representing 302 and 324 pregnancies in former workers and referents respectively). The odds ratio (for pair matching design) of spontaneous abortion among women workers, before beginning to assemble microelectronic components, was 0.9 (chi 2 = 0.04; NS). After the beginning of employment this odds ratio became 5.6 (chi 2 = 9.8; p less than 1%). This estimated odds ratio decreased to 4.0, taking into account the increased risk for spontaneous abortion in previous pregnancies before employment (chi 2 = 5.4; p less than 5%). It was not possible to determine if this effect was reversible owing to the small number of pairs available after employment. The findings of this study corroborate the results of former studies that suggest a potential association between electronic manufacturing activity and risk of spontaneous abortion. Although the organic solvents were suspected of being the potential risk factor, this study was inconclusive from this point of view. Nevertheless, these investigations may provide some insight into reproductive outcomes among female workers exposed to solvents. PMID:2378817

Finding the missing honey bee genes: lessons learned from a genome upgrade.

PubMed

Elsik, Christine G; Worley, Kim C; Bennett, Anna K; Beye, Martin; Camara, Francisco; Childers, Christopher P; de Graaf, Dirk C; Debyser, Griet; Deng, Jixin; Devreese, Bart; Elhaik, Eran; Evans, Jay D; Foster, Leonard J; Graur, Dan; Guigo, Roderic; Hoff, Katharina Jasmin; Holder, Michael E; Hudson, Matthew E; Hunt, Greg J; Jiang, Huaiyang; Joshi, Vandita; Khetani, Radhika S; Kosarev, Peter; Kovar, Christie L; Ma, Jian; Maleszka, Ryszard; Moritz, Robin F A; Munoz-Torres, Monica C; Murphy, Terence D; Muzny, Donna M; Newsham, Irene F; Reese, Justin T; Robertson, Hugh M; Robinson, Gene E; Rueppell, Olav; Solovyev, Victor; Stanke, Mario; Stolle, Eckart; Tsuruda, Jennifer M; Vaerenbergh, Matthias Van; Waterhouse, Robert M; Weaver, Daniel B; Whitfield, Charles W; Wu, Yuanqing; Zdobnov, Evgeny M; Zhang, Lan; Zhu, Dianhui; Gibbs, Richard A

2014-01-30

The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination.

Finding the missing honey bee genes: lessons learned from a genome upgrade

PubMed Central

2014-01-01

Background The first generation of genome sequence assemblies and annotations have had a significant impact upon our understanding of the biology of the sequenced species, the phylogenetic relationships among species, the study of populations within and across species, and have informed the biology of humans. As only a few Metazoan genomes are approaching finished quality (human, mouse, fly and worm), there is room for improvement of most genome assemblies. The honey bee (Apis mellifera) genome, published in 2006, was noted for its bimodal GC content distribution that affected the quality of the assembly in some regions and for fewer genes in the initial gene set (OGSv1.0) compared to what would be expected based on other sequenced insect genomes. Results Here, we report an improved honey bee genome assembly (Amel_4.5) with a new gene annotation set (OGSv3.2), and show that the honey bee genome contains a number of genes similar to that of other insect genomes, contrary to what was suggested in OGSv1.0. The new genome assembly is more contiguous and complete and the new gene set includes ~5000 more protein-coding genes, 50% more than previously reported. About 1/6 of the additional genes were due to improvements to the assembly, and the remaining were inferred based on new RNAseq and protein data. Conclusions Lessons learned from this genome upgrade have important implications for future genome sequencing projects. Furthermore, the improvements significantly enhance genomic resources for the honey bee, a key model for social behavior and essential to global ecology through pollination. PMID:24479613

In-space assembly and servicing infrastructures for the Evolvable Space Telescope (EST)

NASA Astrophysics Data System (ADS)

Lillie, Charles F.; MacEwen, Howard A.

2016-07-01

The concept for EST presented in past SPIE forums will benefit significantly from the current efforts of DARPA, NASA and several commercial organizations to develop an in-space infrastructure that will enable on-orbit assembly, servicing, repair and repurposing of space vehicles. Two documents provide particularly relevant discussions: "NASA's Journey to Mars: Pioneering Next Steps in Space Exploration" provides a recent (2015) outline of NASA's thoughts on human deep space exploration and the tools that will enable it, while the "On-Orbit Satellite Servicing Study: Project Report" details a number of the concepts and technologies that must be developed. In this paper we examine the concepts in these and related documents to explore how systems such as EST will shape and support the infrastructure needed by future space vehicles. In so doing, we address previous examples of on-orbit assembly and servicing of space vehicles; the lessons learned from these efforts and the existing systems and facilities available to execute servicing missions; the EST concept for an LUVOIR telescope designed for in-orbit assembly and servicing and the resulting requirements for a servicing vehicle; the use of heavy lift launch vehicles, including the SLS and Exploration Upper Stage to co-manifest other large payloads along with a crewed Orion mission; Deep Space Habitats (DSHs) in cislunar space as a site for assembly and servicing spacecraft vehicles, and a base for Maneuverable Servicing Vehicles; and how space vehicles need to be designed for in-space assembly and servicing (i.e., commonality of parts, systems, modularity, accessibility, and stable maneuverability).

Reference-guided de novo assembly approach improves genome reconstruction for related species.

PubMed

Lischer, Heidi E L; Shimizu, Kentaro K

2017-11-10

The development of next-generation sequencing has made it possible to sequence whole genomes at a relatively low cost. However, de novo genome assemblies remain challenging due to short read length, missing data, repetitive regions, polymorphisms and sequencing errors. As more and more genomes are sequenced, reference-guided assembly approaches can be used to assist the assembly process. However, previous methods mostly focused on the assembly of other genotypes within the same species. We adapted and extended a reference-guided de novo assembly approach, which enables the usage of a related reference sequence to guide the genome assembly. In order to compare and evaluate de novo and our reference-guided de novo assembly approaches, we used a simulated data set of a repetitive and heterozygotic plant genome. The extended reference-guided de novo assembly approach almost always outperforms the corresponding de novo assembly program even when a reference of a different species is used. Similar improvements can be observed in high and low coverage situations. In addition, we show that a single evaluation metric, like the widely used N50 length, is not enough to properly rate assemblies as it not always points to the best assembly evaluated with other criteria. Therefore, we used the summed z-scores of 36 different statistics to evaluate the assemblies. The combination of reference mapping and de novo assembly provides a powerful tool to improve genome reconstruction by integrating information of a related genome. Our extension of the reference-guided de novo assembly approach enables the application of this strategy not only within but also between related species. Finally, the evaluation of genome assemblies is often not straight forward, as the truth is not known. Thus one should always use a combination of evaluation metrics, which not only try to assess the continuity but also the accuracy of an assembly.

Novel multi-biotin grafted poly(lactic acid) and its self-assembling nanoparticles capable of binding to streptavidin

PubMed Central

Yan, Hao; Jiang, Weimin; Zhang, Yinxing; Liu, Ying; Wang, Bin; Yang, Li; Deng, Lihong; Singh, Gurinder K; Pan, Jun

2012-01-01

Targeted drug delivery requires novel biodegradable, specific binding systems with longer circulation time. The aim of this study was to prepare biotinylated poly(lactic acid) (PLA) nanoparticles (NPs) which can meet regular requirements as well conjugate more biotins in the polymer to provide better binding with streptavidin. A biotin-graft-PLA was synthesized based on previously published biodegradable poly(ethylene glycol) (PEG)-graft-PLA, with one polymer molecule containing three PEG molecules. Newly synthesized biotin-graft-PLA had three biotins per polymer molecule, higher than the previous biotinylated PLA (≤1 biotin per polymer molecule). A PEG with a much lower molecular weight (MW ~1900) than the previous biotinylated PLA (PEG MW ≥ 3800), and thus more biocompatible, was used which supplied good nonspecific protein-resistant property compatible to PEG-graft-PLA, suggesting its possible longer stay in the bloodstream. Biotin-graft-PLA specifically bound to streptavidin and self-assembled into NPs, during which naproxen, a model small molecule (MW 230 Da) and hydrophobic drug, was encapsulated (encapsulation efficiency 51.88%). The naproxen-loaded NPs with particle size and zeta potential of 175 nm and −27.35 mV realized controlled release within 170 hours, comparable to previous studies. The biotin-graft-PLA NPs adhered approximately two-fold more on streptavidin film and on biotin film via a streptavidin arm both in static and dynamic conditions compared with PEG-graft-PLA NPs, the proven nonspecific protein-resistant NPs. The specific binding of biotin-graft-PLA NPs with streptavidin and with biotin using streptavidin arm, as well as its entrapment and controlled release for naproxen, suggest potential applications in targeted drug delivery. PMID:22334778

An Efficient and Versatile Means for Assembling and Manufacturing Systems in Space

NASA Technical Reports Server (NTRS)

Dorsey, John T.; Doggett, William R.; Hafley, Robert A.; Komendera, Erik; Correll, Nikolaus; King, Bruce

2012-01-01

Within NASA Space Science, Exploration and the Office of Chief Technologist, there are Grand Challenges and advanced future exploration, science and commercial mission applications that could benefit significantly from large-span and large-area structural systems. Of particular and persistent interest to the Space Science community is the desire for large (in the 10- 50 meter range for main aperture diameter) space telescopes that would revolutionize space astronomy. Achieving these systems will likely require on-orbit assembly, but previous approaches for assembling large-scale telescope truss structures and systems in space have been perceived as very costly because they require high precision and custom components. These components rely on a large number of mechanical connections and supporting infrastructure that are unique to each application. In this paper, a new assembly paradigm that mitigates these concerns is proposed and described. A new assembly approach, developed to implement the paradigm, is developed incorporating: Intelligent Precision Jigging Robots, Electron-Beam welding, robotic handling/manipulation, operations assembly sequence and path planning, and low precision weldable structural elements. Key advantages of the new assembly paradigm, as well as concept descriptions and ongoing research and technology development efforts for each of the major elements are summarized.

Advanced Stirling Radioisotope Generator Engineering Unit 2 (ASRG EU2) Final Assembly

NASA Technical Reports Server (NTRS)

Oriti, Salvatore M.

2015-01-01

NASA Glenn Research Center (GRC) has recently completed the assembly of a unique Stirling generator test article for laboratory experimentation. Under the Advanced Stirling Radioisotope Generator (ASRG) flight development contract, NASA GRC initiated a task to design and fabricate a flight-like generator for in-house testing. This test article was given the name ASRG Engineering Unit 2 (EU2) as it was effectively the second engineering unit to be built within the ASRG project. The intent of the test article was to duplicate Lockheed Martin's qualification unit ASRG design as much as possible to enable system-level tests not previously possible at GRC. After the cancellation of the ASRG flight development project, the decision was made to continue the EU2 build, and make use of a portion of the hardware from the flight development project. GRC and Lockheed Martin engineers collaborated to develop assembly procedures, leveraging the valuable knowledge gathered by Lockheed Martin during the ASRG development contract. The ASRG EU2 was then assembled per these procedures at GRC with Lockheed Martin engineers on site. The assembly was completed in August 2014. This paper details the components that were used for the assembly, and the assembly process itself.

The SSU processome interactome in Saccharomyces cerevisiae reveals novel protein subcomplexes.

PubMed

Vincent, Nicholas G; Charette, J Michael; Baserga, Susan J

2018-01-01

Ribosome assembly is an evolutionarily conserved and energy intensive process required for cellular growth, proliferation, and maintenance. In yeast, assembly of the small ribosomal subunit (SSU) requires approximately 75 assembly factors that act in coordination to form the SSU processome, a 6 MDa ribonucleoprotein complex. The SSU processome is required for processing, modifying, and folding the preribosomal RNA (rRNA) to prepare it for incorporation into the mature SSU. Although the protein composition of the SSU processome has been known for some time, the interaction network of the proteins required for its assembly has remained poorly defined. Here, we have used a semi-high-throughput yeast two-hybrid (Y2H) assay and coimmunoprecipitation validation method to produce a high-confidence interactome of SSU processome assembly factors (SPAFs), providing essential insight into SSU assembly and ribosome biogenesis. Further, we used glycerol density-gradient sedimentation to reveal the presence of protein subcomplexes that have not previously been observed. Our work not only provides essential insight into SSU assembly and ribosome biogenesis, but also serves as an important resource for future investigations into how defects in biogenesis and assembly cause congenital disorders of ribosomes known as ribosomopathies. © 2018 Vincent et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Chemical controls on uranyl citrate speciation and the self-assembly of nanoscale macrocycles and sandwich complexes in aqueous solutions.

PubMed

Basile, M; Unruh, D K; Gojdas, K; Flores, E; Streicher, L; Forbes, T Z

2015-03-28

Uranyl citrate forms trimeric species at pH > 5.5, but exact structural characteristics of these important oligomers have not previously been reported. Crystallization and structural characterization of the trimers suggests the self-assembly of the 3 : 3 and 3 : 2 U : Cit complexes into larger sandwich and macrocyclic molecules. Raman spectroscopy and ESI-MS have been utilized to investigate the relative abundance of these species in solution under varying pH and citrate concentrations. Additional dynamic light scattering experiments indicate that self-assembly of the larger molecules does occur in aqueous solution.

Gel Filtration Of Dilute Human Embryonic Hemoglobins Reveals Basis For Their Increased Oxygen Binding

PubMed Central

Manning, Lois R.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, James M.

2016-01-01

This report establishes a correlation between two known properties of the human embryonic hemoglobins-- their weak subunit assemblies as demonstrated here by gel filtration at very dilute protein concentrations and their high oxygen affinities and reduced cooperativities reported previously by others but without a mechanistic basis. We demonstrate here that their high oxygen affinities are a consequence of their weak assemblies. Weak vs strong hemoglobin tetramers represent a regulatory mechanism to modulate oxygen binding capacity by altering the equilibrium between the various steps in the assembly process that can be described as an inverse allosteric effect. PMID:27965062

Gel filtration of dilute human embryonic hemoglobins reveals basis for their increased oxygen binding.

PubMed

Manning, Lois R; Popowicz, Anthony M; Padovan, Julio C; Chait, Brian T; Manning, James M

2017-02-15

This report establishes a correlation between two known properties of the human embryonic hemoglobins-- their weak subunit assemblies as demonstrated here by gel filtration at very dilute protein concentrations and their high oxygen affinities and reduced cooperativities reported previously by others but without a mechanistic basis. We demonstrate here that their high oxygen affinities are a consequence of their weak assemblies. Weak vs strong hemoglobin tetramers represent a regulatory mechanism to modulate oxygen binding capacity by altering the equilibrium between the various steps in the assembly process that can be described as an inverse allosteric effect. Copyright © 2016 Elsevier Inc. All rights reserved.

Isothermal DNA origami folding: avoiding denaturing conditions for one-pot, hybrid-component annealing

NASA Astrophysics Data System (ADS)

Kopielski, Andreas; Schneider, Anne; Csáki, Andrea; Fritzsche, Wolfgang

2015-01-01

The DNA origami technique offers great potential for nanotechnology. Using biomolecular self-assembly, defined 2D and 3D nanoscale DNA structures can be realized. DNA origami allows the positioning of proteins, fluorophores or nanoparticles with an accuracy of a few nanometers and enables thereby novel nanoscale devices. Origami assembly usually includes a thermal denaturation step at 90 °C. Additional components used for nanoscale assembly (such as proteins) are often thermosensitive, and possibly damaged by such harsh conditions. They have therefore to be attached in an extra second step to avoid defects. To enable a streamlined one-step nanoscale synthesis - a so called one-pot folding - an adaptation of the folding procedures is required. Here we present a thermal optimization of this process for a 2D DNA rectangle-shaped origami resulting in an isothermal assembly protocol below 60 °C without thermal denaturation. Moreover, a room temperature protocol is presented using the chemical additive betaine, which is biocompatible in contrast to chemical denaturing approaches reported previously.The DNA origami technique offers great potential for nanotechnology. Using biomolecular self-assembly, defined 2D and 3D nanoscale DNA structures can be realized. DNA origami allows the positioning of proteins, fluorophores or nanoparticles with an accuracy of a few nanometers and enables thereby novel nanoscale devices. Origami assembly usually includes a thermal denaturation step at 90 °C. Additional components used for nanoscale assembly (such as proteins) are often thermosensitive, and possibly damaged by such harsh conditions. They have therefore to be attached in an extra second step to avoid defects. To enable a streamlined one-step nanoscale synthesis - a so called one-pot folding - an adaptation of the folding procedures is required. Here we present a thermal optimization of this process for a 2D DNA rectangle-shaped origami resulting in an isothermal assembly protocol below 60 °C without thermal denaturation. Moreover, a room temperature protocol is presented using the chemical additive betaine, which is biocompatible in contrast to chemical denaturing approaches reported previously. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr04176c

Virtual Genome Walking across the 32 Gb Ambystoma mexicanum genome; assembling gene models and intronic sequence.

PubMed

Evans, Teri; Johnson, Andrew D; Loose, Matthew

2018-01-12

Large repeat rich genomes present challenges for assembly using short read technologies. The 32 Gb axolotl genome is estimated to contain ~19 Gb of repetitive DNA making an assembly from short reads alone effectively impossible. Indeed, this model species has been sequenced to 20× coverage but the reads could not be conventionally assembled. Using an alternative strategy, we have assembled subsets of these reads into scaffolds describing over 19,000 gene models. We call this method Virtual Genome Walking as it locally assembles whole genome reads based on a reference transcriptome, identifying exons and iteratively extending them into surrounding genomic sequence. These assemblies are then linked and refined to generate gene models including upstream and downstream genomic, and intronic, sequence. Our assemblies are validated by comparison with previously published axolotl bacterial artificial chromosome (BAC) sequences. Our analyses of axolotl intron length, intron-exon structure, repeat content and synteny provide novel insights into the genic structure of this model species. This resource will enable new experimental approaches in axolotl, such as ChIP-Seq and CRISPR and aid in future whole genome sequencing efforts. The assembled sequences and annotations presented here are freely available for download from https://tinyurl.com/y8gydc6n . The software pipeline is available from https://github.com/LooseLab/iterassemble .

Reversible Self-Assembly of 3D Architectures Actuated by Responsive Polymers.

PubMed

Zhang, Cheng; Su, Jheng-Wun; Deng, Heng; Xie, Yunchao; Yan, Zheng; Lin, Jian

2017-11-29

An assembly of three-dimensional (3D) architectures with defined configurations has important applications in broad areas. Among various approaches of constructing 3D structures, a stress-driven assembly provides the capabilities of creating 3D architectures in a broad range of functional materials with unique merits. However, 3D architectures built via previous methods are simple, irreversible, or not free-standing. Furthermore, the substrates employed for the assembly remain flat, thus not involved as parts of the final 3D architectures. Herein, we report a reversible self-assembly of various free-standing 3D architectures actuated by the self-folding of smart polymer substrates with programmed geometries. The strategically designed polymer substrates can respond to external stimuli, such as organic solvents, to initiate the 3D assembly process and subsequently become the parts of the final 3D architectures. The self-assembly process is highly controllable via origami and kirigami designs patterned by direct laser writing. Self-assembled geometries include 3D architectures such as "flower", "rainbow", "sunglasses", "box", "pyramid", "grating", and "armchair". The reported self-assembly also shows wide applicability to various materials including epoxy, polyimide, laser-induced graphene, and metal films. The device examples include 3D architectures integrated with a micro light-emitting diode and a flex sensor, indicting the potential applications in soft robotics, bioelectronics, microelectromechanical systems, and others.

Regulated Assembly of Vacuolar ATPase Is Increased during Cluster Disruption-induced Maturation of Dendritic Cells through a Phosphatidylinositol 3-Kinase/mTOR-dependent Pathway*

PubMed Central

Liberman, Rachel; Bond, Sarah; Shainheit, Mara G.; Stadecker, Miguel J.; Forgac, Michael

2014-01-01

The vacuolar (H+)-ATPases (V-ATPases) are ATP-driven proton pumps composed of a peripheral V1 domain and a membrane-embedded V0 domain. Regulated assembly of V1 and V0 represents an important regulatory mechanism for controlling V-ATPase activity in vivo. Previous work has shown that V-ATPase assembly increases during maturation of bone marrow-derived dendritic cells induced by activation of Toll-like receptors. This increased assembly is essential for antigen processing, which is dependent upon an acidic lysosomal pH. Cluster disruption of dendritic cells induces a semi-mature phenotype associated with immune tolerance. Thus, semi-mature dendritic cells are able to process and present self-peptides to suppress autoimmune responses. We have investigated V-ATPase assembly in bone marrow-derived, murine dendritic cells and observed an increase in assembly following cluster disruption. This increased assembly is not dependent upon new protein synthesis and is associated with an increase in concanamycin A-sensitive proton transport in FITC-loaded lysosomes. Inhibition of phosphatidylinositol 3-kinase with wortmannin or mTORC1 with rapamycin effectively inhibits the increased assembly observed upon cluster disruption. These results suggest that the phosphatidylinositol 3-kinase/mTOR pathway is involved in controlling V-ATPase assembly during dendritic cell maturation. PMID:24273170

Nanocrystalline Precursors for the Co-Assembly of Crack-Free Metal Oxide Inverse Opals.

PubMed

Phillips, Katherine R; Shirman, Tanya; Shirman, Elijah; Shneidman, Anna V; Kay, Theresa M; Aizenberg, Joanna

2018-05-01

Inorganic microstructured materials are ubiquitous in nature. However, their formation in artificial self-assembly systems is challenging as it involves a complex interplay of competing forces during and after assembly. For example, colloidal assembly requires fine-tuning of factors such as the size and surface charge of the particles and electrolyte strength of the solvent to enable successful self-assembly and minimize crack formation. Co-assembly of templating colloidal particles together with a sol-gel matrix precursor material helps to release stresses that accumulate during drying and solidification, as previously shown for the formation of high-quality inverse opal (IO) films out of amorphous silica. Expanding this methodology to crystalline materials would result in microscale architectures with enhanced photonic, electronic, and catalytic properties. This work describes tailoring the crystallinity of metal oxide precursors that enable the formation of highly ordered, large-area (mm 2 ) crack-free titania, zirconia, and alumina IO films. The same bioinspired approach can be applied to other crystalline materials as well as structures beyond IOs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

New Autonomous Motors of Metal-Organic Framework (MOF) Powered by Reorganization of Self-Assembled Peptides at interfaces

PubMed Central

Ikezoe, Yasuhiro; Washino, Gosuke; Uemura, Takashi; Kitagawa, Susumu; Matsui, Hiroshi

2012-01-01

There have developed a variety of microsystems that harness energy and convert it to mechanical motion. Here we developed new autonomous biochemical motors by integrating metal-organic framework (MOF) and self-assembling peptides. MOF is applied as an energy-storing cell that assembles peptides inside nanoscale pores of the coordination framework. The robust assembling nature of peptides enables reconfiguring their assemblies at the water-MOF interface, which is converted to fuel energy. Re-organization of hydrophobic peptides could create the large surface tension gradient around the MOF and it efficiently powers the translation motion of MOF. As a comparison, the velocity of normalized by volume for the DPA-MOF particle is faster and the kinetic energy per the unit mass of fuel is more than twice as large as the one for previous gel motor systems. This demonstration opens the new application of MOF and reconfigurable molecular self-assembly and it may evolve into the smart autonomous motor that mimic bacteria to swim and harvest target chemicals by integrating recognition units. PMID:23104155

The histone chaperone TAF-I/SET/INHAT is required for transcription in vitro of chromatin templates.

PubMed

Gamble, Matthew J; Erdjument-Bromage, Hediye; Tempst, Paul; Freedman, Leonard P; Fisher, Robert P

2005-01-01

To uncover factors required for transcription by RNA polymerase II on chromatin, we fractionated a mammalian cell nuclear extract. We identified the histone chaperone TAF-I (also known as INHAT [inhibitor of histone acetyltransferase]), which was previously proposed to repress transcription, as a potent activator of chromatin transcription responsive to the vitamin D3 receptor or to Gal4-VP16. TAF-I associates with chromatin in vitro and can substitute for the related protein NAP-1 in assembling chromatin onto cloned DNA templates in cooperation with the remodeling enzyme ATP-dependent chromatin assembly factor (ACF). The chromatin assembly and transcriptional activation functions are distinct, however, and can be dissociated temporally. Efficient transcription of chromatin assembled with TAF-I still requires the presence of TAF-I during the polymerization reaction. Conversely, TAF-I cannot stimulate transcript elongation when added after the other factors necessary for assembly of a preinitiation complex on naked DNA. Thus, TAF-I is required to facilitate transcription at a step after chromatin assembly but before transcript elongation.

Epitaxy: Programmable Atom Equivalents Versus Atoms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Mary X.; Seo, Soyoung E.; Gabrys, Paul A.

The programmability of DNA makes it an attractive structure-directing ligand for the assembly of nanoparticle superlattices in a manner that mimics many aspects of atomic crystallization. However, the synthesis of multilayer single crystals of defined size remains a challenge. Though previous studies considered lattice mismatch as the major limiting factor for multilayer assembly, thin film growth depends on many interlinked variables. Here, a more comprehensive approach is taken to study fundamental elements, such as the growth temperature and the thermodynamics of interfacial energetics, to achieve epitaxial growth of nanoparticle thin films. Under optimized equilibrium conditions, single crystal, multilayer thin filmsmore » can be synthesized over 500 × 500 μm2 areas on lithographically patterned templates. Importantly, these superlattices follow the same patterns of crystal growth demonstrated in thin film atomic deposition, allowing for these processes to be understood in the context of well-studied atomic epitaxy, and potentially enabling a nanoscale model to study fundamental crystallization processes.« less

Harnessing NGS and Big Data Optimally: Comparison of miRNA Prediction from Assembled versus Non-assembled Sequencing Data--The Case of the Grass Aegilops tauschii Complex Genome.

PubMed

Budak, Hikmet; Kantar, Melda

2015-07-01

MicroRNAs (miRNAs) are small, endogenous, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. As high-throughput next generation sequencing (NGS) and Big Data rapidly accumulate for various species, efforts for in silico identification of miRNAs intensify. Surprisingly, the effect of the input genomics sequence on the robustness of miRNA prediction was not evaluated in detail to date. In the present study, we performed a homology-based miRNA and isomiRNA prediction of the 5D chromosome of bread wheat progenitor, Aegilops tauschii, using two distinct sequence data sets as input: (1) raw sequence reads obtained from 454-GS FLX Titanium sequencing platform and (2) an assembly constructed from these reads. We also compared this method with a number of available plant sequence datasets. We report here the identification of 62 and 22 miRNAs from raw reads and the assembly, respectively, of which 16 were predicted with high confidence from both datasets. While raw reads promoted sensitivity with the high number of miRNAs predicted, 55% (12 out of 22) of the assembly-based predictions were supported by previous observations, bringing specificity forward compared to the read-based predictions, of which only 37% were supported. Importantly, raw reads could identify several repeat-related miRNAs that could not be detected with the assembly. However, raw reads could not capture 6 miRNAs, for which the stem-loops could only be covered by the relatively longer sequences from the assembly. In summary, the comparison of miRNA datasets obtained by these two strategies revealed that utilization of raw reads, as well as assemblies for in silico prediction, have distinct advantages and disadvantages. Consideration of these important nuances can benefit future miRNA identification efforts in the current age of NGS and Big Data driven life sciences innovation.

Transcription boundaries of U1 small nuclear RNA.

PubMed Central

Kunkel, G R; Pederson, T

1985-01-01

Transcription-proximal stages of U1 small nuclear RNA biosynthesis were studied by 32P labeling of nascent chains in isolated HeLa cell nuclei. Labeled RNA was hybridized to nitrocellulose-immobilized, single-stranded M13 DNA clones corresponding to regions within or flanking a human U1 RNA gene. Transcription of U1 RNA was inhibited by greater than 95% by alpha-amanitin at 1 microgram/ml, consistent with previous evidence that it is synthesized by RNA polymerase II. No hybridization to DNA immediately adjacent to the 5' end of mature U1 RNA (-6 to -105 nucleotides) was detected, indicating that, like all studied polymerase II initiation, transcription of U1 RNA starts at or very near the cap site. However, in contrast to previously described transcription units for mRNA, in which equimolar transcription occurs for hundreds or thousands of nucleotides beyond the mature 3' end of the mRNA, labeled U1 RNA hybridization dropped off sharply within a very short region (approximately 60 nucleotides) immediately downstream from the 3' end of mature U1 RNA. Also in contrast to pre-mRNA, which is assembled into ribonucleoprotein (RNP) particles while still nascent RNA chains, the U1 RNA transcribed in isolated nuclei did not form RNP complexes by the criterion of reaction with a monoclonal antibody for the small nuclear RNP Sm proteins. This suggests that, unlike pre-mRNA-RNP particle formation, U1 small nuclear RNP assembly does not occur until after the completion of transcription. These results show that, despite their common synthesis by RNA polymerase II, mRNA and U1 small nuclear RNA differ markedly both in their extents of 3' processing and their temporal patterns of RNP assembly. Images PMID:2942763

Non-native three-dimensional block copolymer morphologies

DOE PAGES

Rahman, Atikur; Majewski, Pawel W.; Doerk, Gregory; ...

2016-12-22

Self-assembly is a powerful paradigm, wherein molecules spontaneously form ordered phases exhibiting well-defined nanoscale periodicity and shapes. However, the inherent energy-minimization aspect of self-assembly yields a very limited set of morphologies, such as lamellae or hexagonally packed cylinders. Here, we show how soft self-assembling materials—block copolymer thin films—can be manipulated to form a diverse library of previously unreported morphologies. In this iterative assembly process, each polymer layer acts as both a structural component of the final morphology and a template for directing the order of subsequent layers. Specifically, block copolymer films are immobilized on surfaces, and template successive layers throughmore » subtle surface topography. As a result, this strategy generates an enormous variety of three-dimensional morphologies that are absent in the native block copolymer phase diagram.« less

Non-native three-dimensional block copolymer morphologies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rahman, Atikur; Majewski, Pawel W.; Doerk, Gregory

Self-assembly is a powerful paradigm, wherein molecules spontaneously form ordered phases exhibiting well-defined nanoscale periodicity and shapes. However, the inherent energy-minimization aspect of self-assembly yields a very limited set of morphologies, such as lamellae or hexagonally packed cylinders. Here, we show how soft self-assembling materials—block copolymer thin films—can be manipulated to form a diverse library of previously unreported morphologies. In this iterative assembly process, each polymer layer acts as both a structural component of the final morphology and a template for directing the order of subsequent layers. Specifically, block copolymer films are immobilized on surfaces, and template successive layers throughmore » subtle surface topography. As a result, this strategy generates an enormous variety of three-dimensional morphologies that are absent in the native block copolymer phase diagram.« less

A Theoretical and Experimental Study of DNA Self-assembly

NASA Astrophysics Data System (ADS)

Chandran, Harish

The control of matter and phenomena at the nanoscale is fast becoming one of the most important challenges of the 21st century with wide-ranging applications from energy and health care to computing and material science. Conventional top-down approaches to nanotechnology, having served us well for long, are reaching their inherent limitations. Meanwhile, bottom-up methods such as self-assembly are emerging as viable alternatives for nanoscale fabrication and manipulation. A particularly successful bottom up technique is DNA self-assembly where a set of carefully designed DNA strands form a nanoscale object as a consequence of specific, local interactions among the different components, without external direction. The final product of the self-assembly process might be a static nanostructure or a dynamic nanodevice that performs a specific function. Over the past two decades, DNA self-assembly has produced stunning nanoscale objects such as 2D and 3D lattices, polyhedra and addressable arbitrary shaped substrates, and a myriad of nanoscale devices such as molecular tweezers, computational circuits, biosensors and molecular assembly lines. In this dissertation we study multiple problems in the theory, simulations and experiments of DNA self-assembly. We extend the Turing-universal mathematical framework of self-assembly known as the Tile Assembly Model by incorporating randomization during the assembly process. This allows us to reduce the tile complexity of linear assemblies. We develop multiple techniques to build linear assemblies of expected length N using far fewer tile types than previously possible. We abstract the fundamental properties of DNA and develop a biochemical system, which we call meta-DNA, based entirely on strands of DNA as the only component molecule. We further develop various enzyme-free protocols to manipulate meta-DNA systems and provide strand level details along with abstract notations for these mechanisms. We simulate DNA circuits by providing detailed designs for local molecular computations that involve spatially contiguous molecules arranged on addressable substrates via enzyme-free DNA hybridization reaction cascades. We use the Visual DSD simulation software in conjunction with localized reaction rates obtained from biophysical modeling to create chemical reaction networks of localized hybridization circuits that are then model checked using the PRISM model checking software. We develop a DNA detection system employing the triggered self-assembly of a novel DNA dendritic nanostructure. Detection begins when a specific, single-stranded target DNA strand triggers a hybridization chain reaction between two distinct DNA hairpins. Each hairpin opens and hybridizes up to two copies of the other, and hence each layer of the growing dendritic nanostructure can in principle accommodate an exponentially increasing number of cognate molecules, generating a nanostructure with high molecular weight. We build linear activatable assemblies employing a novel protection/deprotection strategy to strictly enforce the direction of tiling assembly growth to ensure the robustness of the assembly process. Our system consists of two tiles that can form a linear co-polymer. These tiles, which are initially protected such that they do not react with each other, can be activated to form linear co-polymers via the use of a strand displacing enzyme.

KSC-97PC1228

NASA Image and Video Library

1997-08-05

The Advanced Composition Explorer (ACE) spacecraft undergoes a spin test in KSC’s Spacecraft Assembly and Encapsulation Facility-II (SAEF-II). Scheduled for launch on a Delta II rocket from Cape Canaveral Air Station on Aug. 25, ACE will study low-energy particles of solar origin and high-energy galactic particles. The collecting power of instruments aboard ACE is 10 to 1,000 times greater than anything previously flown to collect similar data by NASA

KSC-97PC1227

NASA Image and Video Library

1997-08-05

The Advanced Composition Explorer (ACE) spacecraft undergoes a spin test in KSC’s Spacecraft Assembly and Encapsulation Facility-II (SAEF-II). Scheduled for launch on a Delta II rocket from Cape Canaveral Air Station on Aug. 25, ACE will study low-energy particles of solar origin and high-energy galactic particles. The collecting power of instruments aboard ACE is 10 to 1,000 times greater than anything previously flown to collect similar data by NASA

Large-scale self-assembled zirconium phosphate smectic layers via a simple spray-coating process

NASA Astrophysics Data System (ADS)

Wong, Minhao; Ishige, Ryohei; White, Kevin L.; Li, Peng; Kim, Daehak; Krishnamoorti, Ramanan; Gunther, Robert; Higuchi, Takeshi; Jinnai, Hiroshi; Takahara, Atsushi; Nishimura, Riichi; Sue, Hung-Jue

2014-04-01

The large-scale assembly of asymmetric colloidal particles is used in creating high-performance fibres. A similar concept is extended to the manufacturing of thin films of self-assembled two-dimensional crystal-type materials with enhanced and tunable properties. Here we present a spray-coating method to manufacture thin, flexible and transparent epoxy films containing zirconium phosphate nanoplatelets self-assembled into a lamellar arrangement aligned parallel to the substrate. The self-assembled mesophase of zirconium phosphate nanoplatelets is stabilized by epoxy pre-polymer and exhibits rheology favourable towards large-scale manufacturing. The thermally cured film forms a mechanically robust coating and shows excellent gas barrier properties at both low- and high humidity levels as a result of the highly aligned and overlapping arrangement of nanoplatelets. This work shows that the large-scale ordering of high aspect ratio nanoplatelets is easier to achieve than previously thought and may have implications in the technological applications for similar materials.

PGL germ granule assembly protein is a base-specific, single-stranded RNase

PubMed Central

Aoki, Scott T.; Kershner, Aaron M.; Bingman, Craig A.; Wickens, Marvin; Kimble, Judith

2016-01-01

Cellular RNA-protein (RNP) granules are ubiquitous and have fundamental roles in biology and RNA metabolism, but the molecular basis of their structure, assembly, and function is poorly understood. Using nematode “P-granules” as a paradigm, we focus on the PGL granule scaffold protein to gain molecular insights into RNP granule structure and assembly. We first identify a PGL dimerization domain (DD) and determine its crystal structure. PGL-1 DD has a novel 13 α-helix fold that creates a positively charged channel as a homodimer. We investigate its capacity to bind RNA and discover unexpectedly that PGL-1 DD is a guanosine-specific, single-stranded endonuclease. Discovery of the PGL homodimer, together with previous results, suggests a model in which the PGL DD dimer forms a fundamental building block for P-granule assembly. Discovery of the PGL RNase activity expands the role of RNP granule assembly proteins to include enzymatic activity in addition to their job as structural scaffolds. PMID:26787882

Assembly and intracellular delivery of quantum dot-fluorescent protein bioconjugates

NASA Astrophysics Data System (ADS)

Medintz, Igor L.; Pons, Thomas; Delehanty, James B.; Susumu, Kimihiro; Dawson, Philip E.; Mattoussi, Hedi

2008-02-01

We have previously assembled semiconductor quantum dot (QD)-based fluorescence resonance energy transfer (FRET) sensors that can specifically detect nutrients, explosives or enzymatic activity. These sensors utilized the inherent benefits of QDs as FRET donors to optimize signal transduction. In this report we functionalize QDs with the multi-subunit multi-chromophore b-phycoerythrin (b-PE) light harvesting complex using biotin-Streptavidin binding. FRET and gel electrophoretic analyses were used to characterize and confirm the QD-b-PE self-assembly. We found that immobilizing additional cell-penetrating peptides on the nanocrystal surface along with the b-PE was the key factor allowing the mixed surface QD-cargos to undergo endocytosis and intracellular delivery. Our findings on the intracellular uptake promoted by CPP were compared to those collected using microinjection technique, where QD-assemblies were delivered directly into the cytoplasm; this strategy allows bypassing of the endocytic uptake pathway. Intracellular delivery of multifunctional QD-fluorescent protein assemblies has potential applications for use in protein tracking, sensing and diagnostics.

High coverage fluid-phase floating lipid bilayers supported by ω-thiolipid self-assembled monolayers

PubMed Central

Hughes, Arwel V.; Holt, Stephen A.; Daulton, Emma; Soliakov, Andrei; Charlton, Timothy R.; Roser, Steven J.; Lakey, Jeremy H.

2014-01-01

Large area lipid bilayers, on solid surfaces, are useful in physical studies of biological membranes. It is advantageous to minimize the interactions of these bilayers with the substrate and this can be achieved via the formation of a floating supported bilayer (FSB) upon either a surface bound phospholipid bilayer or monolayer. The FSB's independence is enabled by the continuous water layer (greater than 15 Å) that remains between the two. However, previous FSBs have had limited stability and low density. Here, we demonstrate by surface plasmon resonance and neutron reflectivity, the formation of a complete self-assembled monolayer (SAM) on gold surfaces by a synthetic phosphatidylcholine bearing a thiol group at the end of one fatty acyl chain. Furthermore, a very dense FSB (more than 96%) of saturated phosphatidylcholine can be formed on this SAM by sequential Langmuir–Blodgett and Langmuir–Schaefer procedures. Neutron reflectivity used both isotopic and magnetic contrast to enhance the accuracy of the data fits. This system offers the means to study transmembrane proteins, membrane potential effects (using the gold as an electrode) and even model bacterial outer membranes. Using unsaturated phosphatidylcholines, which have previously failed to form stable FSBs, we achieved a coverage of 73%. PMID:25030385

Assembly of the Caenorhabditis elegans gut microbiota from diverse soil microbial environments

PubMed Central

Berg, Maureen; Stenuit, Ben; Ho, Joshua; Wang, Andrew; Parke, Caitlin; Knight, Matthew; Alvarez-Cohen, Lisa; Shapira, Michael

2016-01-01

It is now well accepted that the gut microbiota contributes to our health. However, what determines the microbiota composition is still unclear. Whereas it might be expected that the intestinal niche would be dominant in shaping the microbiota, studies in vertebrates have repeatedly demonstrated dominant effects of external factors such as host diet and environmental microbial diversity. Hypothesizing that genetic variation may interfere with discerning contributions of host factors, we turned to Caenorhabditis elegans as a new model, offering the ability to work with genetically homogenous populations. Deep sequencing of 16S rDNA was used to characterize the (previously unknown) worm gut microbiota as assembled from diverse produce-enriched soil environments under laboratory conditions. Comparisons of worm microbiotas with those in their soil environment revealed that worm microbiotas resembled each other even when assembled from different microbial environments, and enabled defining a shared core gut microbiota. Community analyses indicated that species assortment in the worm gut was non-random and that assembly rules differed from those in their soil habitat, pointing at the importance of competitive interactions between gut-residing taxa. The data presented fills a gap in C. elegans biology. Furthermore, our results demonstrate a dominant contribution of the host niche in shaping the gut microbiota. PMID:26800234

Deep analysis of wild Vitis flower transcriptome reveals unexplored genome regions associated with sex specification.

PubMed

Ramos, Miguel Jesus Nunes; Coito, João Lucas; Fino, Joana; Cunha, Jorge; Silva, Helena; de Almeida, Patrícia Gomes; Costa, Maria Manuela Ribeiro; Amâncio, Sara; Paulo, Octávio S; Rocheta, Margarida

2017-01-01

RNA-seq of Vitis during early stages of bud development, in male, female and hermaphrodite flowers, identified new loci outside of annotated gene models, suggesting their involvement in sex establishment. The molecular mechanisms responsible for flower sex specification remain unclear for most plant species. In the case of V. vinifera ssp. vinifera, it is not fully understood what determines hermaphroditism in the domesticated subspecies and male or female flowers in wild dioecious relatives (Vitis vinifera ssp. sylvestris). Here, we describe a de novo assembly of the transcriptome of three flower developmental stages from the three Vitis vinifera flower types. The validation of de novo assembly showed a correlation of 0.825. The main goals of this work were the identification of V. v. sylvestris exclusive transcripts and the characterization of differential gene expression during flower development. RNA from several flower developmental stages was used previously to generate Illumina sequence reads. Through a sequential de novo assembly strategy one comprehensive transcriptome comprising 95,516 non-redundant transcripts was assembled. From this dataset 81,064 transcripts were annotated to V. v. vinifera reference transcriptome and 11,084 were annotated against V. v. vinifera reference genome. Moreover, we found 3368 transcripts that could not be mapped to Vitis reference genome. From all the non-redundant transcripts that were assembled, bioinformatics analysis identified 133 specific of V. v. sylvestris and 516 transcripts differentially expressed among the three flower types. The detection of transcription from areas of the genome not currently annotated suggests active transcription of previously unannotated genomic loci during early stages of bud development.

Optical imaging of the rat brain suggests a previously missing link between top-down and bottom-up nervous system function

PubMed Central

Greenfield, Susan A.; Badin, Antoine-Scott; Ferrati, Giovanni; Devonshire, Ian M.

2017-01-01

Abstract. Optical imaging with voltage-sensitive dyes enables the visualization of extensive yet highly transient coalitions of neurons (assemblies) operating throughout the brain on a subsecond time scale. We suggest that operating at the mesoscale level of brain organization, neuronal assemblies may provide a functional link between “bottom-up” cellular mechanisms and “top-down” cognitive ones within anatomically defined regions. We demonstrate in ex vivo rat brain slices how varying spatiotemporal dynamics of assemblies reveal differences not previously appreciated between: different stages of development in cortical versus subcortical brain areas, different sensory modalities (hearing versus vision), different classes of psychoactive drugs (anesthetics versus analgesics), different effects of anesthesia linked to hyperbaric conditions and, in vivo, depths of anesthesia. The strategy of voltage-sensitive dye imaging is therefore as powerful as it is versatile and as such can now be applied to the evaluation of neurochemical signaling systems and the screening of related new drugs, as well as to mathematical modeling and, eventually, even theories of consciousness. PMID:28573153

Optical imaging of the rat brain suggests a previously missing link between top-down and bottom-up nervous system function.

PubMed

Greenfield, Susan A; Badin, Antoine-Scott; Ferrati, Giovanni; Devonshire, Ian M

2017-07-01

Optical imaging with voltage-sensitive dyes enables the visualization of extensive yet highly transient coalitions of neurons (assemblies) operating throughout the brain on a subsecond time scale. We suggest that operating at the mesoscale level of brain organization, neuronal assemblies may provide a functional link between "bottom-up" cellular mechanisms and "top-down" cognitive ones within anatomically defined regions. We demonstrate in ex vivo rat brain slices how varying spatiotemporal dynamics of assemblies reveal differences not previously appreciated between: different stages of development in cortical versus subcortical brain areas, different sensory modalities (hearing versus vision), different classes of psychoactive drugs (anesthetics versus analgesics), different effects of anesthesia linked to hyperbaric conditions and, in vivo , depths of anesthesia. The strategy of voltage-sensitive dye imaging is therefore as powerful as it is versatile and as such can now be applied to the evaluation of neurochemical signaling systems and the screening of related new drugs, as well as to mathematical modeling and, eventually, even theories of consciousness.

Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bloch, Donald B., E-mail: bloch@helix.mgh.harvard.edu; Nobre, Rita A.; Bernstein, Gillian A.

2011-09-10

Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenousmore » nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: {yields} A two-hybrid assay was developed to study interactions in macromolecular complexes. {yields} The assay was applied to interactions between components of mRNA P-bodies. {yields} The assay effectively and efficiently identified protein interaction domains. {yields} P-body assembly in mammalian cells differs from that in other species.« less

Draft genome and reference transcriptomic resources for the urticating pine defoliator Thaumetopoea pityocampa (Lepidoptera: Notodontidae).

PubMed

Gschloessl, B; Dorkeld, F; Berges, H; Beydon, G; Bouchez, O; Branco, M; Bretaudeau, A; Burban, C; Dubois, E; Gauthier, P; Lhuillier, E; Nichols, J; Nidelet, S; Rocha, S; Sauné, L; Streiff, R; Gautier, M; Kerdelhué, C

2018-05-01

The pine processionary moth Thaumetopoea pityocampa (Lepidoptera: Notodontidae) is the main pine defoliator in the Mediterranean region. Its urticating larvae cause severe human and animal health concerns in the invaded areas. This species shows a high phenotypic variability for various traits, such as phenology, fecundity and tolerance to extreme temperatures. This study presents the construction and analysis of extensive genomic and transcriptomic resources, which are an obligate prerequisite to understand their underlying genetic architecture. Using a well-studied population from Portugal with peculiar phenological characteristics, the karyotype was first determined and a first draft genome of 537 Mb total length was assembled into 68,292 scaffolds (N50 = 164 kb). From this genome assembly, 29,415 coding genes were predicted. To circumvent some limitations for fine-scale physical mapping of genomic regions of interest, a 3X coverage BAC library was also developed. In particular, 11 BACs from this library were individually sequenced to assess the assembly quality. Additionally, de novo transcriptomic resources were generated from various developmental stages sequenced with HiSeq and MiSeq Illumina technologies. The reads were de novo assembled into 62,376 and 63,175 transcripts, respectively. Then, a robust subset of the genome-predicted coding genes, the de novo transcriptome assemblies and previously published 454/Sanger data were clustered to obtain a high-quality and comprehensive reference transcriptome consisting of 29,701 bona fide unigenes. These sequences covered 99% of the cegma and 88% of the busco highly conserved eukaryotic genes and 84% of the busco arthropod gene set. Moreover, 90% of these transcripts could be localized on the draft genome. The described information is available via a genome annotation portal (http://bipaa.genouest.org/sp/thaumetopoea_pityocampa/). © 2018 John Wiley & Sons Ltd.

Core microstructure, morphology and chain arrangement of block copolymer self-assemblies as investigated by thermal field-flow fractionation.

PubMed

Muza, U L; Greyling, G; Pasch, H

2018-08-10

The self-assembly of block copolymers (BCPs), as a result of solvent selectivity for one block, has recently received significant attention due to novel applications of BCPs in pharmaceuticals, biomedicine, cosmetics, electronics and nanotechnology. The correlation of BCP microstructure and the structure of the resulting self-assemblies requires advanced analytical methods. However, traditional bulk characterization techniques are limited in the quest of providing detailed information regarding molar mass (M w ), hydrodynamic size (D h ), chemical composition, and morphology for these self-assemblies. In the present study, thermal field-flow fractionation (ThFFF) is utilised to investigate the impact of core microstructure on the resultant solution properties of vesicles prepared from polystyrene-polybutadiene block copolymers (PS-b-PBd) with 1.2- and 1.4-polybutadiene blocks, respectively. As compared to investigations on the impact of the corona microstructure, the impact of core microstructure on micellar properties has largely been neglected in previous work. In N,N-dimethylacetamide (DMAc) these BCPs form vesicles having PS shells and PBd cores. D h , M w , aggregation number, and critical micelle concentration of these micelles are shown to be sensitive to the core microstructure, therefore, demonstrating the potential of microstructural differences to be used for providing tuneable pathways to specific self-assemblies. It is shown that micelles prepared from BCPs of similar PS and PBd block sizes are successfully separated by ThFFF. It is further demonstrated in this study that PS-b-PBd vesicles and PS homopolymers of identical surface chemistry (PS) and comparable D h in DMAc, can be separated by ThFFF. Copyright © 2018 Elsevier B.V. All rights reserved.

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods.

PubMed

Oldfield, Lauren M; Grzesik, Peter; Voorhies, Alexander A; Alperovich, Nina; MacMath, Derek; Najera, Claudia D; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N; Montague, Michael G; Friedman, Robert M; Desai, Prashant J; Vashee, Sanjay

2017-10-17

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOS YA , replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats.

De novo assembly, characterization and functional annotation of pineapple fruit transcriptome through massively parallel sequencing.

PubMed

Ong, Wen Dee; Voo, Lok-Yung Christopher; Kumar, Vijay Subbiah

2012-01-01

Pineapple (Ananas comosus var. comosus), is an important tropical non-climacteric fruit with high commercial potential. Understanding the mechanism and processes underlying fruit ripening would enable scientists to enhance the improvement of quality traits such as, flavor, texture, appearance and fruit sweetness. Although, the pineapple is an important fruit, there is insufficient transcriptomic or genomic information that is available in public databases. Application of high throughput transcriptome sequencing to profile the pineapple fruit transcripts is therefore needed. To facilitate this, we have performed transcriptome sequencing of ripe yellow pineapple fruit flesh using Illumina technology. About 4.7 millions Illumina paired-end reads were generated and assembled using the Velvet de novo assembler. The assembly produced 28,728 unique transcripts with a mean length of approximately 200 bp. Sequence similarity search against non-redundant NCBI database identified a total of 16,932 unique transcripts (58.93%) with significant hits. Out of these, 15,507 unique transcripts were assigned to gene ontology terms. Functional annotation against Kyoto Encyclopedia of Genes and Genomes pathway database identified 13,598 unique transcripts (47.33%) which were mapped to 126 pathways. The assembly revealed many transcripts that were previously unknown. The unique transcripts derived from this work have rapidly increased of the number of the pineapple fruit mRNA transcripts as it is now available in public databases. This information can be further utilized in gene expression, genomics and other functional genomics studies in pineapple.

De Novo Assembly, Characterization and Functional Annotation of Pineapple Fruit Transcriptome through Massively Parallel Sequencing

PubMed Central

Ong, Wen Dee; Voo, Lok-Yung Christopher; Kumar, Vijay Subbiah

2012-01-01

Background Pineapple (Ananas comosus var. comosus), is an important tropical non-climacteric fruit with high commercial potential. Understanding the mechanism and processes underlying fruit ripening would enable scientists to enhance the improvement of quality traits such as, flavor, texture, appearance and fruit sweetness. Although, the pineapple is an important fruit, there is insufficient transcriptomic or genomic information that is available in public databases. Application of high throughput transcriptome sequencing to profile the pineapple fruit transcripts is therefore needed. Methodology/Principal Findings To facilitate this, we have performed transcriptome sequencing of ripe yellow pineapple fruit flesh using Illumina technology. About 4.7 millions Illumina paired-end reads were generated and assembled using the Velvet de novo assembler. The assembly produced 28,728 unique transcripts with a mean length of approximately 200 bp. Sequence similarity search against non-redundant NCBI database identified a total of 16,932 unique transcripts (58.93%) with significant hits. Out of these, 15,507 unique transcripts were assigned to gene ontology terms. Functional annotation against Kyoto Encyclopedia of Genes and Genomes pathway database identified 13,598 unique transcripts (47.33%) which were mapped to 126 pathways. The assembly revealed many transcripts that were previously unknown. Conclusions The unique transcripts derived from this work have rapidly increased of the number of the pineapple fruit mRNA transcripts as it is now available in public databases. This information can be further utilized in gene expression, genomics and other functional genomics studies in pineapple. PMID:23091603

Functional Analysis of the Accessory Protein TapA in Bacillus subtilis Amyloid Fiber Assembly

PubMed Central

Romero, Diego; Vlamakis, Hera; Losick, Richard

2014-01-01

Bacillus subtilis biofilm formation relies on the assembly of a fibrous scaffold formed by the protein TasA. TasA polymerizes into highly stable fibers with biochemical and morphological features of functional amyloids. Previously, we showed that assembly of TasA fibers requires the auxiliary protein TapA. In this study, we investigated the roles of TapA sequences from the C-terminal and N-terminal ends and TapA cysteine residues in its ability to promote the assembly of TasA amyloid-like fibers. We found that the cysteine residues are not essential for the formation of TasA fibers, as their replacement by alanine residues resulted in only minor defects in biofilm formation. Mutating sequences in the C-terminal half had no effect on biofilm formation. However, we identified a sequence of 8 amino acids in the N terminus that is key for TasA fiber formation. Strains expressing TapA lacking these 8 residues were completely defective in biofilm formation. In addition, this TapA mutant protein exhibited a dominant negative effect on TasA fiber formation. Even in the presence of wild-type TapA, the mutant protein inhibited fiber assembly in vitro and delayed biofilm formation in vivo. We propose that this 8-residue sequence is crucial for the formation of amyloid-like fibers on the cell surface, perhaps by mediating the interaction between TapA or TapA and TasA molecules. PMID:24488317

Functional analysis of the accessory protein TapA in Bacillus subtilis amyloid fiber assembly.

PubMed

Romero, Diego; Vlamakis, Hera; Losick, Richard; Kolter, Roberto

2014-04-01

Bacillus subtilis biofilm formation relies on the assembly of a fibrous scaffold formed by the protein TasA. TasA polymerizes into highly stable fibers with biochemical and morphological features of functional amyloids. Previously, we showed that assembly of TasA fibers requires the auxiliary protein TapA. In this study, we investigated the roles of TapA sequences from the C-terminal and N-terminal ends and TapA cysteine residues in its ability to promote the assembly of TasA amyloid-like fibers. We found that the cysteine residues are not essential for the formation of TasA fibers, as their replacement by alanine residues resulted in only minor defects in biofilm formation. Mutating sequences in the C-terminal half had no effect on biofilm formation. However, we identified a sequence of 8 amino acids in the N terminus that is key for TasA fiber formation. Strains expressing TapA lacking these 8 residues were completely defective in biofilm formation. In addition, this TapA mutant protein exhibited a dominant negative effect on TasA fiber formation. Even in the presence of wild-type TapA, the mutant protein inhibited fiber assembly in vitro and delayed biofilm formation in vivo. We propose that this 8-residue sequence is crucial for the formation of amyloid-like fibers on the cell surface, perhaps by mediating the interaction between TapA or TapA and TasA molecules.

Genome-wide engineering of an infectious clone of herpes simplex virus type 1 using synthetic genomics assembly methods

PubMed Central

Grzesik, Peter; Voorhies, Alexander A.; Alperovich, Nina; MacMath, Derek; Najera, Claudia D.; Chandra, Diya Sabrina; Prasad, Sanjana; Noskov, Vladimir N.; Montague, Michael G.; Friedman, Robert M.; Desai, Prashant J.

2017-01-01

Here, we present a transformational approach to genome engineering of herpes simplex virus type 1 (HSV-1), which has a large DNA genome, using synthetic genomics tools. We believe this method will enable more rapid and complex modifications of HSV-1 and other large DNA viruses than previous technologies, facilitating many useful applications. Yeast transformation-associated recombination was used to clone 11 fragments comprising the HSV-1 strain KOS 152 kb genome. Using overlapping sequences between the adjacent pieces, we assembled the fragments into a complete virus genome in yeast, transferred it into an Escherichia coli host, and reconstituted infectious virus following transfection into mammalian cells. The virus derived from this yeast-assembled genome, KOSYA, replicated with kinetics similar to wild-type virus. We demonstrated the utility of this modular assembly technology by making numerous modifications to a single gene, making changes to two genes at the same time and, finally, generating individual and combinatorial deletions to a set of five conserved genes that encode virion structural proteins. While the ability to perform genome-wide editing through assembly methods in large DNA virus genomes raises dual-use concerns, we believe the incremental risks are outweighed by potential benefits. These include enhanced functional studies, generation of oncolytic virus vectors, development of delivery platforms of genes for vaccines or therapy, as well as more rapid development of countermeasures against potential biothreats. PMID:28928148

A clone-free, single molecule map of the domestic cow (Bos taurus) genome.

PubMed

Zhou, Shiguo; Goldstein, Steve; Place, Michael; Bechner, Michael; Patino, Diego; Potamousis, Konstantinos; Ravindran, Prabu; Pape, Louise; Rincon, Gonzalo; Hernandez-Ortiz, Juan; Medrano, Juan F; Schwartz, David C

2015-08-28

The cattle (Bos taurus) genome was originally selected for sequencing due to its economic importance and unique biology as a model organism for understanding other ruminants, or mammals. Currently, there are two cattle genome sequence assemblies (UMD3.1 and Btau4.6) from groups using dissimilar assembly algorithms, which were complemented by genetic and physical map resources. However, past comparisons between these assemblies revealed substantial differences. Consequently, such discordances have engendered ambiguities when using reference sequence data, impacting genomic studies in cattle and motivating construction of a new optical map resource--BtOM1.0--to guide comparisons and improvements to the current sequence builds. Accordingly, our comprehensive comparisons of BtOM1.0 against the UMD3.1 and Btau4.6 sequence builds tabulate large-to-immediate scale discordances requiring mediation. The optical map, BtOM1.0, spanning the B. taurus genome (Hereford breed, L1 Dominette 01449) was assembled from an optical map dataset consisting of 2,973,315 (439 X; raw dataset size before assembly) single molecule optical maps (Rmaps; 1 Rmap = 1 restriction mapped DNA molecule) generated by the Optical Mapping System. The BamHI map spans 2,575.30 Mb and comprises 78 optical contigs assembled by a combination of iterative (using the reference sequence: UMD3.1) and de novo assembly techniques. BtOM1.0 is a high-resolution physical map featuring an average restriction fragment size of 8.91 Kb. Comparisons of BtOM1.0 vs. UMD3.1, or Btau4.6, revealed that Btau4.6 presented far more discordances (7,463) vs. UMD3.1 (4,754). Overall, we found that Btau4.6 presented almost double the number of discordances than UMD3.1 across most of the 6 categories of sequence vs. map discrepancies, which are: COMPLEX (misassembly), DELs (extraneous sequences), INSs (missing sequences), ITs (Inverted/Translocated sequences), ECs (extra restriction cuts) and MCs (missing restriction cuts). Alignments of UMD3.1 and Btau4.6 to BtOM1.0 reveal discordances commensurate with previous reports, and affirm the NCBI's current designation of UMD3.1 sequence assembly as the "reference assembly" and the Btau4.6 as the "alternate assembly." The cattle genome optical map, BtOM1.0, when used as a comprehensive and largely independent guide, will greatly assist improvements to existing sequence builds, and later serve as an accurate physical scaffold for studies concerning the comparative genomics of cattle breeds.

Comparative analysis of international standards for the fatigue testing of posterior spinal fixation systems: the importance of preload in ISO 12189.

PubMed

La Barbera, Luigi; Ottardi, Claudia; Villa, Tomaso

2015-10-01

Preclinical evaluation of the mechanical reliability of fixation devices is a mandatory activity before their introduction into market. There are two standardized protocols for preclinical testing of spinal implants. The American Society for Testing Materials (ASTM) recommends the F1717 standard, which describes a vertebrectomy condition that is relatively simple to implement, whereas the International Organization for Standardization (ISO) suggests the 12189 standard, which describes a more complex physiological anterior support-based setup. Moreover, ASTM F1717 is nowadays well established, whereas ISO 12189 has received little attention: A few studies tried to accurately describe the ISO experimental procedure through numeric models, but these studies totally neglect the recommended precompression step. This study aimed to build up a reliable, validated numeric model capable of describing the stress on the rods of a spinal fixator assembled according to ISO 12189 standard procedure. Such a model would more adequately represent the in vitro testing condition. This study used finite element (FE) simulations and experimental validation testing. An FE model of the ISO setup was built to calculate the stress on the rods. Simulation was validated by comparison with experimental strain gauges measurements. The same fixator has been previously virtually mounted in an L2-L4 FE model of the lumbar spine, and stresses in the rods were calculated when the spine was subjected to physiological forces and moments. The comparison between the FE predictions and experimental measurements is in good agreement, thus confirming the suitability of the FE method to evaluate the stresses in the device. The initial precompression induces a significant extension of the assembled construct. As the applied load increases, the initial extension is gradually compensated, so that at peak load the rods are bent in flexion: The final stress value predicted is thus reduced to about 50%, if compared with the previous model where the precompression was not considered. Neglecting the initial preload due to the assembly of the overall construct according to ISO 12189 standard could lead to an overestimation of the stress on the rods up to 50%. To correctly describe the state of stress on the posterior spinal fixator, tested according to the ISO procedure, it is important to take into account the initial preload due to the assembly of the overall construct. Copyright © 2015 Elsevier Inc. All rights reserved.

Heterologous pathway assembly reveals molecular steps of fungal terreic acid biosynthesis.

PubMed

Kong, Chuixing; Huang, Hezhou; Xue, Ying; Liu, Yiqi; Peng, Qiangqiang; Liu, Qi; Xu, Qin; Zhu, Qiaoyun; Yin, Ying; Zhou, Xiangshan; Zhang, Yuanxing; Cai, Menghao

2018-02-01

Terreic acid is a potential anticancer drug as it inhibits Bruton's tyrosine kinase; however, its biosynthetic molecular steps remain unclear. In this work, the individual reactions of terreic acid biosynthesis were determined by stepwise pathway assembly in a heterologous host, Pichia pastoris, on the basis of previous knockout studies in a native host, Aspergillus terreus. Polyketide synthase AtX was found to catalyze the formation of partially reduced polyketide 6-methylsalicylic acid, followed by 3-methylcatechol synthesis by salicylate 1-monooxygenase AtA-mediated decarboxylative hydroxylation of 6-methylsalicylic acid. Our results show that cytochrome P450 monooxygenase AtE hydroxylates 3-methylcatechol, thus producing the next product, 3-methyl-1,2,4-benzenetriol. A smaller putative cytochrome P450 monooxygenase, AtG, assists with this step. Then, AtD causes epoxidation and hydroxyl oxidation of 3-methyl-1,2,4-benzenetriol and produces a compound terremutin, via which the previously unknown function of AtD was identified as cyclooxygenation. The final step involves an oxidation reaction of a hydroxyl group by a glucose-methanol-choline oxidoreductase, AtC, which leads to the final product: terreic acid. Functions of AtD and AtG were determined for the first time. All the genes were reanalyzed and all intermediates and final products were isolated and identified. Our model fully defines the molecular steps and corrects previous results from the literature.

Ecomorphology of the African felid ensemble: the role of the skull and postcranium in determining species segregation and assembling history.

PubMed

Morales, M M; Giannini, N P

2013-05-01

Morphology of extant felids is regarded as highly conservative. Most previous studies have focussed on skull morphology, so a vacuum exists about morphofunctional variation in postcranium and its role in structuring ensembles of felids in different continents. The African felid ensemble is particularly rich in ecologically specialized felids. We studied the ecomorphology of this ensemble using 31 cranial and 93 postcranial morphometric variables measured in 49 specimens of all 10 African species. We took a multivariate approach controlling for phylogeny, with and without body size correction. Postcranial and skull + postcranial analyses (but not skull-only analyses) allowed for a complete segregation of species in morphospace. Morphofunctional factors segregating species included body size, bite force, zeugopodial lengths and osteological features related to parasagittal leg movement. A general gradient of bodily proportions was recovered: lightly built, long-legged felids with small heads and weak bite forces vs. the opposite. Three loose groups were recognized: small terrestrial felids, mid-to-large sized scansorial felids and specialized Acinonyx jubatus and Leptailurus serval. As predicted from a previous study, the assembling of the African felid ensemble during the Plio-Pleistocene occurred by the arrival of distinct felid lineages that occupied then vacant areas of morphospace, later diversifying in the continent. © 2013 The Authors. Journal of Evolutionary Biology © 2013 European Society For Evolutionary Biology.

Radio frequency glow discharge-induced acidification of fluoropolymers.

PubMed

Krawczyk, Benjamin M; Baltrusaitis, Jonas; Yoder, Colin M; Vargo, Terrence G; Bowden, Ned B; Kader, Khalid N

2011-12-01

Fluoropolymer surfaces are unique in view of the fact that they are quite inert, have low surface energies, and possess high thermal stabilities. Attempts to modify fluoropolymer surfaces have met with difficulties in that it is difficult to control the modification to maintain bulk characteristics of the polymer. In a previously described method, the replacement of a small fraction of surface fluorine by acid groups through radio frequency glow discharge created a surface with unexpected reactivity allowing for attachment of proteins in their active states. The present study demonstrates that 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) reacts with the acid groups on fluoropolymer surfaces in a novel reaction not previously described. This reaction yields an excellent leaving group in which a primary amine on proteins can substitute to form a covalent bond between a protein and these surfaces. In an earlier study, we demonstrated that collagen IV could be deposited on a modified PTFE surface using EDC as a linker. Once collagen IV is attached to the surface, it assembles to form a functional stratum resembling collagen IV in native basement membrane. In this study, we show data suggesting that the fluorine to carbon ratio determines the acidity of the fluoropolymer surfaces and how well collagen IV attaches to and assembles on four different fluoropolymer surfaces. Copyright © 2011 Wiley Periodicals, Inc.

Consensus pan-genome assembly of the specialised wine bacterium Oenococcus oeni.

PubMed

Sternes, Peter R; Borneman, Anthony R

2016-04-27

Oenococcus oeni is a lactic acid bacterium that is specialised for growth in the ecological niche of wine, where it is noted for its ability to perform the secondary, malolactic fermentation that is often required for many types of wine. Expanding the understanding of strain-dependent genetic variations in its small and streamlined genome is important for realising its full potential in industrial fermentation processes. Whole genome comparison was performed on 191 strains of O. oeni; from this rich source of genomic information consensus pan-genome assemblies of the invariant (core) and variable (flexible) regions of this organism were established. Genetic variation in amino acid biosynthesis and sugar transport and utilisation was found to be common between strains. Furthermore, we characterised previously-unreported intra-specific genetic variations in the natural competence of this microbe. By assembling a consensus pan-genome from a large number of strains, this study provides a tool for researchers to readily compare protein-coding genes across strains and infer functional relationships between genes in conserved syntenic regions. This establishes a foundation for further genetic, and thus phenotypic, research of this industrially-important species.

The impact of recent improvements in cryo-electron microscopy technology on the understanding of bacterial ribosome assembly

PubMed Central

Razi, Aida; Britton, Robert A.

2017-01-01

Abstract Cryo-electron microscopy (cryo-EM) had played a central role in the study of ribosome structure and the process of translation in bacteria since the development of this technique in the mid 1980s. Until recently cryo-EM structures were limited to ∼10 Å in the best cases. However, the recent advent of direct electron detectors has greatly improved the resolution of cryo-EM structures to the point where atomic resolution is now achievable. This improved resolution will allow cryo-EM to make groundbreaking contributions in essential aspects of ribosome biology, including the assembly process. In this review, we summarize important insights that cryo-EM, in combination with chemical and genetic approaches, has already brought to our current understanding of the ribosomal assembly process in bacteria using previous detector technology. More importantly, we discuss how the higher resolution structures now attainable with direct electron detectors can be leveraged to propose precise testable models regarding this process. These structures will provide an effective platform to develop new antibiotics that target this fundamental cellular process. PMID:28180306

Self-assembly of [Pt(3n)(CO)(6n)]2- (n = 4-8) carbonyl clusters: from molecules to conducting molecular metal wires.

PubMed

Femoni, Cristina; Iapalucci, Maria Carmela; Longoni, Giuliano; Lovato, Tatiana; Stagni, Stefano; Zacchini, Stefano

2010-07-05

A comprehensive study discussing the different parameters that influence the self-assembly of [Pt(3n)(CO)(6n)](2-) (n = 4-8) clusters with miscellaneous mono- and dications into 0-D, 1-D, 2-D, and 3-D materials is herein reported. As an unexpected bonus, the use of Ru(II) dications allowed the first structural characterization of the previously unknown [Pt(21)(CO)(42)](2-) dianion. 0-D structures, which contain isolated ions, are electrical insulators in solid form. Conversely, as soon as infinite chains of clusters are formed, the electrical resistivity, measured in pressed pellets, decreases to 10(5)-10(6), 10(4), and 10(2) ohms cm for discontinuous, semicontinuous, and continuous chains, respectively. Therefore, the resemblance of these materials to molecular metal wires is not only morphological but also functional. Preliminary results of possible self-assembly phenomena in a solution of [Pt(15)(CO)(30)](2-) and [Pt(18)(CO)(36)](2-) according to dynamic light scattering experiments are also reported.

Patterns in the English language: phonological networks, percolation and assembly models

NASA Astrophysics Data System (ADS)

Stella, Massimo; Brede, Markus

2015-05-01

In this paper we provide a quantitative framework for the study of phonological networks (PNs) for the English language by carrying out principled comparisons to null models, either based on site percolation, randomization techniques, or network growth models. In contrast to previous work, we mainly focus on null models that reproduce lower order characteristics of the empirical data. We find that artificial networks matching connectivity properties of the English PN are exceedingly rare: this leads to the hypothesis that the word repertoire might have been assembled over time by preferentially introducing new words which are small modifications of old words. Our null models are able to explain the ‘power-law-like’ part of the degree distributions and generally retrieve qualitative features of the PN such as high clustering, high assortativity coefficient and small-world characteristics. However, the detailed comparison to expectations from null models also points out significant differences, suggesting the presence of additional constraints in word assembly. Key constraints we identify are the avoidance of large degrees, the avoidance of triadic closure and the avoidance of large non-percolating clusters.

A review on simple assembly line balancing type-e problem

NASA Astrophysics Data System (ADS)

Jusop, M.; Rashid, M. F. F. Ab

2015-12-01

Simple assembly line balancing (SALB) is an attempt to assign the tasks to the various workstations along the line so that the precedence relations are satisfied and some performance measure are optimised. Advanced approach of algorithm is necessary to solve large-scale problems as SALB is a class of NP-hard. Only a few studies are focusing on simple assembly line balancing of Type-E problem (SALB-E) since it is a general and complex problem. SALB-E problem is one of SALB problem which consider the number of workstation and the cycle time simultaneously for the purpose of maximising the line efficiency. This paper review previous works that has been done in order to optimise SALB -E problem. Besides that, this paper also reviewed the Genetic Algorithm approach that has been used to optimise SALB-E. From the reviewed that has been done, it was found that none of the existing works are concern on the resource constraint in the SALB-E problem especially on machine and tool constraints. The research on SALB-E will contribute to the improvement of productivity in real industrial application.

Coarse grained modeling of directed assembly to form functional nanoporous films

NASA Astrophysics Data System (ADS)

Al Khatib, Amir

A coarse-grained (CG) simulation of polyethylene glycol (PEG) and Polymethylsilsesquixane nanoparticle (PMSSQ) referred to as (NP) at different sizes and concentrations were done using the Martini coarse-grained (CG) force field. The interactions between CG PEG and CG NP were parameterized from the chemical compound of each molecule and based on Martini force field. NP particles migrates to the surface of the substrate in an agreement with the experimental output at high temperature of 800K. This demonstration of nanoparticles-polymer film to direct it to self-assemble a systematically spatial pattern using the substrate surface energy as the key gating parameter. Validation of the model comparing molecular dynamics simulations with experimental data collected from previous study. NP interaction with the substrate at low interactions energy using Lennard-Johns potential were able to direct the NP to self-assemble in a hexagonal shape up to 4 layers above the substrate. This thesis established that substrate surface energy is a key gating parameter to direct the collective behavior of functional nanoparticles to form thin nanoporous films with spatially predetermined optical/dielectric constants.

Self-assembled three-dimensional chiral colloidal architecture.

PubMed

Ben Zion, Matan Yah; He, Xiaojin; Maass, Corinna C; Sha, Ruojie; Seeman, Nadrian C; Chaikin, Paul M

2017-11-03

Although stereochemistry has been a central focus of the molecular sciences since Pasteur, its province has previously been restricted to the nanometric scale. We have programmed the self-assembly of micron-sized colloidal clusters with structural information stemming from a nanometric arrangement. This was done by combining DNA nanotechnology with colloidal science. Using the functional flexibility of DNA origami in conjunction with the structural rigidity of colloidal particles, we demonstrate the parallel self-assembly of three-dimensional microconstructs, evincing highly specific geometry that includes control over position, dihedral angles, and cluster chirality. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly

PubMed Central

Schneider, Valerie A.; Graves-Lindsay, Tina; Howe, Kerstin; Bouk, Nathan; Chen, Hsiu-Chuan; Kitts, Paul A.; Murphy, Terence D.; Pruitt, Kim D.; Thibaud-Nissen, Françoise; Albracht, Derek; Fulton, Robert S.; Kremitzki, Milinn; Magrini, Vincent; Markovic, Chris; McGrath, Sean; Steinberg, Karyn Meltz; Auger, Kate; Chow, William; Collins, Joanna; Harden, Glenn; Hubbard, Timothy; Pelan, Sarah; Simpson, Jared T.; Threadgold, Glen; Torrance, James; Wood, Jonathan M.; Clarke, Laura; Koren, Sergey; Boitano, Matthew; Peluso, Paul; Li, Heng; Chin, Chen-Shan; Phillippy, Adam M.; Durbin, Richard; Wilson, Richard K.; Flicek, Paul; Eichler, Evan E.; Church, Deanna M.

2017-01-01

The human reference genome assembly plays a central role in nearly all aspects of today's basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009; it reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures, and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions, and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that although the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health. PMID:28396521

The thermodynamics of Pr55Gag-RNA interaction regulate the assembly of HIV

PubMed Central

Waddington, Lynne; Hijnen, Marcel; Velkov, Tony; McKinstry, William J.

2017-01-01

The interactions that occur during HIV Pr55Gag oligomerization and genomic RNA packaging are essential elements that facilitate HIV assembly. However, mechanistic details of these interactions are not clearly defined. Here, we overcome previous limitations in producing large quantities of full-length recombinant Pr55Gag that is required for isothermal titration calorimetry (ITC) studies, and we have revealed the thermodynamic properties of HIV assembly for the first time. Thermodynamic analysis showed that the binding between RNA and HIV Pr55Gag is an energetically favourable reaction (ΔG<0) that is further enhanced by the oligomerization of Pr55Gag. The change in enthalpy (ΔH) widens sequentially from: (1) Pr55Gag-Psi RNA binding during HIV genome selection; to (2) Pr55Gag-Guanosine Uridine (GU)-containing RNA binding in cytoplasm/plasma membrane; and then to (3) Pr55Gag-Adenosine(A)-containing RNA binding in immature HIV. These data imply the stepwise increments of heat being released during HIV biogenesis may help to facilitate the process of viral assembly. By mimicking the interactions between A-containing RNA and oligomeric Pr55Gag in immature HIV, it was noted that a p6 domain truncated Pr50Gag Δp6 is less efficient than full-length Pr55Gag in this thermodynamic process. These data suggest a potential unknown role of p6 in Pr55Gag-Pr55Gag oligomerization and/or Pr55Gag-RNA interaction during HIV assembly. Our data provide direct evidence on how nucleic acid sequences and the oligomeric state of Pr55Gag regulate HIV assembly. PMID:28222188

Assembling a protein-protein interaction map of the SSU processome from existing datasets.

PubMed

Lim, Young H; Charette, J Michael; Baserga, Susan J

2011-03-10

The small subunit (SSU) processome is a large ribonucleoprotein complex involved in small ribosomal subunit assembly. It consists of the U3 snoRNA and ∼72 proteins. While most of its components have been identified, the protein-protein interactions (PPIs) among them remain largely unknown, and thus the assembly, architecture and function of the SSU processome remains unclear. We queried PPI databases for SSU processome proteins to quantify the degree to which the three genome-wide high-throughput yeast two-hybrid (HT-Y2H) studies, the genome-wide protein fragment complementation assay (PCA) and the literature-curated (LC) datasets cover the SSU processome interactome. We find that coverage of the SSU processome PPI network is remarkably sparse. Two of the three HT-Y2H studies each account for four and six PPIs between only six of the 72 proteins, while the third study accounts for as little as one PPI and two proteins. The PCA dataset has the highest coverage among the genome-wide studies with 27 PPIs between 25 proteins. The LC dataset was the most extensive, accounting for 34 proteins and 38 PPIs, many of which were validated by independent methods, thereby further increasing their reliability. When the collected data were merged, we found that at least 70% of the predicted PPIs have yet to be determined and 26 proteins (36%) have no known partners. Since the SSU processome is conserved in all Eukaryotes, we also queried HT-Y2H datasets from six additional model organisms, but only four orthologues and three previously known interologous interactions were found. This provides a starting point for further work on SSU processome assembly, and spotlights the need for a more complete genome-wide Y2H analysis.

Assembling a Protein-Protein Interaction Map of the SSU Processome from Existing Datasets

PubMed Central

Baserga, Susan J.

2011-01-01

Background The small subunit (SSU) processome is a large ribonucleoprotein complex involved in small ribosomal subunit assembly. It consists of the U3 snoRNA and ∼72 proteins. While most of its components have been identified, the protein-protein interactions (PPIs) among them remain largely unknown, and thus the assembly, architecture and function of the SSU processome remains unclear. Methodology We queried PPI databases for SSU processome proteins to quantify the degree to which the three genome-wide high-throughput yeast two-hybrid (HT-Y2H) studies, the genome-wide protein fragment complementation assay (PCA) and the literature-curated (LC) datasets cover the SSU processome interactome. Conclusions We find that coverage of the SSU processome PPI network is remarkably sparse. Two of the three HT-Y2H studies each account for four and six PPIs between only six of the 72 proteins, while the third study accounts for as little as one PPI and two proteins. The PCA dataset has the highest coverage among the genome-wide studies with 27 PPIs between 25 proteins. The LC dataset was the most extensive, accounting for 34 proteins and 38 PPIs, many of which were validated by independent methods, thereby further increasing their reliability. When the collected data were merged, we found that at least 70% of the predicted PPIs have yet to be determined and 26 proteins (36%) have no known partners. Since the SSU processome is conserved in all Eukaryotes, we also queried HT-Y2H datasets from six additional model organisms, but only four orthologues and three previously known interologous interactions were found. This provides a starting point for further work on SSU processome assembly, and spotlights the need for a more complete genome-wide Y2H analysis. PMID:21423703

Simplifier: a web tool to eliminate redundant NGS contigs.

PubMed

Ramos, Rommel Thiago Jucá; Carneiro, Adriana Ribeiro; Azevedo, Vasco; Schneider, Maria Paula; Barh, Debmalya; Silva, Artur

2012-01-01

Modern genomic sequencing technologies produce a large amount of data with reduced cost per base; however, this data consists of short reads. This reduction in the size of the reads, compared to those obtained with previous methodologies, presents new challenges, including a need for efficient algorithms for the assembly of genomes from short reads and for resolving repetitions. Additionally after abinitio assembly, curation of the hundreds or thousands of contigs generated by assemblers demands considerable time and computational resources. We developed Simplifier, a stand-alone software that selectively eliminates redundant sequences from the collection of contigs generated by ab initio assembly of genomes. Application of Simplifier to data generated by assembly of the genome of Corynebacterium pseudotuberculosis strain 258 reduced the number of contigs generated by ab initio methods from 8,004 to 5,272, a reduction of 34.14%; in addition, N50 increased from 1 kb to 1.5 kb. Processing the contigs of Escherichia coli DH10B with Simplifier reduced the mate-paired library 17.47% and the fragment library 23.91%. Simplifier removed redundant sequences from datasets produced by assemblers, thereby reducing the effort required for finalization of genome assembly in tests with data from Prokaryotic organisms. Simplifier is available at http://www.genoma.ufpa.br/rramos/softwares/simplifier.xhtmlIt requires Sun jdk 6 or higher.

CENP-C and CENP-I are key connecting factors for kinetochore and CENP-A assembly

PubMed Central

Shono, Nobuaki; Ohzeki, Jun-ichirou; Otake, Koichiro; Martins, Nuno M. C.; Nagase, Takahiro; Kimura, Hiroshi; Larionov, Vladimir; Earnshaw, William C.; Masumoto, Hiroshi

2015-01-01

ABSTRACT Although it is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity, the pathways leading to the formation and maintenance of centromere chromatin remain unclear. We previously generated human artificial chromosomes (HACs) whose centromeres contain a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator (alphoidtetO). We also obtained cell lines bearing the alphoidtetO array at ectopic integration sites on chromosomal arms. Here, we have examined the regulation of CENP-A assembly at centromeres as well as de novo assembly on the ectopic arrays by tethering tetracycline repressor (tetR) fusions of substantial centromeric factors and chromatin modifiers. This analysis revealed four classes of factors that influence CENP-A assembly. Interestingly, many kinetochore structural components induced de novo CENP-A assembly at the ectopic site. We showed that these components work by recruiting CENP-C and subsequently recruiting M18BP1. Furthermore, we found that CENP-I can also recruit M18BP1 and, as a consequence, enhances M18BP1 assembly on centromeres in the downstream of CENP-C. Thus, we suggest that CENP-C and CENP-I are key factors connecting kinetochore to CENP-A assembly. PMID:26527398

Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy

PubMed Central

Ridgeway, William K.; Millar, David P.; Williamson, James R.

2012-01-01

The self-assembly of bacterial 30S ribosomes involves a large number of RNA folding and RNA-protein binding steps. The sequence of steps determines the overall assembly mechanism and the structure of the mechanism has ramifications for the robustness of biogenesis and resilience against kinetic traps. Thermodynamic interdependencies of protein binding inferred from omission-reconstitution experiments are thought to preclude certain assembly pathways and thus enforce ordered assembly, but this concept is at odds with kinetic data suggesting a more parallel assembly landscape. A major challenge is deconvolution of the statistical distribution of intermediates that are populated during assembly at high concentrations approaching in vivo assembly conditions. To specifically resolve the intermediates formed by binding of three ribosomal proteins to the full length 16S rRNA, we introduce Fluorescence Triple-Correlation Spectroscopy (F3CS). F3CS identifies specific ternary complexes by detecting coincident fluctuations in three-color fluorescence data. Triple correlation integrals quantify concentrations and diffusion kinetics of triply labeled species, and F3CS data can be fit alongside auto-correlation and cross-correlation data to quantify the populations of 10 specific ribosome assembly intermediates. The distribution of intermediates generated by binding three ribosomal proteins to the entire native 16S rRNA included significant populations of species that were not previously thought to be thermodynamically accessible, questioning the current interpretation of the classic omission-reconstitution experiments. F3CS is a general approach for analyzing assembly and function of macromolecular complexes, especially those too large for traditional biophysical methods. PMID:22869699

Biocatalytic Self-Assembly on Magnetic Nanoparticles.

PubMed

Conte, Maria P; Sahoo, Jugal Kishore; Abul-Haija, Yousef M; Lau, K H Aaron; Ulijn, Rein V

2018-01-24

Combining (bio)catalysis and molecular self-assembly provides an effective approach for the production and processing of self-assembled materials by exploiting catalysis to direct the assembly kinetics and hence controlling the formation of ordered nanostructures. Applications of (bio)catalytic self-assembly in biologically interfacing systems and in nanofabrication have recently been reported. Inspired by self-assembly in biological cells, efforts to confine catalysts on flat or patterned surfaces to exert spatial control over molecular gelator generation and nanostructure self-assembly have also emerged. Building on our previous work in the area, we demonstrate in this report the use of enzymes immobilized onto magnetic nanoparticles (NPs) to spatially localize the initiation of peptide self-assembly into nanofibers around NPs. The concept is generalized for both an equilibrium biocatalytic system that forms stable hydrogels and a nonequilibrium system that normally has a preset lifetime. Characterization of the hydrogels shows that self-assembly occurs at the site of enzyme immobilization on the NPs to give rise to gels with a "hub-and-spoke" morphology, where the nanofibers are linked through the enzyme-NP conjugates. This NP-controlled arrangement of self-assembled nanofibers enables both remarkable enhancements in the shear strength of hydrogel systems and a dramatic extension of the hydrogel stability in the nonequilibrium system. We are also able to show that the use of magnetic NPs enables the external control of both the formation of the hydrogel and its overall structure by application of an external magnetic field. We anticipate that the enhanced properties and stimuli-responsiveness of our NP-enzyme system will have applications ranging from nanomaterial fabrication to biomaterials and biosensing.

Structural Polymorphism in a Self-Assembled Tri-Aromatic Peptide System.

PubMed

Brown, Noam; Lei, Jiangtao; Zhan, Chendi; Shimon, Linda J W; Adler-Abramovich, Lihi; Wei, Guanghong; Gazit, Ehud

2018-04-24

Self-assembly is a process of key importance in natural systems and in nanotechnology. Peptides are attractive building blocks due to their relative facile synthesis, biocompatibility, and other unique properties. Diphenylalanine (FF) and its derivatives are known to form nanostructures of various architectures and interesting and varied characteristics. The larger triphenylalanine peptide (FFF) was found to self-assemble as efficiently as FF, forming related but distinct architectures of plate-like and spherical nanostructures. Here, to understand the effect of triaromatic systems on the self-assembly process, we examined carboxybenzyl-protected diphenylalanine (z-FF) as a minimal model for such an arrangement. We explored different self-assembly conditions by changing solvent compositions and peptide concentrations, generating a phase diagram for the assemblies. We discovered that z-FF can form a variety of structures, including nanowires, fibers, nanospheres, and nanotoroids, the latter were previously observed only in considerably larger or co-assembly systems. Secondary structure analysis revealed that all assemblies possessed a β-sheet conformation. Additionally, in solvent combinations with high water ratios, z-FF formed rigid and self-healing hydrogels. X-ray crystallography revealed a "wishbone" structure, in which z-FF dimers are linked by hydrogen bonds mediated by methanol molecules, with a 2-fold screw symmetry along the c-axis. All-atom molecular dynamics (MD) simulations revealed conformations similar to the crystal structure. Coarse-grained MD simulated the assembly of the peptide into either fibers or spheres in different solvent systems, consistent with the experimental results. This work thus expands the building block library for the fabrication of nanostructures by peptide self-assembly.

Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

PubMed Central

Stransky, Laura A.; Forgac, Michael

2015-01-01

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

Protein-directed self-assembly of a fullerene crystal.

PubMed

Kim, Kook-Han; Ko, Dong-Kyun; Kim, Yong-Tae; Kim, Nam Hyeong; Paul, Jaydeep; Zhang, Shao-Qing; Murray, Christopher B; Acharya, Rudresh; DeGrado, William F; Kim, Yong Ho; Grigoryan, Gevorg

2016-04-26

Learning to engineer self-assembly would enable the precise organization of molecules by design to create matter with tailored properties. Here we demonstrate that proteins can direct the self-assembly of buckminsterfullerene (C60) into ordered superstructures. A previously engineered tetrameric helical bundle binds C60 in solution, rendering it water soluble. Two tetramers associate with one C60, promoting further organization revealed in a 1.67-Å crystal structure. Fullerene groups occupy periodic lattice sites, sandwiched between two Tyr residues from adjacent tetramers. Strikingly, the assembly exhibits high charge conductance, whereas both the protein-alone crystal and amorphous C60 are electrically insulating. The affinity of C60 for its crystal-binding site is estimated to be in the nanomolar range, with lattices of known protein crystals geometrically compatible with incorporating the motif. Taken together, these findings suggest a new means of organizing fullerene molecules into a rich variety of lattices to generate new properties by design.

Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast

PubMed Central

Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R.; Drubin, David G.

2016-01-01

Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin–Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

A super-assembly of Whi3 encodes memory of deceptive encounters by single cells during yeast courtship.

PubMed

Caudron, Fabrice; Barral, Yves

2013-12-05

Cellular behavior is frequently influenced by the cell's history, indicating that single cells may memorize past events. We report that budding yeast permanently escape pheromone-induced cell-cycle arrest when experiencing a deceptive mating attempt, i.e., not reaching their putative partner within reasonable time. This acquired behavior depends on super-assembly and inactivation of the G1/S inhibitor Whi3, which liberates the G1 cyclin Cln3 from translational inhibition. Super-assembly of Whi3 is a slow response to pheromone, driven by polyQ and polyN domains, counteracted by Hsp70, and stable over generations. Unlike prion aggregates, Whi3 super-assemblies are not inherited mitotically but segregate to the mother cell. We propose that such polyQ- and polyN-based elements, termed here mnemons, act as cellular memory devices to encode previous environmental conditions. Copyright © 2013 Elsevier Inc. All rights reserved.

Reversible Assembly of Graphitic Carbon Nitride 3D Network for Highly Selective Dyes Absorption and Regeneration.

PubMed

Zhang, Yuye; Zhou, Zhixin; Shen, Yanfei; Zhou, Qing; Wang, Jianhai; Liu, Anran; Liu, Songqin; Zhang, Yuanjian

2016-09-27

Responsive assembly of 2D materials is of great interest for a range of applications. In this work, interfacial functionalized carbon nitride (CN) nanofibers were synthesized by hydrolyzing bulk CN in sodium hydroxide solution. The reversible assemble and disassemble behavior of the as-prepared CN nanofibers was investigated by using CO2 as a trigger to form a hydrogel network at first. Compared to the most widespread absorbent materials such as active carbon, graphene and previously reported supramolecular gel, the proposed CN hydrogel not only exhibited a competitive absorbing capacity (maximum absorbing capacity of methylene blue up to 402 mg/g) but also overcame the typical deficiencies such as poor selectivity and high energy-consuming regeneration. This work would provide a strategy to construct a 3D CN network and open an avenue for developing smart assembly for potential applications ranging from environment to selective extraction.

Use your head! Perception of action possibilities by means of an object attached to the head.

PubMed

Wagman, Jeffrey B; Hajnal, Alen

2016-03-01

Perceiving any environmental property requires spontaneously assembling a smart perceptual instrument-a task-specific measurement device assembled across potentially independent anatomical units. Previous research has shown that to a large degree, perception of a given environmental property is anatomically independent. We attempted to provide stronger evidence for this proposal by investigating perception by an organization of anatomical and inert components that likely requires the spontaneous assembly of a novel smart perceptual instrument-a rod attached to the head. Specifically, we compared cephalic and manual perception of whether an inclined surface affords standing on. In both conditions, perception reflected the action capabilities of the perceiver and not the appendage used to wield the rod. Such results provide stronger evidence for anatomical independence of perception within a given perceptual system and highlight that flexible task-specific detection units can be assembled across units that span the body and inert objects.

Novel mesostructured inclusions in the epidermal lining of Artemia franciscana ovisacs show optical activity

PubMed Central

Hollergschwandtner, Elena; Schwaha, Thomas; Neumüller, Josef; Kaindl, Ulrich; Gruber, Daniela; Eckhard, Margret; Stöger-Pollach, Michael

2017-01-01

Background Biomineralization, e.g., in sea urchins or mollusks, includes the assembly of mesoscopic superstructures from inorganic crystalline components and biopolymers. The resulting mesocrystals inspire biophysicists and material scientists alike, because of their extraordinary physical properties. Current efforts to replicate mesocrystal synthesis in vitro require understanding the principles of their self-assembly in vivo. One question, not addressed so far, is whether intracellular crystals of proteins can assemble with biopolymers into functional mesocrystal-like structures. During our electron microscopy studies into Artemia franciscana (Crustacea: Branchiopoda), we found initial evidence of such proteinaceous mesostructures. Results EM preparations with high-pressure freezing and accelerated freeze substitution revealed an extraordinary intracellular source of mesostructured inclusions in both the cyto-and nucleoplasm of the epidermal lining of ovisacs of A. franciscana. Confocal reflection microscopy not only confirmed our finding; it also revealed reflective, light dispersing activity of these flake-like structures, their positioning and orientation with respect to the ovisac inside. Both the striation of alternating electron dense and electron-lucent components and the sharp edges of the flakes indicate self-assembly of material of yet unknown origin under supposed participation of crystallization. However, selected area electron diffraction could not verify the status of crystallization. Energy dispersive X-ray analysis measured a marked increase in nitrogen within the flake-like inclusion, and the almost complete absence of elements that are typically involved in inorganic crystallization. This rise in nitrogen could possibility be related to higher package density of proteins, achieved by mesostructure assembly. Conclusions The ovisac lining of A. franciscana is endowed with numerous mesostructured inclusions that have not been previously reported. We hypothesize that their self-assembly was from proteinaceous polycrystalline units and carbohydrates. These mesostructured flakes displayed active optical properties, as an umbrella-like, reflective cover of the ovisac, which suggests a functional role in the reproduction of A. franciscana. In turn, studies into ovisac mesostructured inclusions could help to optimizing rearing Artemia as feed for fish farming. We propose Artemia ovisacs as an in vivo model system for studying mesostructure formation. PMID:29093995

Novel mesostructured inclusions in the epidermal lining of Artemia franciscana ovisacs show optical activity.

PubMed

Hollergschwandtner, Elena; Schwaha, Thomas; Neumüller, Josef; Kaindl, Ulrich; Gruber, Daniela; Eckhard, Margret; Stöger-Pollach, Michael; Reipert, Siegfried

2017-01-01

Biomineralization, e.g., in sea urchins or mollusks, includes the assembly of mesoscopic superstructures from inorganic crystalline components and biopolymers. The resulting mesocrystals inspire biophysicists and material scientists alike, because of their extraordinary physical properties. Current efforts to replicate mesocrystal synthesis in vitro require understanding the principles of their self-assembly in vivo . One question, not addressed so far, is whether intracellular crystals of proteins can assemble with biopolymers into functional mesocrystal-like structures. During our electron microscopy studies into Artemia franciscana (Crustacea: Branchiopoda), we found initial evidence of such proteinaceous mesostructures. EM preparations with high-pressure freezing and accelerated freeze substitution revealed an extraordinary intracellular source of mesostructured inclusions in both the cyto-and nucleoplasm of the epidermal lining of ovisacs of A. franciscana . Confocal reflection microscopy not only confirmed our finding; it also revealed reflective, light dispersing activity of these flake-like structures, their positioning and orientation with respect to the ovisac inside. Both the striation of alternating electron dense and electron-lucent components and the sharp edges of the flakes indicate self-assembly of material of yet unknown origin under supposed participation of crystallization. However, selected area electron diffraction could not verify the status of crystallization. Energy dispersive X-ray analysis measured a marked increase in nitrogen within the flake-like inclusion, and the almost complete absence of elements that are typically involved in inorganic crystallization. This rise in nitrogen could possibility be related to higher package density of proteins, achieved by mesostructure assembly. The ovisac lining of A. franciscana is endowed with numerous mesostructured inclusions that have not been previously reported. We hypothesize that their self-assembly was from proteinaceous polycrystalline units and carbohydrates. These mesostructured flakes displayed active optical properties, as an umbrella-like, reflective cover of the ovisac, which suggests a functional role in the reproduction of A. franciscana . In turn, studies into ovisac mesostructured inclusions could help to optimizing rearing Artemia as feed for fish farming. We propose Artemia ovisacs as an in vivo model system for studying mesostructure formation.

Analysis of a rotating advanced-technology space station for the year 2025

NASA Technical Reports Server (NTRS)

Queijo, M. J.; Butterfield, A. J.; Cuddihy, W. F.; King, C. B.; Stone, R. W.; Garn, P. A.

1988-01-01

An analysis is made of several aspects of an advanced-technology rotating space station configuration generated under a previous study. The analysis includes examination of several modifications of the configuration, interface with proposed launch systems, effects of low-gravity environment on human subjects, and the space station assembly sequence. Consideration was given also to some aspects of space station rotational dynamics, surface charging, and the possible application of tethers.

KSC-97PC1230

NASA Image and Video Library

1997-08-11

Extension of the solar panels is tested on the Advanced Composition Explorer (ACE) spacecraft in KSC’s Spacecraft Assembly and Encapsulation Facility-II (SAEF-II). Scheduled for launch on a Delta II rocket from Cape Canaveral Air Station on Aug. 25, ACE will study low-energy particles of solar origin and high-energy galactic particles. The collecting power of instruments aboard ACE is 10 to 1,000 times greater than anything previously flown to collect similar data by NASA

Fabrication of Organic Thin Film Transistors Using Layer-By-Layer Assembly (Preprint)

DTIC Science & Technology

2007-03-01

thin-film transistors ( TFTs ) have received considerable attention as a low- cost, light-weight, flexible alternative to traditional amorphous silicon...Previous studies have investigated the use of a number of materials for both the active layer and the gate dielectric in various TFT architectures. These...performance. Conjugated small molecules, such as pentacene, or polymers, such as poly(3- hexylthiophene), are commonly used as the active layer in organic TFT

Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly.

PubMed

Wu, Shuo; Zhao, Qiong; Zhang, Pinghu; Kulp, John; Hu, Lydia; Hwang, Nicky; Zhang, Jiming; Block, Timothy M; Xu, Xiaodong; Du, Yanming; Chang, Jinhong; Guo, Ju-Tao

2017-08-15

Chronic hepatitis B virus (HBV) infection is a global public health problem. Although the currently approved medications can reliably reduce the viral load and prevent the progression of liver diseases, they fail to cure the viral infection. In an effort toward discovery of novel antiviral agents against HBV, a group of benzamide (BA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA were discovered. The initial lead optimization efforts identified two BA derivatives with improved antiviral activity for further mechanistic studies. Interestingly, similar to our previously reported sulfamoylbenzamides (SBAs), the BAs promote the formation of empty capsids through specific interaction with HBV core protein but not other viral and host cellular components. Genetic evidence suggested that both SBAs and BAs inhibited HBV nucleocapsid assembly by binding to the heteroaryldihydropyrimidine (HAP) pocket between core protein dimer-dimer interfaces. However, unlike SBAs, BA compounds uniquely induced the formation of empty capsids that migrated more slowly in native agarose gel electrophoresis from A36V mutant than from the wild-type core protein. Moreover, we showed that the assembly of chimeric capsids from wild-type and drug-resistant core proteins was susceptible to multiple capsid assembly modulators. Hence, HBV core protein is a dominant antiviral target that may suppress the selection of drug-resistant viruses during core protein-targeting antiviral therapy. Our studies thus indicate that BAs are a chemically and mechanistically unique type of HBV capsid assembly modulators and warranted for further development as antiviral agents against HBV. IMPORTANCE HBV core protein plays essential roles in many steps of the viral replication cycle. In addition to packaging viral pregenomic RNA (pgRNA) and DNA polymerase complex into nucleocapsids for reverse transcriptional DNA replication to take place, the core protein dimers, existing in several different quaternary structures in infected hepatocytes, participate in and regulate HBV virion assembly, capsid uncoating, and covalently closed circular DNA (cccDNA) formation. It is anticipated that small molecular core protein assembly modulators may disrupt one or multiple steps of HBV replication, depending on their interaction with the distinct quaternary structures of core protein. The discovery of novel core protein-targeting antivirals, such as benzamide derivatives reported here, and investigation of their antiviral mechanism may lead to the identification of antiviral therapeutics for the cure of chronic hepatitis B. Copyright © 2017 American Society for Microbiology.

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dick, Robert A.; Datta, Siddhartha A. K.; Nanda, Hirsh

2016-05-06

Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, whichmore » is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization. Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our results show that RSV Gag is highly flexible and can adopt a folded-over conformation on a lipid bilayer, implicating both the N and C termini in membrane binding. In addition, binding of Gag to membranes is diminished when either terminal domain is truncated. RSV Gag membrane association is significantly less sensitive than HIV Gag membrane association to lipid acyl chain saturation. These findings shed light on Gag assembly and membrane binding, critical steps in the viral life cycle and an untapped target for antiretroviral drugs.« less

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly.

PubMed

Datta, Siddhartha A K; Clark, Patrick K; Fan, Lixin; Ma, Buyong; Harvin, Demetria P; Sowder, Raymond C; Nussinov, Ruth; Wang, Yun-Xing; Rein, Alan

2016-02-15

HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a "spacer" between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it "association competent." Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Dimerization of the SP1 Region of HIV-1 Gag Induces a Helical Conformation and Association into Helical Bundles: Implications for Particle Assembly

PubMed Central

Clark, Patrick K.; Fan, Lixin; Ma, Buyong; Harvin, Demetria P.; Sowder, Raymond C.; Nussinov, Ruth; Wang, Yun-Xing

2015-01-01

ABSTRACT HIV-1 immature particle (virus-like particle [VLP]) assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We previously investigated the role of SP1, a “spacer” between CA and NC, in VLP assembly. We found that small changes in SP1 drastically disrupt assembly and that a peptide representing the sequence around the CA-SP1 junction is helical at high but not low concentrations. We suggested that by virtue of such a concentration-dependent change, this region could act as a molecular switch to activate HIV-1 Gag for VLP assembly. A leucine zipper domain can replace NC in Gag and still lead to the efficient assembly of VLPs. We find that SP1 mutants also disrupt assembly by these Gag-Zip proteins and have now studied a small fragment of this Gag-Zip protein, i.e., the CA-SP1 junction region fused to a leucine zipper. Dimerization of the zipper places SP1 at a high local concentration, even at low total concentrations. In this context, the CA-SP1 junction region spontaneously adopts a helical conformation, and the proteins associate into tetramers. Tetramerization requires residues from both CA and SP1. The data suggest that once this region becomes helical, its propensity to self-associate could contribute to Gag-Gag interactions and thus to particle assembly. There is complete congruence between CA/SP1 sequences that promote tetramerization when fused to zippers and those that permit the proper assembly of full-length Gag; thus, equivalent interactions apparently participate in VLP assembly and in SP1-Zip tetramerization. IMPORTANCE Assembly of HIV-1 Gag into virus-like particles (VLPs) appears to require an interaction with nucleic acid, but replacement of its principal nucleic acid-binding domain with a dimerizing leucine zipper domain leads to the assembly of RNA-free VLPs. It has not been clear how dimerization triggers assembly. Results here show that the SP1 region spontaneously switches to a helical state when fused to a leucine zipper and that these helical molecules further associate into tetramers, mediated by interactions between hydrophobic faces of the helices. Thus, the correct juxtaposition of the SP1 region makes it “association competent.” Residues from both capsid and SP1 contribute to tetramerization, while mutations disrupting proper assembly in Gag also prevent tetramerization. Thus, this region is part of an associating interface within Gag, and its intermolecular interactions evidently help stabilize the immature Gag lattice. These interactions are disrupted by proteolysis of the CA-SP1 junction during virus maturation. PMID:26637452

Improvement of the banana "Musa acuminata" reference sequence using NGS data and semi-automated bioinformatics methods.

PubMed

Martin, Guillaume; Baurens, Franc-Christophe; Droc, Gaëtan; Rouard, Mathieu; Cenci, Alberto; Kilian, Andrzej; Hastie, Alex; Doležel, Jaroslav; Aury, Jean-Marc; Alberti, Adriana; Carreel, Françoise; D'Hont, Angélique

2016-03-16

Recent advances in genomics indicate functional significance of a majority of genome sequences and their long range interactions. As a detailed examination of genome organization and function requires very high quality genome sequence, the objective of this study was to improve reference genome assembly of banana (Musa acuminata). We have developed a modular bioinformatics pipeline to improve genome sequence assemblies, which can handle various types of data. The pipeline comprises several semi-automated tools. However, unlike classical automated tools that are based on global parameters, the semi-automated tools proposed an expert mode for a user who can decide on suggested improvements through local compromises. The pipeline was used to improve the draft genome sequence of Musa acuminata. Genotyping by sequencing (GBS) of a segregating population and paired-end sequencing were used to detect and correct scaffold misassemblies. Long insert size paired-end reads identified scaffold junctions and fusions missed by automated assembly methods. GBS markers were used to anchor scaffolds to pseudo-molecules with a new bioinformatics approach that avoids the tedious step of marker ordering during genetic map construction. Furthermore, a genome map was constructed and used to assemble scaffolds into super scaffolds. Finally, a consensus gene annotation was projected on the new assembly from two pre-existing annotations. This approach reduced the total Musa scaffold number from 7513 to 1532 (i.e. by 80%), with an N50 that increased from 1.3 Mb (65 scaffolds) to 3.0 Mb (26 scaffolds). 89.5% of the assembly was anchored to the 11 Musa chromosomes compared to the previous 70%. Unknown sites (N) were reduced from 17.3 to 10.0%. The release of the Musa acuminata reference genome version 2 provides a platform for detailed analysis of banana genome variation, function and evolution. Bioinformatics tools developed in this work can be used to improve genome sequence assemblies in other species.

Simulating Self-Assembly with Simple Models

NASA Astrophysics Data System (ADS)

Rapaport, D. C.

Results from recent molecular dynamics simulations of virus capsid self-assembly are described. The model is based on rigid trapezoidal particles designed to form polyhedral shells of size 60, together with an atomistic solvent. The underlying bonding process is fully reversible. More extensive computations are required than in previous work on icosahedral shells built from triangular particles, but the outcome is a high yield of closed shells. Intermediate clusters have a variety of forms, and bond counts provide a useful classification scheme

Assembly of micro/nanomaterials into complex, three-dimensional architectures by compressive buckling

NASA Astrophysics Data System (ADS)

Xu, Sheng; Yan, Zheng; Jang, Kyung-In; Huang, Wen; Fu, Haoran; Kim, Jeonghyun; Wei, Zijun; Flavin, Matthew; McCracken, Joselle; Wang, Renhan; Badea, Adina; Liu, Yuhao; Xiao, Dongqing; Zhou, Guoyan; Lee, Jungwoo; Chung, Ha Uk; Cheng, Huanyu; Ren, Wen; Banks, Anthony; Li, Xiuling; Paik, Ungyu; Nuzzo, Ralph G.; Huang, Yonggang; Zhang, Yihui; Rogers, John A.

2015-01-01

Complex three-dimensional (3D) structures in biology (e.g., cytoskeletal webs, neural circuits, and vasculature networks) form naturally to provide essential functions in even the most basic forms of life. Compelling opportunities exist for analogous 3D architectures in human-made devices, but design options are constrained by existing capabilities in materials growth and assembly. We report routes to previously inaccessible classes of 3D constructs in advanced materials, including device-grade silicon. The schemes involve geometric transformation of 2D micro/nanostructures into extended 3D layouts by compressive buckling. Demonstrations include experimental and theoretical studies of more than 40 representative geometries, from single and multiple helices, toroids, and conical spirals to structures that resemble spherical baskets, cuboid cages, starbursts, flowers, scaffolds, fences, and frameworks, each with single- and/or multiple-level configurations.

Phylogenomics from Whole Genome Sequences Using aTRAM.

PubMed

Allen, Julie M; Boyd, Bret; Nguyen, Nam-Phuong; Vachaspati, Pranjal; Warnow, Tandy; Huang, Daisie I; Grady, Patrick G S; Bell, Kayce C; Cronk, Quentin C B; Mugisha, Lawrence; Pittendrigh, Barry R; Leonardi, M Soledad; Reed, David L; Johnson, Kevin P

2017-09-01

Novel sequencing technologies are rapidly expanding the size of data sets that can be applied to phylogenetic studies. Currently the most commonly used phylogenomic approaches involve some form of genome reduction. While these approaches make assembling phylogenomic data sets more economical for organisms with large genomes, they reduce the genomic coverage and thereby the long-term utility of the data. Currently, for organisms with moderate to small genomes ($<$1000 Mbp) it is feasible to sequence the entire genome at modest coverage ($10-30\\times$). Computational challenges for handling these large data sets can be alleviated by assembling targeted reads, rather than assembling the entire genome, to produce a phylogenomic data matrix. Here we demonstrate the use of automated Target Restricted Assembly Method (aTRAM) to assemble 1107 single-copy ortholog genes from whole genome sequencing of sucking lice (Anoplura) and out-groups. We developed a pipeline to extract exon sequences from the aTRAM assemblies by annotating them with respect to the original target protein. We aligned these protein sequences with the inferred amino acids and then performed phylogenetic analyses on both the concatenated matrix of genes and on each gene separately in a coalescent analysis. Finally, we tested the limits of successful assembly in aTRAM by assembling 100 genes from close- to distantly related taxa at high to low levels of coverage.Both the concatenated analysis and the coalescent-based analysis produced the same tree topology, which was consistent with previously published results and resolved weakly supported nodes. These results demonstrate that this approach is successful at developing phylogenomic data sets from raw genome sequencing reads. Further, we found that with coverages above $5-10\\times$, aTRAM was successful at assembling 80-90% of the contigs for both close and distantly related taxa. As sequencing costs continue to decline, we expect full genome sequencing will become more feasible for a wider array of organisms, and aTRAM will enable mining of these genomic data sets for an extensive variety of applications, including phylogenomics. [aTRAM; gene assembly; genome sequencing; phylogenomics.]. © The Author(s) 2017. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

DOE Office of Scientific and Technical Information (OSTI.GOV)

J.C. Ryman

This calculation is a revision of a previous calculation (Ref. 7.5) that bears the same title and has the document identifier BBAC00000-01717-0210-00006 REV 01. The purpose of this revision is to remove TBV (to-be-verified) -41 10 associated with the output files of the previous version (Ref. 7.30). The purpose of this and the previous calculation is to generate source terms for a representative boiling water reactor (BWR) spent nuclear fuel (SNF) assembly for the first one million years after the SNF is discharged from the reactors. This calculation includes an examination of several ways to represent BWR assemblies and operatingmore » conditions in SAS2H in order to quantify the effects these representations may have on source terms. These source terms provide information characterizing the neutron and gamma spectra in particles per second, the decay heat in watts, and radionuclide inventories in curies. Source terms are generated for a range of burnups and enrichments (see Table 2) that are representative of the waste stream and stainless steel (SS) clad assemblies. During this revision, it was determined that the burnups used for the computer runs of the previous revision were actually about 1.7% less than the stated, or nominal, burnups. See Section 6.6 for a discussion of how to account for this effect before using any source terms from this calculation. The source term due to the activation of corrosion products deposited on the surfaces of the assembly from the coolant is also calculated. The results of this calculation support many areas of the Monitored Geologic Repository (MGR), which include thermal evaluation, radiation dose determination, radiological safety analyses, surface and subsurface facility designs, and total system performance assessment. This includes MGR items classified as Quality Level 1, for example, the Uncanistered Spent Nuclear Fuel Disposal Container (Ref. 7.27, page 7). Therefore, this calculation is subject to the requirements of the Quality Assurance Requirements and Description (Ref. 7.28). The performance of the calculation and development of this document are carried out in accordance with AP-3.124, ''Design Calculation and Analyses'' (Ref. 7.29).« less

N-halamine biocidal coatings via a layer-by-layer assembly technique.

PubMed

Cerkez, Idris; Kocer, Hasan B; Worley, S D; Broughton, R M; Huang, T S

2011-04-05

Two N-halamine copolymer precursors, poly(2,2,6,6-tetramethyl-4-piperidyl methacrylate-co-acrylic acid potassium salt) and poly(2,2,6,6-tetramethyl-4-piperidyl methacrylate-co-trimethyl-2-methacryloxyethylammonium chloride) have been synthesized and successfully coated onto cotton fabric via a layer-by-layer (LbL) assembly technique. A multilayer thin film was deposited onto the fiber surfaces by alternative exposure to polyelectrolyte solutions. The coating was rendered biocidal by a dilute household bleach treatment. The biocidal efficacies of tested swatches composed of treated fibers were evaluated against Staphylococcus aureus and Escherichia coli. It was determined that chlorinated samples inactivated both S. aureus and E. coli O157:H7 within 15 min of contact time, whereas the unchlorinated control samples did not exhibit significant biocidal activities. Stabilities of the coatings toward washing and ultraviolet light exposure have also been studied. It was found that the stability toward washing was superior, whereas the UVA light stability was moderate compared to previously studied N-halamine moieties. The layer-by-layer assembly technique can be used to attach N-halamine precursor polymers onto cellulose surfaces without using covalently bonding tethering groups which limit the structure designs. In addition, ionic precursors are very soluble in water, thus promising for biocidal coatings without the use of organic solvents.

Dissecting the Structure-Function Relationship of a Fungicidal Peptide Derived from the Constant Region of Human Immunoglobulins.

PubMed

Ciociola, Tecla; Pertinhez, Thelma A; Giovati, Laura; Sperindè, Martina; Magliani, Walter; Ferrari, Elena; Gatti, Rita; D'Adda, Tiziana; Spisni, Alberto; Conti, Stefania; Polonelli, Luciano

2016-04-01

Synthetic peptides encompassing sequences related to the complementarity-determining regions of antibodies or derived from their constant region (Fc peptides) were proven to exert differential antimicrobial, antiviral, antitumor, and/or immunomodulatory activitiesin vitroand/orin vivo, regardless of the specificity and isotype of the parental antibody. Alanine substitution derivatives of these peptides exhibited unaltered, increased, or decreased candidacidal activitiesin vitro The bioactive IgG-derived Fc N10K peptide (NQVSLTCLVK) spontaneously self-assembles, a feature previously recognized as relevant for the therapeutic activity of another antibody-derived peptide. We evaluated the contribution of each residue to the peptide self-assembling capability by circular-dichroism spectroscopy. The interaction of the N10K peptide and its derivatives withCandida albicanscells was studied by confocal, transmission, and scanning electron microscopy. The apoptosis and autophagy induction profiles in yeast cells treated with the peptides were evaluated by flow cytometry, and the therapeutic efficacy against candidal infection was studied in aGalleria mellonellamodel. Overall, the results indicate a critical role for some residues in the self-assembly process and a correlation of that capability with the candidacidal activities of the peptidesin vitroand their therapeutic effectsin vivo. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Towards an Integrated QR Code Biosensor: Light-Driven Sample Acquisition and Bacterial Cellulose Paper Substrate.

PubMed

Yuan, Mingquan; Jiang, Qisheng; Liu, Keng-Ku; Singamaneni, Srikanth; Chakrabartty, Shantanu

2018-06-01

This paper addresses two key challenges toward an integrated forward error-correcting biosensor based on our previously reported self-assembled quick-response (QR) code. The first challenge involves the choice of the paper substrate for printing and self-assembling the QR code. We have compared four different substrates that includes regular printing paper, Whatman filter paper, nitrocellulose membrane and lab synthesized bacterial cellulose. We report that out of the four substrates bacterial cellulose outperforms the others in terms of probe (gold nanorods) and ink retention capability. The second challenge involves remote activation of the analyte sampling and the QR code self-assembly process. In this paper, we use light as a trigger signal and a graphite layer as a light-absorbing material. The resulting change in temperature due to infrared absorption leads to a temperature gradient that then exerts a diffusive force driving the analyte toward the regions of self-assembly. The working principle has been verified in this paper using assembled biosensor prototypes where we demonstrate higher sample flow rate due to light induced thermal gradients.

Solution Properties of Murine Leukemia Virus Gag Protein: Differences from HIV-1 Gag▿

PubMed Central

Datta, Siddhartha A. K.; Zuo, Xiaobing; Clark, Patrick K.; Campbell, Stephen J.; Wang, Yun-Xing; Rein, Alan

2011-01-01

Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of ∼7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed. PMID:21917964

Haplotype assembly in polyploid genomes and identical by descent shared tracts.

PubMed

Aguiar, Derek; Istrail, Sorin

2013-07-01

Genome-wide haplotype reconstruction from sequence data, or haplotype assembly, is at the center of major challenges in molecular biology and life sciences. For complex eukaryotic organisms like humans, the genome is vast and the population samples are growing so rapidly that algorithms processing high-throughput sequencing data must scale favorably in terms of both accuracy and computational efficiency. Furthermore, current models and methodologies for haplotype assembly (i) do not consider individuals sharing haplotypes jointly, which reduces the size and accuracy of assembled haplotypes, and (ii) are unable to model genomes having more than two sets of homologous chromosomes (polyploidy). Polyploid organisms are increasingly becoming the target of many research groups interested in the genomics of disease, phylogenetics, botany and evolution but there is an absence of theory and methods for polyploid haplotype reconstruction. In this work, we present a number of results, extensions and generalizations of compass graphs and our HapCompass framework. We prove the theoretical complexity of two haplotype assembly optimizations, thereby motivating the use of heuristics. Furthermore, we present graph theory-based algorithms for the problem of haplotype assembly using our previously developed HapCompass framework for (i) novel implementations of haplotype assembly optimizations (minimum error correction), (ii) assembly of a pair of individuals sharing a haplotype tract identical by descent and (iii) assembly of polyploid genomes. We evaluate our methods on 1000 Genomes Project, Pacific Biosciences and simulated sequence data. HapCompass is available for download at http://www.brown.edu/Research/Istrail_Lab/. Supplementary data are available at Bioinformatics online.

Evaluation of GRCh38 and de novo haploid genome assemblies demonstrates the enduring quality of the reference assembly.

PubMed

Schneider, Valerie A; Graves-Lindsay, Tina; Howe, Kerstin; Bouk, Nathan; Chen, Hsiu-Chuan; Kitts, Paul A; Murphy, Terence D; Pruitt, Kim D; Thibaud-Nissen, Françoise; Albracht, Derek; Fulton, Robert S; Kremitzki, Milinn; Magrini, Vincent; Markovic, Chris; McGrath, Sean; Steinberg, Karyn Meltz; Auger, Kate; Chow, William; Collins, Joanna; Harden, Glenn; Hubbard, Timothy; Pelan, Sarah; Simpson, Jared T; Threadgold, Glen; Torrance, James; Wood, Jonathan M; Clarke, Laura; Koren, Sergey; Boitano, Matthew; Peluso, Paul; Li, Heng; Chin, Chen-Shan; Phillippy, Adam M; Durbin, Richard; Wilson, Richard K; Flicek, Paul; Eichler, Evan E; Church, Deanna M

2017-05-01

The human reference genome assembly plays a central role in nearly all aspects of today's basic and clinical research. GRCh38 is the first coordinate-changing assembly update since 2009; it reflects the resolution of roughly 1000 issues and encompasses modifications ranging from thousands of single base changes to megabase-scale path reorganizations, gap closures, and localization of previously orphaned sequences. We developed a new approach to sequence generation for targeted base updates and used data from new genome mapping technologies and single haplotype resources to identify and resolve larger assembly issues. For the first time, the reference assembly contains sequence-based representations for the centromeres. We also expanded the number of alternate loci to create a reference that provides a more robust representation of human population variation. We demonstrate that the updates render the reference an improved annotation substrate, alter read alignments in unchanged regions, and impact variant interpretation at clinically relevant loci. We additionally evaluated a collection of new de novo long-read haploid assemblies and conclude that although the new assemblies compare favorably to the reference with respect to continuity, error rate, and gene completeness, the reference still provides the best representation for complex genomic regions and coding sequences. We assert that the collected updates in GRCh38 make the newer assembly a more robust substrate for comprehensive analyses that will promote our understanding of human biology and advance our efforts to improve health. © 2017 Schneider et al.; Published by Cold Spring Harbor Laboratory Press.

The dual role of fragments in fragment-assembly methods for de novo protein structure prediction

PubMed Central

Handl, Julia; Knowles, Joshua; Vernon, Robert; Baker, David; Lovell, Simon C.

2013-01-01

In fragment-assembly techniques for protein structure prediction, models of protein structure are assembled from fragments of known protein structures. This process is typically guided by a knowledge-based energy function and uses a heuristic optimization method. The fragments play two important roles in this process: they define the set of structural parameters available, and they also assume the role of the main variation operators that are used by the optimiser. Previous analysis has typically focused on the first of these roles. In particular, the relationship between local amino acid sequence and local protein structure has been studied by a range of authors. The correlation between the two has been shown to vary with the window length considered, and the results of these analyses have informed directly the choice of fragment length in state-of-the-art prediction techniques. Here, we focus on the second role of fragments and aim to determine the effect of fragment length from an optimization perspective. We use theoretical analyses to reveal how the size and structure of the search space changes as a function of insertion length. Furthermore, empirical analyses are used to explore additional ways in which the size of the fragment insertion influences the search both in a simulation model and for the fragment-assembly technique, Rosetta. PMID:22095594

Lipid dip-pen nanolithography on self-assembled monolayers

NASA Astrophysics Data System (ADS)

Gavutis, Martynas; Navikas, Vytautas; Rakickas, Tomas; Vaitekonis, Šarūnas; Valiokas, Ramūnas

2016-02-01

Dip-pen nanolithography (DPN) with lipids as an ink enables functional micro/nanopatterning on different substrates at high process speeds. However, only a few studies have addressed the influence of the physicochemical properties of the surface on the structure and phase behavior of DPN-printed lipid assemblies. Therefore, by combining the scanning probe and optical imaging techniques in this work we have analyzed lipid microdomain formation on the self-assembled monolayers (SAMs) on gold as well-defined model surfaces that displayed hydrophilic (protein-repellent) or hydrophobic (protein-adhesive) characteristics. We have found that on the tri(ethylene glycol)-terminated SAM the lipid ink transfer was fast (~10-1 μm3 s-1), quasi-linear and it yielded unstable, sparsely packed lipid microspots. Contrary to this, on the methyl-terminated SAM the lipid transfer was ~20 times slower, nonlinear, and the obtained stable dots of ~1 μm in diameter consisted of lipid multilayers. Our comparative analysis indicated that the measured lipid transfer was consistent with the previously reported so-called polymer transfer model (Felts et al 2012, Nanotechnology 23 215301). Further on, by employing the observed distinct contrast in the DPN ink behavior we constructed confined lipid microdomains on pre-patterned SAMs, in which the lipids assembled either into monolayer or multilamellar phases. Such microdomains can be further utilized for lipid membrane mimetics in microarray and lab-on-a-chip device formats.

On the Selective Packaging of Genomic RNA by HIV-1.

PubMed

Comas-Garcia, Mauricio; Davis, Sean R; Rein, Alan

2016-09-12

Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) selectively packages genomic RNA (gRNA) during virus assembly. However, in the absence of the gRNA, cellular messenger RNAs (mRNAs) are packaged. While the gRNA is selected because of its cis-acting packaging signal, the mechanism of this selection is not understood. The affinity of Gag (the viral structural protein) for cellular RNAs at physiological ionic strength is not much higher than that for the gRNA. However, binding to the gRNA is more salt-resistant, implying that it has a higher non-electrostatic component. We have previously studied the spacer 1 (SP1) region of Gag and showed that it can undergo a concentration-dependent conformational transition. We proposed that this transition represents the first step in assembly, i.e., the conversion of Gag to an assembly-ready state. To explain selective packaging of gRNA, we suggest here that binding of Gag to gRNA, with its high non-electrostatic component, triggers this conversion more readily than binding to other RNAs; thus we predict that a Gag-gRNA complex will nucleate particle assembly more efficiently than other Gag-RNA complexes. New data shows that among cellular mRNAs, those with long 3'-untranslated regions (UTR) are selectively packaged. It seems plausible that the 3'-UTR, a stretch of RNA not occupied by ribosomes, offers a favorable binding site for Gag.

Transcriptome sequencing and annotation of the microalgae Dunaliella tertiolecta: Pathway description and gene discovery for production of next-generation biofuels

PubMed Central

2011-01-01

Background Biodiesel or ethanol derived from lipids or starch produced by microalgae may overcome many of the sustainability challenges previously ascribed to petroleum-based fuels and first generation plant-based biofuels. The paucity of microalgae genome sequences, however, limits gene-based biofuel feedstock optimization studies. Here we describe the sequencing and de novo transcriptome assembly for the non-model microalgae species, Dunaliella tertiolecta, and identify pathways and genes of importance related to biofuel production. Results Next generation DNA pyrosequencing technology applied to D. tertiolecta transcripts produced 1,363,336 high quality reads with an average length of 400 bases. Following quality and size trimming, ~ 45% of the high quality reads were assembled into 33,307 isotigs with a 31-fold coverage and 376,482 singletons. Assembled sequences and singletons were subjected to BLAST similarity searches and annotated with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology (KO) identifiers. These analyses identified the majority of lipid and starch biosynthesis and catabolism pathways in D. tertiolecta. Conclusions The construction of metabolic pathways involved in the biosynthesis and catabolism of fatty acids, triacylglycrols, and starch in D. tertiolecta as well as the assembled transcriptome provide a foundation for the molecular genetics and functional genomics required to direct metabolic engineering efforts that seek to enhance the quantity and character of microalgae-based biofuel feedstock. PMID:21401935

Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly.

PubMed

Rebane, Aleksander A; Wang, Bigeng; Ma, Lu; Qu, Hong; Coleman, Jeff; Krishnakumar, Shyam; Rothman, James E; Zhang, Yongli

2018-02-16

Synaptic exocytosis relies on assembly of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins into a parallel four-helix bundle to drive membrane fusion. SNARE assembly occurs by stepwise zippering of the vesicle-associated SNARE (v-SNARE) onto a binary SNARE complex on the target plasma membrane (t-SNARE). Zippering begins with slow N-terminal association followed by rapid C-terminal zippering, which serves as a power stroke to drive membrane fusion. SNARE mutations have been associated with numerous diseases, especially neurological disorders. It remains unclear how these mutations affect SNARE zippering, partly due to difficulties to quantify the energetics and kinetics of SNARE assembly. Here, we used single-molecule optical tweezers to measure the assembly energy and kinetics of SNARE complexes containing single mutations I67T/N in neuronal SNARE synaptosomal-associated protein of 25kDa (SNAP-25B), which disrupt neurotransmitter release and have been implicated in neurological disorders. We found that both mutations significantly reduced the energy of C-terminal zippering by ~10 k B T, but did not affect N-terminal assembly. In addition, we observed that both mutations lead to unfolding of the C-terminal region in the t-SNARE complex. Our findings suggest that both SNAP-25B mutations impair synaptic exocytosis by destabilizing SNARE assembly, rather than stabilizing SNARE assembly as previously proposed. Therefore, our measurements provide insights into the molecular mechanism of the disease caused by SNARE mutations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modular multiaperatures for light sensors

NASA Technical Reports Server (NTRS)

Rizzo, A. A.

1977-01-01

Process involves electroplating multiaperature masks as unit, eliminating alinement and assembly difficulties previously encountered. Technique may be applied to masks in automated and surveillance light systems, when precise, wide angle field of view is needed.

When is Analysis Sufficient? A Study of how Professional Intelligence Analysts Judge Rigor

DTIC Science & Technology

2007-05-01

investors, the marketing researcher assembling an analysis of a competitor’s new products for a corporate executive, and the military analyst preparing a...previously mentioned. In all instances of analysis, the risk of shallowness is fundamental—for both the middle school student and the marketing researcher...natural gas energy policy to respond to the changing consumption of a limited resource in a dynamic energy market . The next critical facet of the

De novo assembly and phasing of a Korean human genome.

PubMed

Seo, Jeong-Sun; Rhie, Arang; Kim, Junsoo; Lee, Sangjin; Sohn, Min-Hwan; Kim, Chang-Uk; Hastie, Alex; Cao, Han; Yun, Ji-Young; Kim, Jihye; Kuk, Junho; Park, Gun Hwa; Kim, Juhyeok; Ryu, Hanna; Kim, Jongbum; Roh, Mira; Baek, Jeonghun; Hunkapiller, Michael W; Korlach, Jonas; Shin, Jong-Yeon; Kim, Changhoon

2016-10-13

Advances in genome assembly and phasing provide an opportunity to investigate the diploid architecture of the human genome and reveal the full range of structural variation across population groups. Here we report the de novo assembly and haplotype phasing of the Korean individual AK1 (ref. 1) using single-molecule real-time sequencing, next-generation mapping, microfluidics-based linked reads, and bacterial artificial chromosome (BAC) sequencing approaches. Single-molecule sequencing coupled with next-generation mapping generated a highly contiguous assembly, with a contig N50 size of 17.9 Mb and a scaffold N50 size of 44.8 Mb, resolving 8 chromosomal arms into single scaffolds. The de novo assembly, along with local assemblies and spanning long reads, closes 105 and extends into 72 out of 190 euchromatic gaps in the reference genome, adding 1.03 Mb of previously intractable sequence. High concordance between the assembly and paired-end sequences from 62,758 BAC clones provides strong support for the robustness of the assembly. We identify 18,210 structural variants by direct comparison of the assembly with the human reference, identifying thousands of breakpoints that, to our knowledge, have not been reported before. Many of the insertions are reflected in the transcriptome and are shared across the Asian population. We performed haplotype phasing of the assembly with short reads, long reads and linked reads from whole-genome sequencing and with short reads from 31,719 BAC clones, thereby achieving phased blocks with an N50 size of 11.6 Mb. Haplotigs assembled from single-molecule real-time reads assigned to haplotypes on phased blocks covered 89% of genes. The haplotigs accurately characterized the hypervariable major histocompatability complex region as well as demonstrating allele configuration in clinically relevant genes such as CYP2D6. This work presents the most contiguous diploid human genome assembly so far, with extensive investigation of unreported and Asian-specific structural variants, and high-quality haplotyping of clinically relevant alleles for precision medicine.

Tabulation as a high-resolution alternative to coarse-graining protein interactions: Initial application to virus capsid subunits

NASA Astrophysics Data System (ADS)

Spiriti, Justin; Zuckerman, Daniel M.

2015-12-01

Traditional coarse-graining based on a reduced number of interaction sites often entails a significant sacrifice of chemical accuracy. As an alternative, we present a method for simulating large systems composed of interacting macromolecules using an energy tabulation strategy previously devised for small rigid molecules or molecular fragments [S. Lettieri and D. M. Zuckerman, J. Comput. Chem. 33, 268-275 (2012); J. Spiriti and D. M. Zuckerman, J. Chem. Theory Comput. 10, 5161-5177 (2014)]. We treat proteins as rigid and construct distance and orientation-dependent tables of the interaction energy between them. Arbitrarily detailed interactions may be incorporated into the tables, but as a proof-of-principle, we tabulate a simple α-carbon Gō-like model for interactions between dimeric subunits of the hepatitis B viral capsid. This model is significantly more structurally realistic than previous models used in capsid assembly studies. We are able to increase the speed of Monte Carlo simulations by a factor of up to 6700 compared to simulations without tables, with only minimal further loss in accuracy. To obtain further enhancement of sampling, we combine tabulation with the weighted ensemble (WE) method, in which multiple parallel simulations are occasionally replicated or pruned in order to sample targeted regions of a reaction coordinate space. In the initial study reported here, WE is able to yield pathways of the final ˜25% of the assembly process.

Cellular response of human neuroblastoma cells to α-synuclein fibrils, the main constituent of Lewy bodies.

PubMed

Pieri, Laura; Chafey, Philippe; Le Gall, Morgane; Clary, Guilhem; Melki, Ronald; Redeker, Virginie

2016-01-01

α-Synuclein (α-Syn) fibrils are the main constituent of Lewy bodies and a neuropathological hallmark of Parkinson's disease (PD). The propagation of α-Syn assemblies from cell to cell suggests that they are involved in PD progression. We previously showed that α-Syn fibrils are toxic because of their ability to bind and permeabilize cell membranes. Here, we document the cellular response in terms of proteome changes of SH-SY5Y cells exposed to exogenous α-Syn fibrils. We compare the proteomes of cells of neuronal origin exposed or not either to oligomeric or fibrillar α-Syn using two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Only α-Syn fibrils induce significant changes in the proteome of SH-SY5Y cells. In addition to proteins associated to apoptosis and toxicity, or proteins previously linked to neurodegenerative diseases, we report an overexpression of proteins involved in intracellular vesicle trafficking. We also report a remarkable increase in fibrillar α-Syn heterogeneity, mainly due to C-terminal truncations. Our results show that cells of neuronal origin adapt their proteome to exogenous α-Syn fibrils and actively modify those assemblies. Cells of neuronal origin adapt their proteome to exogenous toxic α-Syn fibrils and actively modify those assemblies. Our results bring insights into the cellular response and clearance events the cells implement to face the propagation of α-Syn assemblies associated to pathology.

γ-Aminobutyric Acid Type A α4, β2, and δ Subunits Assemble to Produce More Than One Functionally Distinct Receptor Type

PubMed Central

Eaton, Megan M.; Bracamontes, John; Shu, Hong-Jin; Li, Ping; Mennerick, Steven; Steinbach, Joe Henry

2014-01-01

Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1–3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes. PMID:25238745

Coarse Grained Modeling of Block Copolymer Lithography: The Effects of Pattern Design on the Thermodynamics and Kinetics of the Directed Self Assembly of Block Copolymers

NASA Astrophysics Data System (ADS)

Garner, Grant Parker

The directed self assembly of block copolymers is an exciting complimentary technique for the fabrication of nanoscale structures for lithographic applications. Typically a directed self assembly process is driven through substrates with chemical (chemoepitaxy) or topographical (graphoepitaxy) guiding features. These patterning strategies have led to the ability to assemble structures with a high degree of perfection over large areas. However, a guiding pattern has not been created which assembles the desired features with a defect density that is commensurate with industrial standards of 1 defect/100cm 2. This work focuses on using molecular simulations on the Theoretically Informed Coarse Grained model to provide design rules for substrate patterns which drive the assembly of desired, device-oriented morphologies. Prior to the work presented in Chapter 2, the TICG model has been used in conjunction with a chemical pattern that is approximated as a hard-impenetrable surface. As many experimental systems use polymer brushes to help guide the polymer melt deposited on the substrate, this work analyzes the consequences of such an assumption by comparing a model where the polymer brush is explicitly implemented to the hard-wall substrate used in the past. Then, a methodology which utilizes a evolutionary optimization method is used to map the parameters of the more detailed model to the hard-surface model. This provides a qualitative understanding of how to interpret the model parameters used in previous works in the context of real experimental pattern designs. Chapter 3 discuss the concept of competitive assemblies in regards to defining a thermodynamic processing window in design space for assembling lines-and-spaces. The most competitive assembly to the desired orientation of the lamella is defined as a rotation of assembled lamella to the underlying pattern. Thermodynamic integration is used to calculate the free-energy difference between these assemblies over chemical patterns with varied design parameters. Local maximums in the free-energy difference are observed over pattern designs that are in qualitatively agreement with the pattern designs which produce the most perfect assemblies in experiments. The analysis is extended to study how choice of chemistry impacts this thermodynamic selection for the desired morphology. Finally, Chapter 4 provides insight into the kinetics of patterned directed self-assembly by investigating cylinder forming block copolymers within cylindrical confinements. Through the use of the string method, the minimum free-energy path between a defective state and the desired assembled morphology is calculated and clear transition states are highlighted. The effects of key parameters of the confinement design on the calculated minimum free energy path are calculated to identify design rules which should lead to a better understanding of optimal connement design for eliminating defects. In addition, a specific modification to existing cylindrical confinements is discussed as a possibility for tackling the problem of placement accuracy for a cylinder that is assembled within the confinement.

Simulation of differential die-away instrument’s response to asymmetrically burned spent nuclear fuel

DOE PAGES

Martinik, Tomas; Henzl, Vladimir; Grape, Sophie; ...

2015-03-04

Here, previous simulation studies of Differential Die–Away (DDA) instrument’s response to active interrogation of spent nuclear fuel from a pressurized water reactor (PWR) yielded promising results in terms of its capability to accurately measure or estimate basic spent fuel assembly (SFA) characteristics, such as multiplication, initial enrichment (IE) and burn-up (BU) as well as the total plutonium content. These studies were however performed only for a subset of idealized SFAs with a symmetric BU with respect to its longitudinal axis. Therefore, to complement the previous results, additional simulations have been performed of the DDA instrument’s response to interrogation of asymmetricallymore » burned spent nuclear fuel in order to determine whether detailed assay of SFAs from all 4 sides will be necessary in real life applications or whether a cost and time saving single sided assay could be used to achieve results of similar quality as previously reported in case of symmetrically burned SFAs.« less

Simulation of differential die-away instrument’s response to asymmetrically burned spent nuclear fuel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martinik, Tomas; Henzl, Vladimir; Grape, Sophie

Here, previous simulation studies of Differential Die–Away (DDA) instrument’s response to active interrogation of spent nuclear fuel from a pressurized water reactor (PWR) yielded promising results in terms of its capability to accurately measure or estimate basic spent fuel assembly (SFA) characteristics, such as multiplication, initial enrichment (IE) and burn-up (BU) as well as the total plutonium content. These studies were however performed only for a subset of idealized SFAs with a symmetric BU with respect to its longitudinal axis. Therefore, to complement the previous results, additional simulations have been performed of the DDA instrument’s response to interrogation of asymmetricallymore » burned spent nuclear fuel in order to determine whether detailed assay of SFAs from all 4 sides will be necessary in real life applications or whether a cost and time saving single sided assay could be used to achieve results of similar quality as previously reported in case of symmetrically burned SFAs.« less

Nondeterministic self-assembly of two tile types on a lattice.

PubMed

Tesoro, S; Ahnert, S E

2016-04-01

Self-assembly is ubiquitous in nature, particularly in biology, where it underlies the formation of protein quaternary structure and protein aggregation. Quaternary structure assembles deterministically and performs a wide range of important functions in the cell, whereas protein aggregation is the hallmark of a number of diseases and represents a nondeterministic self-assembly process. Here we build on previous work on a lattice model of deterministic self-assembly to investigate nondeterministic self-assembly of single lattice tiles and mixtures of two tiles at varying relative concentrations. Despite limiting the simplicity of the model to two interface types, which results in 13 topologically distinct single tiles and 106 topologically distinct sets of two tiles, we observe a wide variety of concentration-dependent behaviors. Several two-tile sets display critical behaviors in the form of a sharp transition from bound to unbound structures as the relative concentration of one tile to another increases. Other sets exhibit gradual monotonic changes in structural density, or nonmonotonic changes, while again others show no concentration dependence at all. We catalog this extensive range of behaviors and present a model that provides a reasonably good estimate of the critical concentrations for a subset of the critical transitions. In addition, we show that the structures resulting from these tile sets are fractal, with one of two different fractal dimensions.

Music is Fun.

ERIC Educational Resources Information Center

Reynolds, Charles

1983-01-01

In Anchorage, Alaska, resource teachers in the Classroom Music Program trained 450 elementary teachers to handle music in their own classrooms by means of packet-type lesson formats, a Music Jamboree cycle, and an assembly that reinforces previous teaching. (AM)

Self-assembly kinetics of microscale components: A parametric evaluation

NASA Astrophysics Data System (ADS)

Carballo, Jose M.

The goal of the present work is to develop, and evaluate a parametric model of a basic microscale Self-Assembly (SA) interaction that provides scaling predictions of process rates as a function of key process variables. At the microscale, assembly by "grasp and release" is generally challenging. Recent research efforts have proposed adapting nanoscale self-assembly (SA) processes to the microscale. SA offers the potential for reduced equipment cost and increased throughput by harnessing attractive forces (most commonly, capillary) to spontaneously assemble components. However, there are challenges for implementing microscale SA as a commercial process. The existing lack of design tools prevents simple process optimization. Previous efforts have characterized a specific aspect of the SA process. However, the existing microscale SA models do not characterize the inter-component interactions. All existing models have simplified the outcome of SA interactions as an experimentally-derived value specific to a particular configuration, instead of evaluating it outcome as a function of component level parameters (such as speed, geometry, bonding energy and direction). The present study parameterizes the outcome of interactions, and evaluates the effect of key parameters. The present work closes the gap between existing microscale SA models to add a key piece towards a complete design tool for general microscale SA process modeling. First, this work proposes a simple model for defining the probability of assembly of basic SA interactions. A basic SA interaction is defined as the event where a single part arrives on an assembly site. The model describes the probability of assembly as a function of kinetic energy, binding energy, orientation and incidence angle for the component and the assembly site. Secondly, an experimental SA system was designed, and implemented to create individual SA interactions while controlling process parameters independently. SA experiments measured the outcome of SA interactions, while studying the independent effects of each parameter. As a first step towards a complete scaling model, experiments were performed to evaluate the effects of part geometry and part travel direction under low kinetic energy conditions. Experimental results show minimal dependence of assembly yield on the incidence angle of the parts, and significant effects induced by changes in part geometry. The results from this work indicate that SA could be modeled as an energy-based process due to the small path dependence effects. Assembly probability is linearly related to the orientation probability. The proportionality constant is based on the area fraction of the sites with an amplification factor. This amplification factor accounts for the ability of capillary forces to align parts with only very small areas of contact when they have a low kinetic energy. Results provide unprecedented insight about SA interactions. The present study is a key step towards completing a basic model of a general SA process. Moreover, the outcome from this work can complement existing SA process models, in order to create a complete design tool for microscale SA systems. In addition to SA experiments, Monte Carlo simulations of experimental part-site interactions were conducted. This study confirmed that a major contributor to experimental variation is the stochastic nature of experimental SA interactions and the limited sample size of the experiments. Furthermore, the simulations serve as a tool for defining an optimum sampling strategy to minimize the uncertainty in future SA experiments.

Initial condition of stochastic self-assembly

NASA Astrophysics Data System (ADS)

Davis, Jason K.; Sindi, Suzanne S.

2016-02-01

The formation of a stable protein aggregate is regarded as the rate limiting step in the establishment of prion diseases. In these systems, once aggregates reach a critical size the growth process accelerates and thus the waiting time until the appearance of the first critically sized aggregate is a key determinant of disease onset. In addition to prion diseases, aggregation and nucleation is a central step of many physical, chemical, and biological process. Previous studies have examined the first-arrival time at a critical nucleus size during homogeneous self-assembly under the assumption that at time t =0 the system was in the all-monomer state. However, in order to compare to in vivo biological experiments where protein constituents inherited by a newly born cell likely contain intermediate aggregates, other possibilities must be considered. We consider one such possibility by conditioning the unique ergodic size distribution on subcritical aggregate sizes; this least-informed distribution is then used as an initial condition. We make the claim that this initial condition carries fewer assumptions than an all-monomer one and verify that it can yield significantly different averaged waiting times relative to the all-monomer condition under various models of assembly.

Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas reinhardtii.

PubMed

Wilson, Nedra F; Lefebvre, Paul A

2004-10-01

Chlamydomonas reinhardtii controls flagellar assembly such that flagella are of an equal and predetermined length. Previous studies demonstrated that lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), induced flagellar elongation, suggesting that a lithium-sensitive signal transduction pathway regulated flagellar length (S. Nakamura, H. Takino, and M. K. Kojima, Cell Struct. Funct. 12:369-374, 1987). Here, we demonstrate that lithium treatment depletes the pool of flagellar proteins from the cell body and that the heterotrimeric kinesin Fla10p accumulates in flagella. We identify GSK3 in Chlamydomonas and demonstrate that its kinase activity is inhibited by lithium in vitro. The tyrosine-phosphorylated, active form of GSK3 was enriched in flagella and GSK3 associated with the axoneme in a phosphorylation-dependent manner. The level of active GSK3 correlated with flagellar length; early during flagellar regeneration, active GSK3 increased over basal levels. This increase in active GSK3 was rapidly lost within 30 min of regeneration as the level of active GSK3 decreased relative to the predeflagellation level. Taken together, these results suggest a possible role for GSK3 in regulating the assembly and length of flagella.

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates*

PubMed Central

Bojja, Ravi Shankar; Andrake, Mark D.; Merkel, George; Weigand, Steven; Dunbrack, Roland L.; Skalka, Anna Marie

2013-01-01

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the “reaching dimer” previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition. PMID:23322775

Exploiting NH···Cl Hydrogen Bonding Interactions in Cooperative Metallosupramolecular Polymerization.

PubMed

Langenstroer, Anja; Dorca, Yeray; Kartha, Kalathil K; Mayoral, Maria Jose; Stepanenko, Vladimir; Fernández, Gustavo; Sánchez, Luis

2018-05-10

The self-assembly features of hydrophobic bispyridyldichlorido Pd(II) complexes, equipped with an extended aromatic surface derived from oligophenyleneethynylene (OPE) and polarizable amide functional groups, are reported. The cooperative supramolecular polymerization of these complexes results in bundles of thin fibers in which the monomer units are arranged in a translationally displaced or slipped fashion. Spectroscopic and microscopy studies reveal that these assemblies are held together by simultaneous π-stacking of the OPE moieties and NH···ClPd hydrogen bonds. These unconventional forces are often observed in crystal engineering but remain largely unexploited in supramolecular polymers. Both steric and electronic effects (the presence of bulky and polarizable metal-bound Cl ligands as well as hydrogen bonding donor NH units) prevent the establishment of short Pd-Pd contacts and strongly condition the aggregation mode of the reported complexes, in close analogy to the previously reported amphiphilic Pd(II) complex 4. The results presented herein shed light on the subtle interplay between different noncovalent interactions and their impact on the self-assembly of metallosupramolecular systems. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analytically tractable model for community ecology with many species

NASA Astrophysics Data System (ADS)

Dickens, Benjamin; Fisher, Charles K.; Mehta, Pankaj

2016-08-01

A fundamental problem in community ecology is understanding how ecological processes such as selection, drift, and immigration give rise to observed patterns in species composition and diversity. Here, we analyze a recently introduced, analytically tractable, presence-absence (PA) model for community assembly, and we use it to ask how ecological traits such as the strength of competition, the amount of diversity, and demographic and environmental stochasticity affect species composition in a community. In the PA model, species are treated as stochastic binary variables that can either be present or absent in a community: species can immigrate into the community from a regional species pool and can go extinct due to competition and stochasticity. Building upon previous work, we show that, despite its simplicity, the PA model reproduces the qualitative features of more complicated models of community assembly. In agreement with recent studies of large, competitive Lotka-Volterra systems, the PA model exhibits distinct ecological behaviors organized around a special ("critical") point corresponding to Hubbell's neutral theory of biodiversity. These results suggest that the concepts of ecological "phases" and phase diagrams can provide a powerful framework for thinking about community ecology, and that the PA model captures the essential ecological dynamics of community assembly.

The Role Of Mergers In Galaxy Formation And Transformations

NASA Astrophysics Data System (ADS)

Conselice, Christopher J.; Mundy, Carl; Duncan, Kenneth

2017-06-01

Baryonic assembly of galaxies is one of the largest questions in extragalactic studies, which relates to many other issues, including environment, feedback, star formation, gas accretion and merging. In fact, all of these processes are related and must be accounted for and understood to paint a full picture of galaxy assembly. Perhaps the most straightforward of these processes to measure are the merging and star formation histories. I will present results of combining in a new reanalysis of the three deepest and large NIR surveys take to date: UDS, Ultra-VISTA and VIDEO as part of the REFINE project. Using consistently measured stellar masses and photometric redshifts for galaxies in these fields up to z =3, I will show how the major and minor merger rate can consistently be measured across these fields. Our new method involves a full use of the PDF for photo-zs and stellar masses. We show how the merger fraction and rate are lower than previous results and the implications for this for other methods of galaxy assembly and feedback mechanisms. Invited Talk presented at the conference Galaxy Evolution Across Time, 12-16 June, Paris, France

Endogenous avian leukosis viral loci in the Red Jungle Fowl genome assembly.

PubMed

Benkel, Bernhard; Rutherford, Katherine

2014-12-01

The current build (galGal4) of the genome of the ancestor of the modern chicken, the Red Jungle Fowl, contains a single endogenous avian leukosis viral element (ALVE) on chromosome 1 (designated RSV-LTR; family ERVK). The assembly shows the ALVE provirus juxtaposed with a member of a second family of avian endogenous retroviruses (designated GGERV20; family ERVL); however, the status of the 3' end of the ALVE element as well as its flanking region remain unclear due to a gap in the reference genome sequence. In this study, we filled the gap in the assembly using a combination of long-range PCR (LR-PCR) and a short contig present in the unassembled portion of the reference genome database. Our results demonstrate that the ALVE element (ALVE-JFevB) is inserted into the putative envelope region of a GGERV20 element, roughly 1 kbp from its 3' end, and that ALVE-JFevB is complete, and depending on its expression status, potentially capable of directing the production of virus. Moreover, the unassembled portion of the genome database contains junction fragments for a second, previously characterized endogenous proviral element, ALVE-6. ©2014 Poultry Science Association Inc.

Ground states of linear rotor chains via the density matrix renormalization group

NASA Astrophysics Data System (ADS)

Iouchtchenko, Dmitri; Roy, Pierre-Nicholas

2018-04-01

In recent years, experimental techniques have enabled the creation of ultracold optical lattices of molecules and endofullerene peapod nanomolecular assemblies. It was previously suggested that the rotor model resulting from the placement of dipolar linear rotors in one-dimensional lattices at low temperature has a transition between ordered and disordered phases. We use the density matrix renormalization group (DMRG) to compute ground states of chains of up to 100 rotors and provide further evidence of the phase transition in the form of a diverging entanglement entropy. We also propose two methods and present some first steps toward rotational spectra of such molecular assemblies using DMRG. The present work showcases the power of DMRG in this new context of interacting molecular rotors and opens the door to the study of fundamental questions regarding criticality in systems with continuous degrees of freedom.

Energy level alignment of self-assembled linear chains of benzenediamine on Au(111) from first principles

DOE PAGES

Li, Guo; Rangel, Tonatiuh; Liu, Zhen -Fei; ...

2016-03-24

Using density functional theory (DFT) with van der Waals functionals, we calculate the adsorption energetics and geometry of benzenediamine (BDA) molecules on Au(111) surfaces. Our results demonstrate that the reported self-assembled linear chain structure of BDA, stabilized via hydrogen bonds between amine groups, is energetically favored over previously-studied monomeric phases. Moreover, using a model based on many-body perturbation theory within the GW approximation, we obtain approximate self-energy corrections to the DFT highest occupied molecular orbital (HOMO) energy associated with BDA adsorbate phases. As a result, we find that, independent of coverage, the HOMO energy of the linear chain phase ismore » lower relative to the Fermi energy than that of the monomer phase, and in good agreement with values measured with ultraviolet photoelectron spectroscopy and X-ray photoelectron spectroscopy.« less

Energy level alignment of self-assembled linear chains of benzenediamine on Au(111) from first principles

NASA Astrophysics Data System (ADS)

Li, Guo; Rangel, Tonatiuh; Liu, Zhen-Fei; Cooper, Valentino R.; Neaton, Jeffrey B.

2016-03-01

Using density functional theory (DFT) with a van der Waals density functional, we calculate the adsorption energetics and geometry of benzenediamine (BDA) molecules on Au(111) surfaces. Our results demonstrate that the reported self-assembled linear chain structure of BDA, stabilized via hydrogen bonds between amine groups, is energetically favored over previously studied monomeric phases. Moreover, using a model, which includes nonlocal polarization effects from the substrate and the neighboring molecules and incorporates many-body perturbation theory calculations within the GW approximation, we obtain approximate self-energy corrections to the DFT highest occupied molecular orbital (HOMO) energy associated with BDA adsorbate phases. We find that, independent of coverage, the HOMO energy of the linear chain phase is lower relative to the Fermi energy than that of the monomer phase, and in good agreement with values measured with ultraviolet photoelectron spectroscopy and x-ray photoelectron spectroscopy.

Materials science. Assembly of micro/nanomaterials into complex, three-dimensional architectures by compressive buckling.

PubMed

Xu, Sheng; Yan, Zheng; Jang, Kyung-In; Huang, Wen; Fu, Haoran; Kim, Jeonghyun; Wei, Zijun; Flavin, Matthew; McCracken, Joselle; Wang, Renhan; Badea, Adina; Liu, Yuhao; Xiao, Dongqing; Zhou, Guoyan; Lee, Jungwoo; Chung, Ha Uk; Cheng, Huanyu; Ren, Wen; Banks, Anthony; Li, Xiuling; Paik, Ungyu; Nuzzo, Ralph G; Huang, Yonggang; Zhang, Yihui; Rogers, John A

2015-01-09

Complex three-dimensional (3D) structures in biology (e.g., cytoskeletal webs, neural circuits, and vasculature networks) form naturally to provide essential functions in even the most basic forms of life. Compelling opportunities exist for analogous 3D architectures in human-made devices, but design options are constrained by existing capabilities in materials growth and assembly. We report routes to previously inaccessible classes of 3D constructs in advanced materials, including device-grade silicon. The schemes involve geometric transformation of 2D micro/nanostructures into extended 3D layouts by compressive buckling. Demonstrations include experimental and theoretical studies of more than 40 representative geometries, from single and multiple helices, toroids, and conical spirals to structures that resemble spherical baskets, cuboid cages, starbursts, flowers, scaffolds, fences, and frameworks, each with single- and/or multiple-level configurations. Copyright © 2015, American Association for the Advancement of Science.

Label-free optical imaging of membrane patches for atomic force microscopy

PubMed Central

Churnside, Allison B.; King, Gavin M.; Perkins, Thomas T.

2010-01-01

In atomic force microscopy (AFM), finding sparsely distributed regions of interest can be difficult and time-consuming. Typically, the tip is scanned until the desired object is located. This process can mechanically or chemically degrade the tip, as well as damage fragile biological samples. Protein assemblies can be detected using the back-scattered light from a focused laser beam. We previously used back-scattered light from a pair of laser foci to stabilize an AFM. In the present work, we integrate these techniques to optically image patches of purple membranes prior to AFM investigation. These rapidly acquired optical images were aligned to the subsequent AFM images to ~40 nm, since the tip position was aligned to the optical axis of the imaging laser. Thus, this label-free imaging efficiently locates sparsely distributed protein assemblies for subsequent AFM study while simultaneously minimizing degradation of the tip and the sample. PMID:21164738

Fine golden rings: Tunable surface plasmon resonance from assembled nanorods in topological defects of liquid crystals

DOE PAGES

Lee, Elaine; Xia, Yu; Ferrier, Jr., Robert C.; ...

2016-02-08

Unprecedented, reversible, and dynamic control over an assembly of gold nanorods dispersed in liquid crystals (LC) is demonstrated. The LC director field is dynamically tuned at the nanoscale using microscale ring confinement through the interplay of elastic energy at different temperatures, thus fine-tuning its core replacement energy to reversibly sequester nanoscale inclusions at the microscale. As a result, this leads to shifts of 100 nm or more in the surface plasmon resonance peak, an order of magnitude greater than any previous work with AuNR composites.

Final Scientific Report - Electromagnetic Interactions in Self-Assembled Metallo-Dielectric Biomaterials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dragnea, Bogdan G.

Achievements which resulted from previous DOE funding include: templated virus-like particle assembly thermodynamics, development of single particle photothermal absorption spectroscopy and dark- field spectroscopy instrumentation for the measurement of optical properties of virus-like nanoparticles, electromagnetic simulations of coupled nanoparticle cluster systems, virus contact mechanics, energy transfer and fluorescence quenching in multichromophore systems supported on biomolecular templates, and photo physical work on virus-aptamer systems. A current total of eight published research articles and a book chapter are acknowledging DOE support for the period 2013-2016.

Hybrid reduced order modeling for assembly calculations

DOE PAGES

Bang, Youngsuk; Abdel-Khalik, Hany S.; Jessee, Matthew A.; ...

2015-08-14

While the accuracy of assembly calculations has greatly improved due to the increase in computer power enabling more refined description of the phase space and use of more sophisticated numerical algorithms, the computational cost continues to increase which limits the full utilization of their effectiveness for routine engineering analysis. Reduced order modeling is a mathematical vehicle that scales down the dimensionality of large-scale numerical problems to enable their repeated executions on small computing environment, often available to end users. This is done by capturing the most dominant underlying relationships between the model's inputs and outputs. Previous works demonstrated the usemore » of the reduced order modeling for a single physics code, such as a radiation transport calculation. This paper extends those works to coupled code systems as currently employed in assembly calculations. Finally, numerical tests are conducted using realistic SCALE assembly models with resonance self-shielding, neutron transport, and nuclides transmutation/depletion models representing the components of the coupled code system.« less

The BOEING 777 - concurrent engineering and digital pre-assembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abarbanel, B.

The processes created on the 777 for checking designs were called {open_quotes}digital pre-assembly{close_quotes}. Using FlyThru(tm), a spin-off of a Boeing advanced computing research project, engineers were able to view up to 1500 models (15000 solids) in 3d traversing that data at high speed. FlyThru(tm) was rapidly deployed in 1991 to meet the needs of the 777 for large scale product visualization and verification. The digital pre-assembly process has bad fantastic results. The 777 has had far fewer assembly and systems problems compared to previous airplane programs. Today, FlyThru(tm) is installed on hundreds of workstations on almost every airplane program, andmore » is being used on Space Station, F22, AWACS, and other defense projects. It`s applications have gone far beyond just design review. In many ways, FlyThru is a Data Warehouse supported by advanced tools for analysis. It is today being integrated with Knowledge Based Engineering geometry generation tools.« less

In-field Calibration of a Fast Neutron Collar for the Measurement of Fresh PWR Fuel Assemblies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Swinhoe, Martyn Thomas; De Baere, Paul

2015-04-17

A new neutron collar has been designed for the measurement of fresh LEU fuel assemblies. This collar uses “fast mode” measurement to reduce the effect of burnable poison rods on the assay and thus reduce the dependence on the operator’s declaration. The new collar design reduces effect of poison rods considerably. Instead of 12 pins of 5.2% Gd causing a 20.4% effect, as in the standard thermal mode collar, they only cause a 3.2% effect in the new collar. However it has higher efficiency so that reasonably precise measurements can be made in 25 minutes, rather than the 1 hourmore » of previous collars. The new collar is fully compatible with the use of the standard data collection and analysis code INCC. This report describes the calibration that was made with a mock-up assembly at Los Alamos National Laboratory and with actual assemblies at the AREVA Fuel fabrication Plant in Lingen, Germany.« less

Architecture of the pontin/reptin complex, essential in the assembly of several macromolecular complexes

PubMed Central

Torreira, Eva; Jha, Sudhakar; López-Blanco, José R.; Arias-Palomo, Ernesto; Chacón, Pablo; Cañas, Cristina; Ayora, Sylvia; Dutta, Anindya; Llorca, Oscar

2008-01-01

Summary Pontin and reptin belong to the AAA+ family and they are essential for the structural integrity and catalytic activity of several chromatin remodeling complexes. They are also indispensable for the assembly of several ribonucleoprotein complexes, including telomerase. Here, we propose a structural model of the yeast pontin/reptin complex based on a cryo-electron microscopy reconstruction at 13 Å. Pontin/reptin hetero-dodecamers were purified from in vivo assembled complexes forming a double ring. Two rings interact through flexible domains projecting from each hexamer, constituting an atypical asymmetric form of oligomerization. These flexible domains and the AAA+ cores reveal significant conformational changes when compared to the crystal structure of human pontin that generate enlarged channels. This structure of endogenously assembled pontin/reptin complexes is different to previously described structures, suggesting that pontin and reptin could acquire distinct structural states to regulate their broad functions as molecular motors and scaffolds for nucleic acids and proteins. PMID:18940606

Alteration of in vivo cellulose ribbon assembly by carboxymethylcellulose and other cellulose derivatives.

PubMed

Haigler, C H; White, A R; Brown, R M; Cooper, K M

1982-07-01

In vivo cellulose ribbon assembly by the Gram-negative bacterium Acetobacter xylinum can be altered by incubation in carboxymethylcellulose (CMC), a negatively charged water-soluble cellulose derivative, and also by incubation in a variety of neutral, water-soluble cellulose derivatives. In the presence of all of these substituted celluloses, normal fasciation of microfibril bundles to form the typical twisting ribbon is prevented. Alteration of ribbon assembly is most extensive in the presence of CMC, which often induces synthesis of separate, intertwining bundles of microfibrils. Freeze-etch preparations of the bacterial outer membrane suggest that particles that are thought to be associated with cellulose synthesis or extrusion may be specifically organized to mediate synthesis of microfibril bundles. These data support the previous hypothesis that the cellulose ribbon of A. xylinum is formed by a hierarchical, cell-directed, self-assembly process. The relationship of these results to the regulation of cellulose microfibril size and wall extensibility in plant cell walls is discussed.

Enabling large-scale next-generation sequence assembly with Blacklight

PubMed Central

Couger, M. Brian; Pipes, Lenore; Squina, Fabio; Prade, Rolf; Siepel, Adam; Palermo, Robert; Katze, Michael G.; Mason, Christopher E.; Blood, Philip D.

2014-01-01

Summary A variety of extremely challenging biological sequence analyses were conducted on the XSEDE large shared memory resource Blacklight, using current bioinformatics tools and encompassing a wide range of scientific applications. These include genomic sequence assembly, very large metagenomic sequence assembly, transcriptome assembly, and sequencing error correction. The data sets used in these analyses included uncategorized fungal species, reference microbial data, very large soil and human gut microbiome sequence data, and primate transcriptomes, composed of both short-read and long-read sequence data. A new parallel command execution program was developed on the Blacklight resource to handle some of these analyses. These results, initially reported previously at XSEDE13 and expanded here, represent significant advances for their respective scientific communities. The breadth and depth of the results achieved demonstrate the ease of use, versatility, and unique capabilities of the Blacklight XSEDE resource for scientific analysis of genomic and transcriptomic sequence data, and the power of these resources, together with XSEDE support, in meeting the most challenging scientific problems. PMID:25294974

Light-assisted, templated self-assembly of gold nanoparticle chains.

PubMed

Jaquay, Eric; Martínez, Luis Javier; Huang, Ningfeng; Mejia, Camilo A; Sarkar, Debarghya; Povinelli, Michelle L

2014-09-10

We experimentally demonstrate the technique of light-assisted, templated self-assembly (LATS) to trap and assemble 200 nm diameter gold nanoparticles. We excite a guided-resonance mode of a photonic-crystal slab with 1.55 μm laser light to create an array of optical traps. Unlike our previous demonstration of LATS with polystyrene particles, we find that the interparticle interactions play a significant role in the resulting particle patterns. Despite a two-dimensionally periodic intensity profile in the slab, the particles form one-dimensional chains whose orientations can be controlled by the incident polarization of the light. The formation of chains can be understood in terms of a competition between the gradient force due to the excitation of the mode in the slab and optical binding between particles.

Tunable porous nanoallotropes prepared by post-assembly etching of binary nanoparticle superlattices

NASA Astrophysics Data System (ADS)

Udayabhaskararao, Thumu; Altantzis, Thomas; Houben, Lothar; Coronado-Puchau, Marc; Langer, Judith; Popovitz-Biro, Ronit; Liz-Marzán, Luis M.; Vuković, Lela; Král, Petr; Bals, Sara; Klajn, Rafal

2017-10-01

Self-assembly of inorganic nanoparticles has been used to prepare hundreds of different colloidal crystals, but almost invariably with the restriction that the particles must be densely packed. Here, we show that non-close-packed nanoparticle arrays can be fabricated through the selective removal of one of two components comprising binary nanoparticle superlattices. First, a variety of binary nanoparticle superlattices were prepared at the liquid-air interface, including several arrangements that were previously unknown. Molecular dynamics simulations revealed the particular role of the liquid in templating the formation of superlattices not achievable through self-assembly in bulk solution. Second, upon stabilization, all of these binary superlattices could be transformed into distinct “nanoallotropes”—nanoporous materials having the same chemical composition but differing in their nanoscale architectures.

Development of a modularized two-step (M2S) chromosome integration technique for integration of multiple transcription units in Saccharomyces cerevisiae.

PubMed

Li, Siwei; Ding, Wentao; Zhang, Xueli; Jiang, Huifeng; Bi, Changhao

2016-01-01

Saccharomyces cerevisiae has already been used for heterologous production of fuel chemicals and valuable natural products. The establishment of complicated heterologous biosynthetic pathways in S. cerevisiae became the research focus of Synthetic Biology and Metabolic Engineering. Thus, simple and efficient genomic integration techniques of large number of transcription units are demanded urgently. An efficient DNA assembly and chromosomal integration method was created by combining homologous recombination (HR) in S. cerevisiae and Golden Gate DNA assembly method, designated as modularized two-step (M2S) technique. Two major assembly steps are performed consecutively to integrate multiple transcription units simultaneously. In Step 1, Modularized scaffold containing a head-to-head promoter module and a pair of terminators was assembled with two genes. Thus, two transcription units were assembled with Golden Gate method into one scaffold in one reaction. In Step 2, the two transcription units were mixed with modules of selective markers and integration sites and transformed into S. cerevisiae for assembly and integration. In both steps, universal primers were designed for identification of correct clones. Establishment of a functional β-carotene biosynthetic pathway in S. cerevisiae within 5 days demonstrated high efficiency of this method, and a 10-transcriptional-unit pathway integration illustrated the capacity of this method. Modular design of transcription units and integration elements simplified assembly and integration procedure, and eliminated frequent designing and synthesis of DNA fragments in previous methods. Also, by assembling most parts in Step 1 in vitro, the number of DNA cassettes for homologous integration in Step 2 was significantly reduced. Thus, high assembly efficiency, high integration capacity, and low error rate were achieved.

Single-molecule sequencing and Hi-C-based proximity-guided assembly of amaranth (Amaranthus hypochondriacus) chromosomes provide insights into genome evolution.

PubMed

Lightfoot, D J; Jarvis, D E; Ramaraj, T; Lee, R; Jellen, E N; Maughan, P J

2017-08-31

Amaranth (Amaranthus hypochondriacus) was a food staple among the ancient civilizations of Central and South America that has recently received increased attention due to the high nutritional value of the seeds, with the potential to help alleviate malnutrition and food security concerns, particularly in arid and semiarid regions of the developing world. Here, we present a reference-quality assembly of the amaranth genome which will assist the agronomic development of the species. Utilizing single-molecule, real-time sequencing (Pacific Biosciences) and chromatin interaction mapping (Hi-C) to close assembly gaps and scaffold contigs, respectively, we improved our previously reported Illumina-based assembly to produce a chromosome-scale assembly with a scaffold N50 of 24.4 Mb. The 16 largest scaffolds contain 98% of the assembly and likely represent the haploid chromosomes (n = 16). To demonstrate the accuracy and utility of this approach, we produced physical and genetic maps and identified candidate genes for the betalain pigmentation pathway. The chromosome-scale assembly facilitated a genome-wide syntenic comparison of amaranth with other Amaranthaceae species, revealing chromosome loss and fusion events in amaranth that explain the reduction from the ancestral haploid chromosome number (n = 18) for a tetraploid member of the Amaranthaceae. The assembly method reported here minimizes cost by relying primarily on short-read technology and is one of the first reported uses of in vivo Hi-C for assembly of a plant genome. Our analyses implicate chromosome loss and fusion as major evolutionary events in the 2n = 32 amaranths and clearly establish the homoeologous relationship among most of the subgenome chromosomes, which will facilitate future investigations of intragenomic changes that occurred post polyploidization.

Cytoplasmic Motifs in the Nipah Virus Fusion Protein Modulate Virus Particle Assembly and Egress.

PubMed

Johnston, Gunner P; Contreras, Erik M; Dabundo, Jeffrey; Henderson, Bryce A; Matz, Keesha M; Ortega, Victoria; Ramirez, Alfredo; Park, Arnold; Aguilar, Hector C

2017-05-15

Nipah virus (NiV), a paramyxovirus in the genus Henipavirus , has a mortality rate in humans of approximately 75%. While several studies have begun our understanding of NiV particle formation, the mechanism of this process remains to be fully elucidated. For many paramyxoviruses, M proteins drive viral assembly and egress; however, some paramyxoviral glycoproteins have been reported as important or essential in budding. For NiV the matrix protein (M), the fusion glycoprotein (F) and, to a much lesser extent, the attachment glycoprotein (G) autonomously induce the formation of virus-like particles (VLPs). However, functional interactions between these proteins during assembly and egress remain to be fully understood. Moreover, if the F-driven formation of VLPs occurs through interactions with host cell machinery, the cytoplasmic tail (CT) of F is a likely interactive domain. Therefore, we analyzed NiV F CT deletion and alanine mutants and report that several but not all regions of the F CT are necessary for efficient VLP formation. Two of these regions contain YXXØ or dityrosine motifs previously shown to interact with cellular machinery involved in F endocytosis and transport. Importantly, our results showed that F-driven, M-driven, and M/F-driven viral particle formation enhanced the recruitment of G into VLPs. By identifying key motifs, specific residues, and functional viral protein interactions important for VLP formation, we improve our understanding of the viral assembly/egress process and point to potential interactions with host cell machinery. IMPORTANCE Henipaviruses can cause deadly infections of medical, veterinary, and agricultural importance. With recent discoveries of new henipa-like viruses, understanding the mechanisms by which these viruses reproduce is paramount. We have focused this study on identifying the functional interactions of three Nipah virus proteins during viral assembly and particularly on the role of one of these proteins, the fusion glycoprotein, in the incorporation of other viral proteins into viral particles. By identifying several regions in the fusion glycoprotein that drive viral assembly, we further our understanding of how these viruses assemble and egress from infected cells. The results presented will likely be useful toward designing treatments targeting this aspect of the viral life cycle and for the production of new viral particle-based vaccines. Copyright © 2017 American Society for Microbiology.

iDoComp: a compression scheme for assembled genomes

PubMed Central

Ochoa, Idoia; Hernaez, Mikel; Weissman, Tsachy

2015-01-01

Motivation: With the release of the latest next-generation sequencing (NGS) machine, the HiSeq X by Illumina, the cost of sequencing a Human has dropped to a mere $4000. Thus we are approaching a milestone in the sequencing history, known as the $1000 genome era, where the sequencing of individuals is affordable, opening the doors to effective personalized medicine. Massive generation of genomic data, including assembled genomes, is expected in the following years. There is crucial need for compression of genomes guaranteed of performing well simultaneously on different species, from simple bacteria to humans, which will ease their transmission, dissemination and analysis. Further, most of the new genomes to be compressed will correspond to individuals of a species from which a reference already exists on the database. Thus, it is natural to propose compression schemes that assume and exploit the availability of such references. Results: We propose iDoComp, a compressor of assembled genomes presented in FASTA format that compresses an individual genome using a reference genome for both the compression and the decompression. In terms of compression efficiency, iDoComp outperforms previously proposed algorithms in most of the studied cases, with comparable or better running time. For example, we observe compression gains of up to 60% in several cases, including H.sapiens data, when comparing with the best compression performance among the previously proposed algorithms. Availability: iDoComp is written in C and can be downloaded from: http://www.stanford.edu/~iochoa/iDoComp.html (We also provide a full explanation on how to run the program and an example with all the necessary files to run it.). Contact: iochoa@stanford.edu Supplementary information: Supplementary Data are available at Bioinformatics online. PMID:25344501

Integrin alphaIIb-subunit cytoplasmic domain mutations demonstrate a requirement for tyrosine phosphorylation of beta3-subunits in actin cytoskeletal organization.

PubMed

Yamodo, Innocent H; Blystone, Scott D

2004-01-01

Using truncated or mutated alphaIIb integrin cytoplasmic domains fused to the alphaV extracellular domain and expressed with the beta3 integrin subunit, we demonstrate that the double mutation of proline residues 998 and 999 to alanine (PP998/999AA), previously shown to disturb the C-terminal conformation of the alphaIIb integrin cytoplasmic domain, prevents tyrosine phosphorylation of beta3 integrin induced by Arg-Gly-Asp peptide ligation. This mutation also inhibits integrin mediated actin assembly and cell adhesion to vitronectin. In contrast, progressive truncation of the alphaIIb-subunit cytoplasmic domain did not reproduce these effects. Interestingly, the PP998/999AA mutations of alphaIIb did not affect beta3 tyrosine phosphorylation, cell adhesion, or actin polymerization induced by manganese. Exogenous addition of manganese was sufficient to rescue beta3 phosphorylation, cell adhesion, and actin assembly in cells expressing the PP998/999AA mutation when presented with a vitronectin substrate. Further, induction of the high affinity conformation of this mutant beta3 integrin by incubation with either Arg-Gly-Asp peptide or exogenous manganese was equivalent. These results suggest that the extracellular structure of beta3 integrins in the high affinity conformation is not directly related to the structure of the cytoplasmic face of the integrin. Moreover, the requirement for beta3 phosphorylation is demonstrated without mutation of the beta3 subunit. In support of our previous hypothesis of a role for beta3 phosphorylation in adhesion, these studies demonstrate a strong correlation between beta3 tyrosine phosphorylation and assembly of a cytoskeleton competent to support firm cell adhesion.

Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

PubMed Central

Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

2016-01-01

The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096

The Mechanisms of Aberrant Protein Aggregation

NASA Astrophysics Data System (ADS)

Cohen, Samuel; Vendruscolo, Michele; Dobson, Chris; Knowles, Tuomas

2012-02-01

We discuss the development of a kinetic theory for understanding the aberrant loss of solubility of proteins. The failure to maintain protein solubility results often in the assembly of organized linear structures, commonly known as amyloid fibrils, the formation of which is associated with over 50 clinical disorders including Alzheimer's and Parkinson's diseases. A true microscopic understanding of the mechanisms that drive these aggregation processes has proved difficult to achieve. To address this challenge, we apply the methodologies of chemical kinetics to the biomolecular self-assembly pathways related to protein aggregation. We discuss the relevant master equation and analytical approaches to studying it. In particular, we derive the underlying rate laws in closed-form using a self-consistent solution scheme; the solutions that we obtain reveal scaling behaviors that are very generally present in systems of growing linear aggregates, and, moreover, provide a general route through which to relate experimental measurements to mechanistic information. We conclude by outlining a study of the aggregation of the Alzheimer's amyloid-beta peptide. The study identifies the dominant microscopic mechanism of aggregation and reveals previously unidentified therapeutic strategies.

Mars exploration study workshop 2

NASA Astrophysics Data System (ADS)

Duke, Michael B.; Budden, Nancy Ann

1993-11-01

A year-long NASA-wide study effort has led to the development of an innovative strategy for the human exploration of Mars. The latest Mars Exploration Study Workshop 2 advanced a design reference mission (DRM) that significantly reduces the perceived high costs, complex infrastructure, and long schedules associated with previous Mars scenarios. This surface-oriented philosophy emphasizes the development of high-leveraging surface technologies in lieu of concentrating exclusively on space transportation technologies and development strategies. As a result of the DRM's balanced approach to mission and crew risk, element commonality, and technology development, human missions to Mars can be accomplished without the need for complex assembly operations in low-Earth orbit. This report, which summarizes the Mars Exploration Study Workshop held at the Ames Research Center on May 24-25, 1993, provides an overview of the status of the Mars Exploration Study, material presented at the workshop, and discussions of open items being addressed by the study team. The workshop assembled three teams of experts to discuss cost, dual-use technology, and international involvement, and to generate a working group white paper addressing these issues. The three position papers which were generated are included in section three of this publication.

Mars exploration study workshop 2

NASA Technical Reports Server (NTRS)

Duke, Michael B.; Budden, Nancy Ann

1993-01-01

A year-long NASA-wide study effort has led to the development of an innovative strategy for the human exploration of Mars. The latest Mars Exploration Study Workshop 2 advanced a design reference mission (DRM) that significantly reduces the perceived high costs, complex infrastructure, and long schedules associated with previous Mars scenarios. This surface-oriented philosophy emphasizes the development of high-leveraging surface technologies in lieu of concentrating exclusively on space transportation technologies and development strategies. As a result of the DRM's balanced approach to mission and crew risk, element commonality, and technology development, human missions to Mars can be accomplished without the need for complex assembly operations in low-Earth orbit. This report, which summarizes the Mars Exploration Study Workshop held at the Ames Research Center on May 24-25, 1993, provides an overview of the status of the Mars Exploration Study, material presented at the workshop, and discussions of open items being addressed by the study team. The workshop assembled three teams of experts to discuss cost, dual-use technology, and international involvement, and to generate a working group white paper addressing these issues. The three position papers which were generated are included in section three of this publication.

Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing.

PubMed

Jäger, Marten; Ott, Claus-Eric; Grünhagen, Johannes; Hecht, Jochen; Schell, Hanna; Mundlos, Stefan; Duda, Georg N; Robinson, Peter N; Lienau, Jasmin

2011-03-24

The sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences. Prior to our study, mRNA sequences for only 1,556 partial or complete ovine genes were publicly available. Therefore, we developed a composite de novo transcriptome assembly method for next-generation sequence data to combine known ovine mRNA and EST sequences, mRNA sequences from mouse and cow, and sequences assembled de novo from short read RNA-Seq data into a composite reference transcriptome, and identified transcripts from over 12 thousand previously undescribed ovine genes. Gene expression analysis based on these data revealed substantially different expression profiles in standard versus delayed bone healing in an ovine tibial osteotomy model. Hundreds of transcripts were differentially expressed between standard and delayed healing and between the time points of the standard and delayed healing groups. We used the sheep sequences to design quantitative RT-PCR assays with which we validated the differential expression of 26 genes that had been identified by RNA-seq analysis. A number of clusters of characteristic expression profiles could be identified, some of which showed striking differences between the standard and delayed healing groups. Gene Ontology (GO) analysis showed that the differentially expressed genes were enriched in terms including extracellular matrix, cartilage development, contractile fiber, and chemokine activity. Our results provide a first atlas of gene expression profiles and differentially expressed genes in standard and delayed bone healing in a large-animal model and provide a number of clues as to the shifts in gene expression that underlie delayed bone healing. In the course of our study, we identified transcripts of 13,987 ovine genes, including 12,431 genes for which no sequence information was previously available. This information will provide a basis for future molecular research involving the sheep as a model organism.

Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing

PubMed Central

2011-01-01

Background The sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences. Results Prior to our study, mRNA sequences for only 1,556 partial or complete ovine genes were publicly available. Therefore, we developed a composite de novo transcriptome assembly method for next-generation sequence data to combine known ovine mRNA and EST sequences, mRNA sequences from mouse and cow, and sequences assembled de novo from short read RNA-Seq data into a composite reference transcriptome, and identified transcripts from over 12 thousand previously undescribed ovine genes. Gene expression analysis based on these data revealed substantially different expression profiles in standard versus delayed bone healing in an ovine tibial osteotomy model. Hundreds of transcripts were differentially expressed between standard and delayed healing and between the time points of the standard and delayed healing groups. We used the sheep sequences to design quantitative RT-PCR assays with which we validated the differential expression of 26 genes that had been identified by RNA-seq analysis. A number of clusters of characteristic expression profiles could be identified, some of which showed striking differences between the standard and delayed healing groups. Gene Ontology (GO) analysis showed that the differentially expressed genes were enriched in terms including extracellular matrix, cartilage development, contractile fiber, and chemokine activity. Conclusions Our results provide a first atlas of gene expression profiles and differentially expressed genes in standard and delayed bone healing in a large-animal model and provide a number of clues as to the shifts in gene expression that underlie delayed bone healing. In the course of our study, we identified transcripts of 13,987 ovine genes, including 12,431 genes for which no sequence information was previously available. This information will provide a basis for future molecular research involving the sheep as a model organism. PMID:21435219

Formation and properties of surface-anchored polymer assemblies with tunable physico-chemical characteristics

NASA Astrophysics Data System (ADS)

Wu, Tao

We describe two new methodologies leading to the formation of novel surface-anchored polymer assemblies on solid substrates. While the main goal is to understand the fundamentals pertaining to the preparation and properties of the surface-bound polymer assemblies (including neutral and chargeable polymers), several examples also are mentioned throughout the Thesis that point out to practical applications of such structures. The first method is based on generating assemblies comprising anchored polymers with a gradual variation of grafting densities on solid substrates. These structures are prepared by first covering the substrate with a molecular gradient of the polymerization initiator, followed by polymerization from these substrate-bound initiator centers ("grafting from"). We apply this technique to prepare grafting density gradients of poly(acryl amide) (PAAm) and poly(acrylic acid) (PAA) on silica-covered substrates. We show that using the grafting density gradient geometry, the characteristics of surface-anchored polymers in both the low grafting density ("mushroom") regime as well as the high grafting density ("brush") regime can be accessed conveniently on a single sample. We use a battery of experimental methods, including Fourier transform infrared spectroscopy (FTIR), Near-edge absorption fine structure spectroscopy (NEXAFS), contact angle, ellipsometry, to study the characteristics of the surface-bound polymer layers. We also probe the scaling laws of neutral polymer as a function of grafting density, and for weak polyelectrolyte, in addition to the grafting density, we study the affect of solution ionic strength and pH values. In the second novel method, which we coined as "mechanically assisted polymer assembly" (MAPA), we form surface anchored polymers by "grafting from" polymerization initiators deposited on elastic surfaces that have been previously extended uniaxially by a certain length increment, Deltax. Upon releasing the strain in the substrate after completion of polymerization, we show the grafting density of the polymers grafted to flexible substrates can be tuned as a function of Deltax.

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro.

PubMed

Cherwa, James E; Tyson, Joshua; Bedwell, Gregory J; Brooke, Dewey; Edwards, Ashton G; Dokland, Terje; Prevelige, Peter E; Fane, Bentley A

2017-01-01

During ϕX174 morphogenesis, 240 copies of the external scaffolding protein D organize 12 pentameric assembly intermediates into procapsids, a reaction reconstituted in vitro In previous studies, ϕX174 strains resistant to exogenously expressed dominant lethal D genes were experimentally evolved. Resistance was achieved by the stepwise acquisition of coat protein mutations. Once resistance was established, a stimulatory D protein mutation that greatly increased strain fitness arose. In this study, in vitro biophysical and biochemical methods were utilized to elucidate the mechanistic details and evolutionary trade-offs created by the resistance mutations. The kinetics of procapsid formation was analyzed in vitro using wild-type, inhibitory, and experimentally evolved coat and scaffolding proteins. Our data suggest that viral fitness is correlated with in vitro assembly kinetics and demonstrate that in vivo experimental evolution can be analyzed within an in vitro biophysical context. Experimental evolution is an extremely valuable tool. Comparisons between ancestral and evolved genotypes suggest hypotheses regarding adaptive mechanisms. However, it is not always possible to rigorously test these hypotheses in vivo We applied in vitro biophysical and biochemical methods to elucidate the mechanistic details that allowed an experimentally evolved virus to become resistant to an antiviral protein and then evolve a productive use for that protein. Moreover, our results indicate that the respective roles of scaffolding and coat proteins may have been redistributed during the evolution of a two-scaffolding-protein system. In one-scaffolding-protein virus assembly systems, coat proteins promiscuously interact to form heterogeneous aberrant structures in the absence of scaffolding proteins. Thus, the scaffolding protein controls fidelity. During ϕX174 assembly, the external scaffolding protein acts like a coat protein, self-associating into large aberrant spherical structures in the absence of coat protein, whereas the coat protein appears to control fidelity. Copyright © 2016 American Society for Microbiology.

Soil Type Has a Stronger Role than Dipterocarp Host Species in Shaping the Ectomycorrhizal Fungal Community in a Bornean Lowland Tropical Rain Forest

PubMed Central

Essene, Adam L.; Shek, Katherine L.; Lewis, J. D.; Peay, Kabir G.; McGuire, Krista L.

2017-01-01

The role that mycorrhizal fungal associations play in the assembly of long-lived tree communities is poorly understood, especially in tropical forests, which have the highest tree diversity of any ecosystem. The lowland tropical rain forests of Southeast Asia are characterized by high levels of species richness within the family Dipterocarpaceae, the entirety of which has been shown to form obligate ectomycorrhizal (ECM) fungal associations. Differences in ECM assembly between co-occurring species of dipterocarp have been suggested, but never tested in adult trees, as a mechanism for maintaining the coexistence of closely related tree species in this family. Testing this hypothesis has proven difficult because the assembly of both dipterocarps and their ECM associates co-varies with the same edaphic variables. In this study, we used high-throughput DNA sequencing of soils and Sanger sequencing of root tips to evaluate how ECM fungi were structured within and across a clay–sand soil nutrient ecotone in a mixed-dipterocarp rain forest in Malaysian Borneo. We compared assembly patterns of ECM fungi in bulk soil to ECM root tips collected from three ecologically distinct species of dipterocarp. This design allowed us to test whether ECM fungi are more strongly structured by soil type or host specificity. As with previous studies of ECM fungi on this plot, we observed that clay vs. sand soil type strongly structured both the bulk soil and root tip ECM fungal communities. However, we also observed significantly different ECM communities associated with two of the three dipterocarp species evaluated on this plot. These results suggest that ECM fungal assembly on these species is shaped by a combination of biotic and abiotic factors, and that the soil edaphic niche occupied by different dipterocarp species may be mediated by distinct ECM fungal assemblages. PMID:29163567

Lessons Learned from the California Drought (1987-1992). National Study of Water Management During Drought

DTIC Science & Technology

1993-09-01

are followed (p. 192). * Market forces are an effective wa) of reallocating restricted water supplies (p. 193). xix TABLE 1 (Continued) SUMMARY OF...LESSNS LEARNED Confirmed Lessons of Previous Droughts "* Water in the aquifers continues to be the most effective strategic weapon against drought (p. 194...Water Code. This act was known as Assembly Bill (AB) 797 and became effective on January 1, 1984. The 300 urban agencies that have to prepare and adopt

Extension of the ACE solar panels is tested in SAEF-II

NASA Technical Reports Server (NTRS)

1997-01-01

Extension of the solar panels is tested on the Advanced Composition Explorer (ACE) spacecraft in KSC's Spacecraft Assembly and Encapsulation Facility-II (SAEF-II). Scheduled for launch on a Delta II rocket from Cape Canaveral Air Station on Aug. 25, ACE will study low-energy particles of solar origin and high-energy galactic particles. The collecting power of instruments aboard ACE is 10 to 1,000 times greater than anything previously flown to collect similar data by NASA.

KSC-97PC1232

NASA Image and Video Library

1997-08-13

In KSC’s Spacecraft Assembly and Encapsulation Facility-II (SAEF-II), the Advanced Composition Explorer (ACE) spacecraft is encapsulated and placed into the transporter which will move it to Launch Complex 17A. Scheduled for launch on a Delta II rocket from Cape Canaveral Air Station on Aug. 24, ACE will study low-energy particles of solar origin and high-energy galactic particles. The collecting power of instruments aboard ACE is 10 to 1,000 times greater than anything previously flown to collect similar data by NASA

KSC-97PC1234

NASA Image and Video Library

1997-08-13

In KSC’s Spacecraft Assembly and Encapsulation Facility-II (SAEF-II), the Advanced Composition Explorer (ACE) spacecraft is encapsulated and placed into the transporter which will move it to Launch Complex 17A. Scheduled for launch on a Delta II rocket from Cape Canaveral Air Station on Aug. 24, ACE will study low-energy particles of solar origin and high-energy galactic particles. The collecting power of instruments aboard ACE is 10 to 1,000 times greater than anything previously flown to collect similar data by NASA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krauss, Sharon Wald; Chen, Cynthia; Penman, Sheldon

Structural protein 4.1, which has crucial interactions within the spectin-actin lattice of the human red cell membrane skeleton, also is widely distributed at diverse intracellular sites in nucleated cells. We previously showed that 4.1 is essential for assembly of functional nuclei in vitro and that the capacity of 4.1 to bind actin is required. Here we report that 4.1 and actin colocalize in mammalian cell nuclei using fluorescence microscopy and, by higher resolution cell whole mount electron microscopy, are associated on nuclear filaments. We also devised a cell-free assay using Xenopus egg extract containing fluorescent actin to follow actin duringmore » nuclear assembly. By directly imaging actin under non-perturbing conditions, the total nuclear actin population is retained and is visualized in situ relative to intact chromatin. We detected actin initially when chromatin and nuclear pores began assembling. As the nuclear lamina assembled, but preceding DNA synthesis, a discrete actin network formed throughout the nucleus. Protein 4.1 epitopes also were detected when actin began to accumulate in nuclei, producing a diffuse coincident pattern. As nuclei matured, actin was detected both coincident with and also independent of 4.1 epitopes. To test whether acquisition of nuclear actin is required for nuclear assembly, the actin inhibitor latrunculin A was added to Xenopus egg extracts during nuclear assembly. Latrunculin A strongly perturbed nuclear assembly and produced distorted nuclear structures containing neither actin nor protein 4.1. Our results suggest that actin as well as 4.1 is necessary for nuclear assembly and that 4.1-actin interactions may be critical.« less

Peptide assemblies: from cell scaffolds to immune adjuvants

NASA Astrophysics Data System (ADS)

Collier, Joel

2011-03-01

This talk will discuss two interrelated aspects of peptide self-assemblies in biological applications: their use as matrices for regenerative medicine, and their use as chemically defined adjuvants for directing immune responses against engineered antigens. In the first half of the presentation, the design of peptide self-assemblies as analogues for the extracellular matrix will be described, with a focus on self-assemblies displaying multiple different cell-binding peptides. We conducted multi-factorial investigations of peptide co-assemblies containing several different ligand-bearing peptides using statistical ``design of experiments'' (DoE). Using the DoE techniques of factorial experimentation and response surface modeling, we systematically explored how precise combinations of ligand-bearing peptides modulated endothelial cell growth, in the process finding interactions between ligands not previously appreciated. By investigating immune responses against the materials intended for tissue engineering applications, we discovered that the basic self-assembling peptides were minimally immunogenic or non-immunogenic, even when delivered in strong adjuvants. -But when they were appended to an appropriately restricted epitope peptide, these materials raised strong and persistent antibody responses. These responses were dependent on covalent conjugation between the epitope and self-assembling domains of the peptides, were mediated by T cells, and could be directed towards both peptide epitopes and conjugated protein antigens. In addition to their demonstrated utility as scaffolds for regenerative medicine, peptide self-assemblies may also be useful as chemically defined adjuvants for vaccines and immunotherapies. This work was funded by NIH/NIDCR (1 R21 DE017703-03), NIH/NIBIB (1 R01 EB009701-01), and NSF (CHE-0802286).

This paper is the winner of an SFB Award in the Hospital Intern, Residency category: Peptide biomaterials raising adaptive immune responses in wound healing contexts.

PubMed

Vigneswaran, Yalini; Han, Huifang; De Loera, Roberto; Wen, Yi; Zhang, Xing; Sun, Tao; Mora-Solano, Carolina; Collier, Joel H

2016-08-01

Biomaterials used in the context of tissue engineering or wound repair are commonly designed to be "nonimmunogenic." However, previously it has been observed that self-assembled peptide nanofiber materials are noninflammatory despite their immunogenicity, suggesting that they may be appropriate for use in wound-healing contexts. To test this hypothesis, mice were immunized with epitope-containing peptide self-assemblies until they maintained high antibody titers against the material, then gels of the same peptide assemblies were applied within full-thickness dermal wounds. In three different murine dermal-wounding models with different baseline healing rates, even significantly immunogenic peptide assemblies did not delay healing. Conversely, adjuvanted peptide assemblies, while raising similar antibody titers to unadjuvanted assemblies, did delay wound healing. Analysis of the healing wounds indicated that compared to adjuvanted peptide assemblies, the unadjuvanted assemblies exhibited a progression of the dominant T-cell subset from CD4(+) to CD8(+) cells in the wound, and CD4(+) cell populations displayed a more Th2-slanted response. These findings illustrate an example of a significant antibiomaterial adaptive immune response that does not adversely affect wound healing despite ongoing antibody production. This material would thus be considered "immunologically compatible" in this specific context rather than "nonimmunogenic," a designation that is expected to apply to a range of other protein- and peptide-based biomaterials in wound-healing and tissue-engineering applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1853-1862, 2016. © 2016 Wiley Periodicals, Inc.

Effect of Control Blade History, and Axial Coolant Density and Burnup Profiles on BWR Burnup Credit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marshall, William BJ J

2016-01-01

A technical basis for peak reactivity boiling water reactor (BWR) burnup credit (BUC) methods was recently generated, and the technical basis for extended BWR BUC is now being developed. In this paper, a number of effects related to extended BWR BUC are analyzed, including three major operational effects in BWRs: the coolant density axial distribution, the use of control blades during operation, and the axial burnup profile. Specifically, uniform axial moderator density profiles are analyzed and compared to previous results and an additional temporal fidelity study combing moderator density profiles for three different fuel assemblies is presented. Realistic control blademore » histories and cask criticality results are compared to previously generated constructed control blade histories. Finally, a preliminary study of the axial burnup profile is provided.« less

Hamaker constants of iron oxide nanoparticles.

PubMed

Faure, Bertrand; Salazar-Alvarez, German; Bergström, Lennart

2011-07-19

The Hamaker constants for iron oxide nanoparticles in various media have been calculated using Lifshitz theory. Expressions for the dielectric responses of three iron oxide phases (magnetite, maghemite, and hematite) were derived from recently published optical data. The nonretarded Hamaker constants for the iron oxide nanoparticles interacting across water, A(1w1) = 33 - 39 zJ, correlate relatively well with previous reports, whereas the calculated values in nonpolar solvents (hexane and toluene), A(131) = 9 - 29 zJ, are much lower than the previous estimates, particularly for magnetite. The magnitude of van der Waals interactions varies significantly between the studied phases (magnetite < maghemite < hematite), which highlights the importance of a thorough characterization of the particles. The contribution of magnetic dispersion interactions for particle sizes in the superparamagnetic regime was found to be negligible. Previous conjectures related to colloidal stability and self-assembly have been revisited on the basis of the new Lifshitz values of the Hamaker constants.

Self-assembled multilayers and photoluminescence properties of a new water-soluble poly(para-phenylene)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, X.; Li, D.; Luett, M.

1998-07-01

This paper reports the synthesis and characterizations of a new water-soluble poly(paraphenylene) (PPP) and its applications in preparing self-assembled multi-layer films. This new water-soluble conducting polymer was prepared through the sulfonation reaction of poly(p-quarterphenylene-2,2{prime}-dicarboxylic acid). The incorporation of sulfonate groups has dramatically improved PPP's solubility in water at a wide pH range, whereas previous PPP is only slightly soluble in basic solutions. Dilute aqueous solutions of this polymer with acidic, neutral or basic pH emit brilliant blue light while irradiated with UV light. The sulfonated PPP emits from 350 nm to 455 nm with a maximum intensity at 380 nm.more » Self-assembled multilayers of this sulfonated PPP were constructed with a positively charged polymer poly(diallyl dimethyl ammonium chloride) and characterized with various surface analyses. Conductive (RuO{sub 2} and ITO), semiconductive (Si wafer), and non-conductive (SiO{sub 2}) substrates were used in the preparation of self-assembled multilayers. Electrical, optical and structural properties of these novel self-assembled thin films will be discussed.« less

Self-assembled multilayers and photoluminescence properties of a new water-soluble poly(para-phenylene)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, X.; Li, D.Q.; Luett, M.

1998-03-01

This paper reports the synthesis and characterizations of a new water-soluble poly(para-phenylene) (PPP) and its applications in preparing self-assembled multilayer films. This new water-soluble conducting polymer was prepared through the sulfonation reaction of poly(p-quarterphenylene-2,2{prime}-dicarboxylic acid). The incorporation of sulfonate groups has dramatically improved PPP`s solubility in water at a wide pH range, whereas previous PPP is only slightly soluble in basic solutions. Dilute aqueous solutions of this polymer with acidic, neutral or basic pH emit brilliant blue light while irradiated with UV light. The sulfonated PPP emits from 350 nm to 455 nm with a maximum intensity at 380 nm.more » Self-assembled multilayers of this sulfonated PPP were constructed with a positively charged polymer poly(diallyl dimethyl ammonium chloride) and characterized with various surface analyses. Conductive (RuO{sub 2} and ITO), semiconductive (Si wafer), and non-conductive (SiO{sub 2}) substrates were used in the preparation of self-assembled multilayers. Electrical, optical and structural properties of these novel self-assembled thin films will be discussed.« less

Towards automatic planning for manufacturing generative processes

DOE Office of Scientific and Technical Information (OSTI.GOV)

CALTON,TERRI L.

2000-05-24

Generative process planning describes methods process engineers use to modify manufacturing/process plans after designs are complete. A completed design may be the result from the introduction of a new product based on an old design, an assembly upgrade, or modified product designs used for a family of similar products. An engineer designs an assembly and then creates plans capturing manufacturing processes, including assembly sequences, component joining methods, part costs, labor costs, etc. When new products originate as a result of an upgrade, component geometry may change, and/or additional components and subassemblies may be added to or are omitted from themore » original design. As a result process engineers are forced to create new plans. This is further complicated by the fact that the process engineer is forced to manually generate these plans for each product upgrade. To generate new assembly plans for product upgrades, engineers must manually re-specify the manufacturing plan selection criteria and re-run the planners. To remedy this problem, special-purpose assembly planning algorithms have been developed to automatically recognize design modifications and automatically apply previously defined manufacturing plan selection criteria and constraints.« less

The amyloid architecture provides a scaffold for enzyme-like catalysts.

PubMed

Al-Garawi, Z S; McIntosh, B A; Neill-Hall, D; Hatimy, A A; Sweet, S M; Bagley, M C; Serpell, L C

2017-08-03

Natural biological enzymes possess catalytic sites that are generally surrounded by a large three-dimensional scaffold. However, the proportion of the protein molecule that participates in the catalytic reaction is relatively small. The generation of artificial or miniature enzymes has long been a focus of research because enzyme mimetics can be produced with high activity at low cost. These enzymes aim to mimic the active sites without the additional architecture contributed by the protein chain. Previous work has shown that amyloidogenic peptides are able to self-assemble to create an active site that is capable of binding zinc and catalysing an esterase reaction. Here, we describe the structural characterisation of a set of designed peptides that form an amyloid-like architecture and reveal that their capability to mimic carbonic anhydrase and serve as enzyme-like catalysts is related to their ability to self-assemble. These amyloid fibril structures can bind the metal ion Zn 2+ via a three-dimensional arrangement of His residues created by the amyloid architecture. Our results suggest that the catalytic efficiency of amyloid-like assembly is not only zinc-dependent but also depends on an active centre created by the peptides which is, in turn, dependent on the ordered architecture. These fibrils have good esterase activity, and they may serve as good models for the evolution of modern-day enzymes. Furthermore, they may be useful in designing self-assembling fibrils for applications as metal ion catalysts. This study also demonstrates that the ligands surrounding the catalytic site affect the affinity of the zinc-binding site to bind the substrate contributing to the enzymatic activity of the assembled peptides.

Halo assembly bias and the tidal anisotropy of the local halo environment

NASA Astrophysics Data System (ADS)

Paranjape, Aseem; Hahn, Oliver; Sheth, Ravi K.

2018-05-01

We study the role of the local tidal environment in determining the assembly bias of dark matter haloes. Previous results suggest that the anisotropy of a halo's environment (i.e. whether it lies in a filament or in a more isotropic region) can play a significant role in determining the eventual mass and age of the halo. We statistically isolate this effect, using correlations between the large-scale and small-scale environments of simulated haloes at z = 0 with masses between 1011.6 ≲ (m/h-1 M⊙) ≲ 1014.9. We probe the large-scale environment, using a novel halo-by-halo estimator of linear bias. For the small-scale environment, we identify a variable αR that captures the tidal anisotropy in a region of radius R = 4R200b around the halo and correlates strongly with halo bias at fixed mass. Segregating haloes by αR reveals two distinct populations. Haloes in highly isotropic local environments (αR ≲ 0.2) behave as expected from the simplest, spherically averaged analytical models of structure formation, showing a negative correlation between their concentration and large-scale bias at all masses. In contrast, haloes in anisotropic, filament-like environments (αR ≳ 0.5) tend to show a positive correlation between bias and concentration at any mass. Our multiscale analysis cleanly demonstrates how the overall assembly bias trend across halo mass emerges as an average over these different halo populations, and provides valuable insights towards building analytical models that correctly incorporate assembly bias. We also discuss potential implications for the nature and detectability of galaxy assembly bias.

Dynamics of Human Telomerase Holoenzyme Assembly and Subunit Exchange across the Cell Cycle*

PubMed Central

Vogan, Jacob M.; Collins, Kathleen

2015-01-01

Human telomerase acts on telomeres during the genome synthesis phase of the cell cycle, accompanied by its concentration in Cajal bodies and transient colocalization with telomeres. Whether the regulation of human telomerase holoenzyme assembly contributes to the cell cycle restriction of telomerase function is unknown. We investigated the steady-state levels, assembly, and exchange dynamics of human telomerase subunits with quantitative in vivo cross-linking and other methods. We determined the physical association of telomerase subunits in cells blocked or progressing through the cell cycle as synchronized by multiple protocols. The total level of human telomerase RNA (hTR) was invariant across the cell cycle. In vivo snapshots of telomerase holoenzyme composition established that hTR remains bound to human telomerase reverse transcriptase (hTERT) throughout all phases of the cell cycle, and subunit competition assays suggested that hTERT-hTR interaction is not readily exchangeable. In contrast, the telomerase holoenzyme Cajal body-associated protein, TCAB1, was released from hTR in mitotic cells coincident with TCAB1 delocalization from Cajal bodies. This telomerase holoenzyme disassembly was reversible with cell cycle progression without any change in total TCAB1 protein level. Consistent with differential cell cycle regulation of hTERT-hTR and TCAB1-hTR protein-RNA interactions, overexpression of hTERT or TCAB1 had limited if any influence on hTR assembly of the other subunit. Overall, these findings revealed a cell cycle regulation that disables human telomerase association with telomeres while preserving the co-folded hTERT-hTR ribonucleoprotein catalytic core. Studies here, integrated with previous work, led to a unifying model for telomerase subunit assembly and trafficking in human cells. PMID:26170453

Fluctuation Effects on Propagating Waves of Self-Assembly in Organosilane Monolayers.

NASA Astrophysics Data System (ADS)

Douglas, Jack

2008-03-01

Wavefronts associated with reaction--diffusion and self-assembly processes are ubiquitous in the natural world. For example, propagating fronts arise in crystallization and diverse other thermodynamic ordering processes, in polymerization fronts involved in cell movement and division, as well as in the competitive social interactions and population dynamics of animals at much larger scales. Although it is often claimed that self-sustaining or autocatalytic front propagation is well described by mean-field ``reaction-- diffusion'' or ``phase field'' ordering models, it has recently become appreciated from simulations and theoretical arguments that fluctuation effects in lower spatial dimensions can lead to appreciable deviations from the classical mean-field theory (MFT) of this type of front propagation. The present work explores these fluctuation effects in a real physical system. In particular, we consider a high-resolution near-edge x-ray absorption fine structure spectroscopy (NEXAFS) study of the spontaneous frontal self-assembly of organosilane (OS) molecules into self-assembled monolayer (SAM) surface-energy gradients on oxidized silicon wafers. We find that these layers organize from the wafer edge as propagating wavefronts having well defined velocities. In accordance with two-dimensional simulations of this type of front propagation that take fluctuation effects into account, we find that the interfacial widths w(t) of these SAM self-assembly fronts exhibit a power-law broadening of in time w(t) ˜ t^β, rather than the constant width predicted by MFT. Moreover, the observed exponent values accord rather well with previous simulation and theoretical estimates. These observations have significant implications for diverse types of ordering fronts that occur under confinement conditions in biological or materials-processing contexts.

Assembly of the Arp5 (Actin-related Protein) Subunit Involved in Distinct INO80 Chromatin Remodeling Activities*

PubMed Central

Yao, Wei; Beckwith, Sean L.; Zheng, Tina; Young, Thomas; Dinh, Van T.; Ranjan, Anand; Morrison, Ashby J.

2015-01-01

ATP-dependent chromatin remodeling, which repositions and restructures nucleosomes, is essential to all DNA-templated processes. The INO80 chromatin remodeling complex is an evolutionarily conserved complex involved in diverse cellular processes, including transcription, DNA repair, and replication. The functional diversity of the INO80 complex can, in part, be attributed to specialized activities of distinct subunits that compose the complex. Furthermore, structural analyses have identified biochemically discrete subunit modules that assemble along the Ino80 ATPase scaffold. Of particular interest is the Saccharomyces cerevisiae Arp5-Ies6 module located proximal to the Ino80 ATPase and the Rvb1-Rvb2 helicase module needed for INO80-mediated in vitro activity. In this study we demonstrate that the previously uncharacterized Ies2 subunit is required for Arp5-Ies6 association with the catalytic components of the INO80 complex. In addition, Arp5-Ies6 module assembly with the INO80 complex is dependent on distinct conserved domains within Arp5, Ies6, and Ino80, including the spacer region within the Ino80 ATPase domain. Arp5-Ies6 interacts with chromatin via assembly with the INO80 complex, as IES2 and INO80 deletion results in loss of Arp5-Ies6 chromatin association. Interestingly, ectopic addition of the wild-type Arp5-Ies6 module stimulates INO80-mediated ATP hydrolysis and nucleosome sliding in vitro. However, the addition of mutant Arp5 lacking unique insertion domains facilitates ATP hydrolysis in the absence of nucleosome sliding. Collectively, these results define the requirements of Arp5-Ies6 assembly, which are needed to couple ATP hydrolysis to productive nucleosome movement. PMID:26306040

Frataxin Accelerates [2Fe-2S] Cluster Formation on the Human Fe–S Assembly Complex

PubMed Central

Fox, Nicholas G.; Das, Deepika; Chakrabarti, Mrinmoy; Lindahl, Paul A.; Barondeau, David P.

2015-01-01

Iron–sulfur (Fe–S) clusters function as protein cofactors for a wide variety of critical cellular reactions. In human mitochondria, a core Fe–S assembly complex [called SDUF and composed of NFS1, ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes Fe–S clusters from iron, cysteine sulfur, and reducing equivalents and then transfers these intact clusters to target proteins. In vitro assays have relied on reducing the complexity of this complicated Fe–S assembly process by using surrogate electron donor molecules and monitoring simplified reactions. Recent studies have concluded that FXN promotes the synthesis of [4Fe-4S] clusters on the mammalian Fe–S assembly complex. Here the kinetics of Fe–S synthesis reactions were determined using different electron donation systems and by monitoring the products with circular dichroism and absorbance spectroscopies. We discovered that common surrogate electron donor molecules intercepted Fe–S cluster intermediates and formed high-molecular weight species (HMWS). The HMWS are associated with iron, sulfide, and thiol-containing proteins and have properties of a heterogeneous solubilized mineral with spectroscopic properties remarkably reminiscent of those of [4Fe-4S] clusters. In contrast, reactions using physiological reagents revealed that FXN accelerates the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as previously reported. In the preceding paper [Fox, N. G., et al. (2015) Biochemistry 54, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates on the SDUF complex were shown to readily transfer to uncomplexed ISCU2 or apo acceptor proteins, depending on the reaction conditions. Our results indicate that FXN accelerates a rate-limiting sulfur transfer step in the synthesis of [2Fe-2S] clusters on the human Fe–S assembly complex. PMID:26016518

Frataxin Accelerates [2Fe-2S] Cluster Formation on the Human Fe-S Assembly Complex.

PubMed

Fox, Nicholas G; Das, Deepika; Chakrabarti, Mrinmoy; Lindahl, Paul A; Barondeau, David P

2015-06-30

Iron-sulfur (Fe-S) clusters function as protein cofactors for a wide variety of critical cellular reactions. In human mitochondria, a core Fe-S assembly complex [called SDUF and composed of NFS1, ISD11, ISCU2, and frataxin (FXN) proteins] synthesizes Fe-S clusters from iron, cysteine sulfur, and reducing equivalents and then transfers these intact clusters to target proteins. In vitro assays have relied on reducing the complexity of this complicated Fe-S assembly process by using surrogate electron donor molecules and monitoring simplified reactions. Recent studies have concluded that FXN promotes the synthesis of [4Fe-4S] clusters on the mammalian Fe-S assembly complex. Here the kinetics of Fe-S synthesis reactions were determined using different electron donation systems and by monitoring the products with circular dichroism and absorbance spectroscopies. We discovered that common surrogate electron donor molecules intercepted Fe-S cluster intermediates and formed high-molecular weight species (HMWS). The HMWS are associated with iron, sulfide, and thiol-containing proteins and have properties of a heterogeneous solubilized mineral with spectroscopic properties remarkably reminiscent of those of [4Fe-4S] clusters. In contrast, reactions using physiological reagents revealed that FXN accelerates the formation of [2Fe-2S] clusters rather than [4Fe-4S] clusters as previously reported. In the preceding paper [Fox, N. G., et al. (2015) Biochemistry 54, DOI: 10.1021/bi5014485], [2Fe-2S] intermediates on the SDUF complex were shown to readily transfer to uncomplexed ISCU2 or apo acceptor proteins, depending on the reaction conditions. Our results indicate that FXN accelerates a rate-limiting sulfur transfer step in the synthesis of [2Fe-2S] clusters on the human Fe-S assembly complex.

Three-dimensional fit-to-flow microfluidic assembly.

PubMed

Chen, Arnold; Pan, Tingrui

2011-12-01

Three-dimensional microfluidics holds great promise for large-scale integration of versatile, digitalized, and multitasking fluidic manipulations for biological and clinical applications. Successful translation of microfluidic toolsets to these purposes faces persistent technical challenges, such as reliable system-level packaging, device assembly and alignment, and world-to-chip interface. In this paper, we extended our previously established fit-to-flow (F2F) world-to-chip interconnection scheme to a complete system-level assembly strategy that addresses the three-dimensional microfluidic integration on demand. The modular F2F assembly consists of an interfacial chip, pluggable alignment modules, and multiple monolithic layers of microfluidic channels, through which convoluted three-dimensional microfluidic networks can be easily assembled and readily sealed with the capability of reconfigurable fluid flow. The monolithic laser-micromachining process simplifies and standardizes the fabrication of single-layer pluggable polymeric modules, which can be mass-produced as the renowned Lego(®) building blocks. In addition, interlocking features are implemented between the plug-and-play microfluidic chips and the complementary alignment modules through the F2F assembly, resulting in facile and secure alignment with average misalignment of 45 μm. Importantly, the 3D multilayer microfluidic assembly has a comparable sealing performance as the conventional single-layer devices, providing an average leakage pressure of 38.47 kPa. The modular reconfigurability of the system-level reversible packaging concept has been demonstrated by re-routing microfluidic flows through interchangeable modular microchannel layers.

Huntingtin-interacting protein 1 (Hip1) and Hip1-related protein (Hip1R) bind the conserved sequence of clathrin light chains and thereby influence clathrin assembly in vitro and actin distribution in vivo.

PubMed

Chen, Chih-Ying; Brodsky, Frances M

2005-02-18

Clathrin heavy and light chains form triskelia, which assemble into polyhedral coats of membrane vesicles that mediate transport for endocytosis and organelle biogenesis. Light chain subunits regulate clathrin assembly in vitro by suppressing spontaneous self-assembly of the heavy chains. The residues that play this regulatory role are at the N terminus of a conserved 22-amino acid sequence that is shared by all vertebrate light chains. Here we show that these regulatory residues and others in the conserved sequence mediate light chain interaction with Hip1 and Hip1R. These related proteins were previously found to be enriched in clathrin-coated vesicles and to promote clathrin assembly in vitro. We demonstrate Hip1R binding preference for light chains associated with clathrin heavy chain and show that Hip1R stimulation of clathrin assembly in vitro is blocked by mutations in the conserved sequence of light chains that abolish interaction with Hip1 and Hip1R. In vivo overexpression of a fragment of clathrin light chain comprising the Hip1R-binding region affected cellular actin distribution. Together these results suggest that the roles of Hip1 and Hip1R in affecting clathrin assembly and actin distribution are mediated by their interaction with the conserved sequence of clathrin light chains.

The 38K-Mediated Specific Dephosphorylation of the Viral Core Protein P6.9 Plays an Important Role in the Nucleocapsid Assembly of Autographa californica Multiple Nucleopolyhedrovirus.

PubMed

Lai, Qingying; Wu, Wenbi; Li, Ao; Wang, Wei; Yuan, Meijin; Yang, Kai

2018-05-01

Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene 38K ( ac98 ) interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes by an unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene ( ac100 ) and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only an unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in 38K -deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrate that 38K mediates the dephosphorylation of specific sites at the C terminus of P6.9, which is essential for viral genome encapsidation. IMPORTANCE Genome packaging is a fundamental process in the virus life cycle, and viruses have different strategies to perform this step. For several double-stranded DNA (dsDNA) viruses, the procapsid is formed before genome encapsidation, which may require basic proteins that help to neutralize the nucleic acid charge repulsion to facilitate the compaction of the genome within the confined capsid space. Baculovirus encodes a small basic protein, P6.9, which is required for a variety of processes in the virus infection cycle. The phosphorylation of P6.9 is thought to result in nucleocapsid uncoating, while the dephosphorylation of P6.9 is involved in viral DNA encapsidation during nucleocapsid assembly. Here, we demonstrate that a haloacid dehalogenase homolog encoded by baculovirus core gene 38K is involved in nucleocapsid assembly by mediating the dephosphorylation of 5 specific sites at the C terminus of P6.9. This finding contributes to the understanding of the mechanisms of virus nucleocapsid assembly. Copyright © 2018 Lai et al.

The 38K-Mediated Specific Dephosphorylation of the Viral Core Protein P6.9 Plays an Important Role in the Nucleocapsid Assembly of Autographa californica Multiple Nucleopolyhedrovirus

PubMed Central

Lai, Qingying; Li, Ao; Wang, Wei; Yuan, Meijin

2018-01-01

ABSTRACT Encapsidation of the viral genomes, leading to the assembly of the nucleocapsids to form infectious progeny virions, is a key step in many virus life cycles. Baculovirus nucleocapsid assembly is a complex process that involves many proteins. Our previous studies showed that the deletion of the core gene 38K (ac98) interrupted the nucleocapsid assembly by producing capsid sheaths devoid of viral genomes by an unknown mechanism. All homologs of 38K contain conserved motifs of the haloacid dehalogenase superfamily, which are involved in phosphoryl transfer. The requirements of these motifs for nucleocapsid assembly, confirmed in the present study, suggest that 38K may be a functioning haloacid dehalogenase. P6.9 is also encoded by a core gene (ac100) and is required for viral genome encapsidation. It has been reported that multiple phosphorylated species of P6.9 are present in virus-infected cells, while only an unphosphorylated species is detected in the budded virus. Therefore, whether 38K mediates the dephosphorylation of P6.9 was investigated. An additional phosphorylated species of P6.9 in 38K-deleted or -mutated virus-transfected cells was detected, and the dephosphorylated sites mediated by 38K were determined by mass spectrometry. To assess the effects of dephosphorylation of P6.9 mediated by 38K on virus replication, these sites were mutated to glutamic acids (phosphorylation-mimic mutant) or to alanines (phosphorylation-deficient mutant). Studies showed that the nucleocapsid assembly was interrupted in phosphorylation-mimic mutant virus-transfected cells. Taken together, our findings demonstrate that 38K mediates the dephosphorylation of specific sites at the C terminus of P6.9, which is essential for viral genome encapsidation. IMPORTANCE Genome packaging is a fundamental process in the virus life cycle, and viruses have different strategies to perform this step. For several double-stranded DNA (dsDNA) viruses, the procapsid is formed before genome encapsidation, which may require basic proteins that help to neutralize the nucleic acid charge repulsion to facilitate the compaction of the genome within the confined capsid space. Baculovirus encodes a small basic protein, P6.9, which is required for a variety of processes in the virus infection cycle. The phosphorylation of P6.9 is thought to result in nucleocapsid uncoating, while the dephosphorylation of P6.9 is involved in viral DNA encapsidation during nucleocapsid assembly. Here, we demonstrate that a haloacid dehalogenase homolog encoded by baculovirus core gene 38K is involved in nucleocapsid assembly by mediating the dephosphorylation of 5 specific sites at the C terminus of P6.9. This finding contributes to the understanding of the mechanisms of virus nucleocapsid assembly. PMID:29444944

Macroinvertebrate community assembly in pools created during peatland restoration.

PubMed

Brown, Lee E; Ramchunder, Sorain J; Beadle, Jeannie M; Holden, Joseph

2016-11-01

Many degraded ecosystems are subject to restoration attempts, providing new opportunities to unravel the processes of ecological community assembly. Restoration of previously drained northern peatlands, primarily to promote peat and carbon accumulation, has created hundreds of thousands of new open water pools. We assessed the potential benefits of this wetland restoration for aquatic biodiversity, and how communities reassemble, by comparing pool ecosystems in regions of the UK Pennines on intact (never drained) versus restored (blocked drainage-ditches) peatland. We also evaluated the conceptual idea that comparing reference ecosystems in terms of their compositional similarity to null assemblages (and thus the relative importance of stochastic versus deterministic assembly) can guide evaluations of restoration success better than analyses of community composition or diversity. Community composition data highlighted some differences in the macroinvertebrate composition of restored pools compared to undisturbed peatland pools, which could be used to suggest that alternative end-points to restoration were influenced by stochastic processes. However, widely used diversity metrics indicated no differences between undisturbed and restored pools. Novel evaluations of restoration using null models confirmed the similarity of deterministic assembly processes from the national species pool across all pools. Stochastic elements were important drivers of between-pool differences at the regional-scale but the scale of these effects was also similar across most of the pools studied. The amalgamation of assembly theory into ecosystem restoration monitoring allows us to conclude with more certainty that restoration has been successful from an ecological perspective in these systems. Evaluation of these UK findings compared to those from peatlands across Europe and North America further suggests that restoring peatland pools delivers significant benefits for aquatic fauna by providing extensive new habitat that is largely equivalent to natural pools. More generally, we suggest that assembly theory could provide new benchmarks for planning and evaluating ecological restoration success. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

A Multi-Platform Draft de novo Genome Assembly and Comparative Analysis for the Scarlet Macaw (Ara macao)

PubMed Central

Seabury, Christopher M.; Dowd, Scot E.; Seabury, Paul M.; Raudsepp, Terje; Brightsmith, Donald J.; Liboriussen, Poul; Halley, Yvette; Fisher, Colleen A.; Owens, Elaine; Viswanathan, Ganesh; Tizard, Ian R.

2013-01-01

Data deposition to NCBI Genomes This Whole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession AMXX00000000 (SMACv1.0, unscaffolded genome assembly). The version described in this paper is the first version (AMXX01000000). The scaffolded assembly (SMACv1.1) has been deposited at DDBJ/EMBL/GenBank under the accession AOUJ00000000, and is also the first version (AOUJ01000000). Strong biological interest in traits such as the acquisition and utilization of speech, cognitive abilities, and longevity catalyzed the utilization of two next-generation sequencing platforms to provide the first-draft de novo genome assembly for the large, new world parrot Ara macao (Scarlet Macaw). Despite the challenges associated with genome assembly for an outbred avian species, including 951,507 high-quality putative single nucleotide polymorphisms, the final genome assembly (>1.035 Gb) includes more than 997 Mb of unambiguous sequence data (excluding N’s). Cytogenetic analyses including ZooFISH revealed complex rearrangements associated with two scarlet macaw macrochromosomes (AMA6, AMA7), which supports the hypothesis that translocations, fusions, and intragenomic rearrangements are key factors associated with karyotype evolution among parrots. In silico annotation of the scarlet macaw genome provided robust evidence for 14,405 nuclear gene annotation models, their predicted transcripts and proteins, and a complete mitochondrial genome. Comparative analyses involving the scarlet macaw, chicken, and zebra finch genomes revealed high levels of nucleotide-based conservation as well as evidence for overall genome stability among the three highly divergent species. Application of a new whole-genome analysis of divergence involving all three species yielded prioritized candidate genes and noncoding regions for parrot traits of interest (i.e., speech, intelligence, longevity) which were independently supported by the results of previous human GWAS studies. We also observed evidence for genes and noncoding loci that displayed extreme conservation across the three avian lineages, thereby reflecting their likely biological and developmental importance among birds. PMID:23667475

Self-Assembly Strategies for Integrating Light Harvesting and Charge Separation in Artificial Photosynthetic Systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wasielewski, Michael R.

In natural photosynthesis, organisms optimize solar energy conversion through organized assemblies of photofunctional chromophores and catalysts within proteins that provide specifically tailored environments for chemical reactions. As with their natural counterparts, artificial photosynthetic systems for practical solar fuels production must collect light energy, separate charge, and transport charge to catalytic sites where multielectron redox processes will occur. While encouraging progress has been made on each aspect of this complex problem, researchers have not yet developed self-ordering and self-assembling components and the tailored environments necessary to realize a fully-functional artificial system. Previously researchers have used complex, covalent molecular systems comprised ofmore » chromophores, electron donors, and electron acceptors to mimic both the light-harvesting and the charge separation functions of photosynthetic proteins. These systems allow for study of the dependencies of electron transfer rate constants on donor?acceptor distance and orientation, electronic interaction, and the free energy of the reaction. The most useful and informative systems are those in which structural constraints control both the distance and the orientation between the electron donors and acceptors. Self-assembly provides a facile means for organizing large numbers of molecules into supramolecular structures that can bridge length scales from nanometers to macroscopic dimensions. The resulting structures must provide pathways for migration of light excitation energy among antenna chromophores, and from antennas to reaction centers. They also must incorporate charge conduits, that is, molecular 'wires' that can efficiently move electrons and holes between reaction centers and catalytic sites. The central scientific challenge is to develop small, functional building blocks with a minimum number of covalent linkages, which also have the appropriate molecular recognition properties to facilitate self-assembly of complete, functional artificial photosynthetic systems. In this Account, we explore how self-assembly strategies involving ?-stacking can be used to integrate light harvesting with charge separation and transport.« less

Rational design of bioactive, modularly assembled aminoglycosides targeting the RNA that causes myotonic dystrophy type 1.

PubMed

Childs-Disney, Jessica L; Parkesh, Raman; Nakamori, Masayuki; Thornton, Charles A; Disney, Matthew D

2012-12-21

Myotonic dystrophy type 1 (DM1) is caused when an expanded r(CUG) repeat (r(CUG)(exp)) binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1) as well as other proteins. Previously, we reported that modularly assembled small molecules displaying a 6'-N-5-hexynoate kanamycin A RNA-binding module (K) on a peptoid backbone potently inhibit the binding of MBNL1 to r(CUG)(exp). However, these parent compounds are not appreciably active in cell-based models of DM1. The lack of potency was traced to suboptimal cellular permeability and localization. To improve these properties, second-generation compounds that are conjugated to a d-Arg(9) molecular transporter were synthesized. These modified compounds enter cells in higher concentrations than the parent compounds and are efficacious in cell-based DM1 model systems at low micromolar concentrations. In particular, they improve three defects that are the hallmarks of DM1: a translational defect due to nuclear retention of transcripts containing r(CUG)(exp); pre-mRNA splicing defects due to inactivation of MBNL1; and the formation of nuclear foci. The best compound in cell-based studies was tested in a mouse model of DM1. Modest improvement of pre-mRNA splicing defects was observed. These studies suggest that a modular assembly approach can afford bioactive compounds that target RNA.

Rational Design of Bioactive, Modularly Assembled Aminoglycosides Targeting the RNA that Causes Myotonic Dystrophy Type 1

PubMed Central

Childs-Disney, Jessica L.; Parkesh, Raman; Nakamori, Masayuki; Thornton, Charles A.; Disney, Matthew D.

2012-01-01

Myotonic dystrophy type 1 (DM1) is caused when an expanded r(CUG) repeat (r(CUG)exp) binds the RNA splicing regulator muscleblind-like 1 protein (MBNL1) as well as other proteins. Previously, we reported that modularly assembled small molecules displaying a 6′-N-5-hexynoate kanamycin A RNA-binding module (K) on a peptoid backbone potently inhibit the binding of MBNL1 to r(CUG)exp. However, these parent compounds are not appreciably active in cell-based models of DM1. The lack of potency was traced to suboptimal cellular permeability and localization. To improve these properties, second-generation compounds that are conjugated to a D-Arg9 molecular transporter were synthesized. These modified compounds enter cells in higher concentrations than the parent compounds and are efficacious in cell-based DM1 model systems at low micromolar concentrations. In particular, they improve three defects that are the hallmarks of DM1: a translational defect due to nuclear retention of transcripts containing r(CUG)exp; pre-mRNA splicing defects due to inactivation of MBNL1; and the formation of nuclear foci. The best compound in cell-based studies was tested in a mouse model of DM1. Modest improvement of pre-mRNA splicing defects was observed. These studies suggest that a modular assembly approach can afford bioactive compounds that target RNA. PMID:23130637

Basis of aggravated hepatic lipid metabolism by chronic stress in high-fat diet-fed rat.

PubMed

Han, Ying; Lin, Min; Wang, Xiaobin; Guo, Keke; Wang, Shanshan; Sun, Mengfei; Wang, Jiao; Han, Xiaoyu; Fu, Ting; Hu, Yang; Fu, Jihua

2015-03-01

Our previous study has demonstrated that long-term stress, known as chronic stress (CS), can aggravate nonalcoholic fatty liver disease in high-fat diet (HFD)-fed rat. In this study, we tried to figure out which lipid metabolic pathways were impacted by CS in the HFD-fed rat. Male Sprague-Dawley rats (6 weeks of age, n = 8 per group) were fed with either standard diet or HFD with or without CS exposure for 8 weeks. Hepatic lipidosis, biochemical, hormonal, and lipid profile markers in serum and liver, and enzymes involved in de novo lipogenesis (DNL) of fatty acids (FAs) and cholesterol, β-oxidation, FAs uptake, triglycerides synthesis, and very low-density lipoprotein (VLDL) assembly in the liver were detected. CS exposure reduced hepatic lipidosis but further elevated hepatic VLDL content with aggravated dyslipidemia in the HFD-fed rats. There was a synergism between CS and HFD on VLDL production and dyslipidemia. PCR and western blot assays showed that CS exposure significantly promoted hepatic VLDL assembly in rats, especially in the HFD-fed rats, while it had little impact on DNL, β-oxidation, FAs uptake, and triglycerides synthesis in the HFD-fed rats. This phenomenon was in accordance with elevated serum glucocorticoid level. The critical influence of CS exposure on hepatic lipid metabolism in the HFD-fed rats is VLDL assembly which might be regulated by glucocorticoid.

A Temporospatial Map That Defines Specific Steps at Which Critical Surfaces in the Gag MA and CA Domains Act during Immature HIV-1 Capsid Assembly in Cells

PubMed Central

Robinson, Bridget A.; Reed, Jonathan C.; Geary, Clair D.; Swain, J. Victor

2014-01-01

ABSTRACT During HIV-1 assembly, Gag polypeptides target to the plasma membrane, where they multimerize to form immature capsids that undergo budding and maturation. Previous mutational analyses identified residues within the Gag matrix (MA) and capsid (CA) domains that are required for immature capsid assembly, and structural studies showed that these residues are clustered on four exposed surfaces in Gag. Exactly when and where the three critical surfaces in CA function during assembly are not known. Here, we analyzed how mutations in these four critical surfaces affect the formation and stability of assembly intermediates in cells expressing the HIV-1 provirus. The resulting temporospatial map reveals that critical MA residues act during membrane targeting, residues in the C-terminal CA subdomain (CA-CTD) dimer interface are needed for the stability of the first membrane-bound assembly intermediate, CA-CTD base residues are necessary for progression past the first membrane-bound intermediate, and residues in the N-terminal CA subdomain (CA-NTD) stabilize the last membrane-bound intermediate. Importantly, we found that all four critical surfaces act while Gag is associated with the cellular facilitators of assembly ABCE1 and DDX6. When correlated with existing structural data, our findings suggest the following model: Gag dimerizes via the CA-CTD dimer interface just before or during membrane targeting, individual CA-CTD hexamers form soon after membrane targeting, and the CA-NTD hexameric lattice forms just prior to capsid release. This model adds an important new dimension to current structural models by proposing the potential order in which key contacts within the immature capsid lattice are made during assembly in cells. IMPORTANCE While much is known about the structure of the completed HIV-1 immature capsid and domains of its component Gag proteins, less is known about the sequence of events leading to formation of the HIV-1 immature capsid. Here we used biochemical and ultrastructural analyses to generate a temporospatial map showing the precise order in which four critical surfaces in Gag act during immature capsid formation in provirus-expressing cells. Because three of these surfaces make important contacts in the hexameric lattices that are found in the completed immature capsid, these data allow us to propose a model for the sequence of events leading to formation of the hexameric lattices. By providing a dynamic view of when and where critical Gag-Gag contacts form during the assembly process and how those contacts function in the nascent capsid, our study provides novel insights into how an immature capsid is built in infected cells. PMID:24623418

1.8 Å structure of murine GITR ligand dimer expressed in Drosophila melanogaster S2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chattopadhyay, Kausik; Ramagopal, Udupi A.; Nathenson, Stanley G., E-mail: nathenso@aecom.yu.edu

2009-05-01

1.8 Å X-ray crystal structure of mouse GITRL expressed in D. melanogaster S2 cells shows an identical ‘strand-exchanged’ dimeric assembly similar to that observed previously for the E. coli-expressed protein. Glucocorticoid-induced TNF receptor ligand (GITRL), a prominent member of the TNF superfamily, activates its receptor on both effector and regulatory T cells to generate critical costimulatory signals that have been implicated in a wide range of T-cell immune functions. The crystal structures of murine and human orthologs of GITRL recombinantly expressed in Escherichia coli have previously been determined. In contrast to all classical TNF structures, including the human GITRL structure,more » murine GITRL demonstrated a unique ‘strand-exchanged’ dimeric organization. Such a novel assembly behavior indicated a dramatic impact on receptor activation as well as on the signaling mechanism associated with the murine GITRL costimulatory system. In this present work, the 1.8 Å resolution crystal structure of murine GITRL expressed in Drosophila melanogaster S2 cells is reported. The eukaryotic protein-expression system allows transport of the recombinant protein into the extracellular culture medium, thus maximizing the possibility of obtaining correctly folded material devoid of any folding/assembly artifacts that are often suspected with E. coli-expressed proteins. The S2 cell-expressed murine GITRL adopts an identical ‘strand-exchanged’ dimeric structure to that observed for the E. coli-expressed protein, thus conclusively demonstrating the novel quaternary structure assembly behavior of murine GITRL.« less

Solution Properties of Murine Leukemia Virus Gag Protein: Differences from HIV-1 Gag

DOE Office of Scientific and Technical Information (OSTI.GOV)

Datta, Siddhartha A.K.; Zuo, Xiaobing; Clark, Patrick K.

2012-05-09

Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV),more » a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of {approx}7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed.« less

Isopeptide bonds of the major pilin protein BcpA influence pilus structure and bundle formation on the surface of Bacillus cereus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hendrickx, Antoni P.A.; Poor, Catherine B.; Jureller, Justin E.

Bacillus cereus strains elaborate pili on their surface using a mechanism of sortase-mediated cross-linking of major and minor pilus components. Here we used a combination of electron microscopy and atomic force microscopy to visualize these structures. Pili occur as single, double or higher order assemblies of filaments formed from monomers of the major pilin, BcpA, capped by the minor pilin, BcpB. Previous studies demonstrated that within assembled pili, four domains of BcpA -- CNA{sub 1}, CNA{sub 2}, XNA and CNA{sub 3} -- each acquire intramolecular lysine-asparagine isopeptide bonds formed via catalytic glutamic acid or aspartic acid residues. Here we showedmore » that mutants unable to form the intramolecular isopeptide bonds in the CNA2 or CNA3 domains retain the ability to form pilus bundles. A mutant lacking the CNA{sub 1} isopeptide bond assembled deformed pilin subunits that failed to associate as bundles. X-ray crystallography revealed that the BcpA variant Asp{sup 312}Ala, lacking an aspartyl catalyst, did not generate the isopeptide bond within the jelly-roll structure of XNA. The Asp{sup 312}Ala mutant was also unable to form bundles and promoted the assembly of deformed pili. Thus, structural integrity of the CNA{sub 1} and XNA domains are determinants for the association of pili into higher order bundle structures and determine native pilus structure.« less

Building America Case Study: Initial and Long-Term Movement of Cladding Installed Over Exterior Rigid Insulation (Fact Sheet)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Not Available

Changes in the International Energy Conservation Code (IECC) from 2009 to 2012 have resulted in the use of exterior rigid insulation becoming part of the prescriptive code requirements. With more jurisdictions adopting the 2012 IECC builders are going to finding themselves required to incorporate exterior insulation in the construction of their exterior wall assemblies. For thick layers of exterior insulation (levels greater than 1.5 inches), the use wood furring strips attached through the insulation back to the structure has been used by many contractors and designers as a means to provide a convenient cladding attachment location. However, there has beenmore » a significant resistance to its widespread implementation due to a lack of research and understanding of the mechanisms involved and potential creep effects of the assembly under the sustained dead load of a cladding. This research was an extension on previous research conducted by BSC in 2011, and 2012. Each year the understanding of the system discrete load component interactions, as well as impacts of environmental loading has increased. The focus of the research was to examine more closely the impacts of screw fastener bending on the total system capacity, effects of thermal expansion and contraction of materials on the compressive forces in the assembly, as well as to analyze a full years worth of cladding movement data from assemblies constructed in an exposed outdoor environment.« less

Stimulatory effects of advanced glycation endproducts (AGEs) on fibronectin matrix assembly.

PubMed

Pastino, Alexandra K; Greco, Todd M; Mathias, Rommel A; Cristea, Ileana M; Schwarzbauer, Jean E

2017-05-01

Advanced glycation endproducts (AGEs) are a heterogeneous group of compounds that form via non-enzymatic glycation of proteins throughout our lifespan and at a higher rate in certain chronic diseases such as diabetes. AGEs contribute to the progression of fibrosis, in part by stimulating cellular pathways that affect gene expression. Long-lived ECM proteins are targets for non-enzymatic glycation but the question of whether the AGE-modified ECM leads to excess ECM accumulation and fibrosis remains unanswered. In this study, cellular changes due to AGE accretion in the ECM were investigated. Non-enzymatic glycation of proteins in a decellularized fibroblast ECM was achieved by incubating the ECM in a solution of methylglyoxal (MGO). Mass spectrometry of fibronectin (FN) isolated from the glycated matrix identified twenty-eight previously unidentified MGO-derived AGE modification sites including functional sites such as the RGD integrin-binding sequence. Mesangial cells grown on the glycated, decellularized matrix assembled increased amounts of FN matrix. Soluble AGE-modified bovine serum albumin (BSA) also stimulated FN matrix assembly and this effect was reduced by function-blocking antibodies against the receptor for AGE (RAGE). These results indicate that cells respond to AGEs by increasing matrix assembly and that RAGE is involved in this response. This raises the possibility that the accumulation of ECM during the progression of fibrosis may be enhanced by cell interactions with AGEs on a glycated ECM. Copyright © 2016 Elsevier B.V. All rights reserved.

Two-axis movable concentrating solar energy collector

NASA Technical Reports Server (NTRS)

Perkins, G. S.

1977-01-01

Proposed solar-tracker collector assembly with boiler in fixed position, allows use of hard line connections, capable of withstanding optimum high temperature fluid flow. System thereby eliminates need for flexible or slip connection previously used with solar collector systems.

From NGS assembly challenges to instability of fungal mitochondrial genomes: A case study in genome complexity.

PubMed

Misas, Elizabeth; Muñoz, José Fernando; Gallo, Juan Esteban; McEwen, Juan Guillermo; Clay, Oliver Keatinge

2016-04-01

The presence of repetitive or non-unique DNA persisting over sizable regions of a eukaryotic genome can hinder the genome's successful de novo assembly from short reads: ambiguities in assigning genome locations to the non-unique subsequences can result in premature termination of contigs and thus overfragmented assemblies. Fungal mitochondrial (mtDNA) genomes are compact (typically less than 100 kb), yet often contain short non-unique sequences that can be shown to impede their successful de novo assembly in silico. Such repeats can also confuse processes in the cell in vivo. A well-studied example is ectopic (out-of-register, illegitimate) recombination associated with repeat pairs, which can lead to deletion of functionally important genes that are located between the repeats. Repeats that remain conserved over micro- or macroevolutionary timescales despite such risks may indicate functionally or structurally (e.g., for replication) important regions. This principle could form the basis of a mining strategy for accelerating discovery of function in genome sequences. We present here our screening of a sample of 11 fully sequenced fungal mitochondrial genomes by observing where exact k-mer repeats occurred several times; initial analyses motivated us to focus on 17-mers occurring more than three times. Based on the diverse repeats we observe, we propose that such screening may serve as an efficient expedient for gaining a rapid but representative first insight into the repeat landscapes of sparsely characterized mitochondrial chromosomes. Our matching of the flagged repeats to previously reported regions of interest supports the idea that systems of persisting, non-trivial repeats in genomes can often highlight features meriting further attention. Copyright © 2016 Elsevier Ltd. All rights reserved.

The cacao Criollo genome v2.0: an improved version of the genome for genetic and functional genomic studies.

PubMed

Argout, X; Martin, G; Droc, G; Fouet, O; Labadie, K; Rivals, E; Aury, J M; Lanaud, C

2017-09-15

Theobroma cacao L., native to the Amazonian basin of South America, is an economically important fruit tree crop for tropical countries as a source of chocolate. The first draft genome of the species, from a Criollo cultivar, was published in 2011. Although a useful resource, some improvements are possible, including identifying misassemblies, reducing the number of scaffolds and gaps, and anchoring un-anchored sequences to the 10 chromosomes. We used a NGS-based approach to significantly improve the assembly of the Belizian Criollo B97-61/B2 genome. We combined four Illumina large insert size mate paired libraries with 52x of Pacific Biosciences long reads to correct misassembled regions and reduced the number of scaffolds. We then used genotyping by sequencing (GBS) methods to increase the proportion of the assembly anchored to chromosomes. The scaffold number decreased from 4,792 in assembly V1 to 554 in V2 while the scaffold N50 size has increased from 0.47 Mb in V1 to 6.5 Mb in V2. A total of 96.7% of the assembly was anchored to the 10 chromosomes compared to 66.8% in the previous version. Unknown sites (Ns) were reduced from 10.8% to 5.7%. In addition, we updated the functional annotations and performed a new RefSeq structural annotation based on RNAseq evidence. Theobroma cacao Criollo genome version 2 will be a valuable resource for the investigation of complex traits at the genomic level and for future comparative genomics and genetics studies in cacao tree. New functional tools and annotations are available on the Cocoa Genome Hub ( http://cocoa-genome-hub.southgreen.fr ).

SOPRA: Scaffolding algorithm for paired reads via statistical optimization.

PubMed

Dayarian, Adel; Michael, Todd P; Sengupta, Anirvan M

2010-06-24

High throughput sequencing (HTS) platforms produce gigabases of short read (<100 bp) data per run. While these short reads are adequate for resequencing applications, de novo assembly of moderate size genomes from such reads remains a significant challenge. These limitations could be partially overcome by utilizing mate pair technology, which provides pairs of short reads separated by a known distance along the genome. We have developed SOPRA, a tool designed to exploit the mate pair/paired-end information for assembly of short reads. The main focus of the algorithm is selecting a sufficiently large subset of simultaneously satisfiable mate pair constraints to achieve a balance between the size and the quality of the output scaffolds. Scaffold assembly is presented as an optimization problem for variables associated with vertices and with edges of the contig connectivity graph. Vertices of this graph are individual contigs with edges drawn between contigs connected by mate pairs. Similar graph problems have been invoked in the context of shotgun sequencing and scaffold building for previous generation of sequencing projects. However, given the error-prone nature of HTS data and the fundamental limitations from the shortness of the reads, the ad hoc greedy algorithms used in the earlier studies are likely to lead to poor quality results in the current context. SOPRA circumvents this problem by treating all the constraints on equal footing for solving the optimization problem, the solution itself indicating the problematic constraints (chimeric/repetitive contigs, etc.) to be removed. The process of solving and removing of constraints is iterated till one reaches a core set of consistent constraints. For SOLiD sequencer data, SOPRA uses a dynamic programming approach to robustly translate the color-space assembly to base-space. For assessing the quality of an assembly, we report the no-match/mismatch error rate as well as the rates of various rearrangement errors. Applying SOPRA to real data from bacterial genomes, we were able to assemble contigs into scaffolds of significant length (N50 up to 200 Kb) with very few errors introduced in the process. In general, the methodology presented here will allow better scaffold assemblies of any type of mate pair sequencing data.

A novel life cycle modeling system for Ebola virus shows a genome length-dependent role of VP24 in virus infectivity.

PubMed

Watt, Ari; Moukambi, Felicien; Banadyga, Logan; Groseth, Allison; Callison, Julie; Herwig, Astrid; Ebihara, Hideki; Feldmann, Heinz; Hoenen, Thomas

2014-09-01

Work with infectious Ebola viruses is restricted to biosafety level 4 (BSL4) laboratories, presenting a significant barrier for studying these viruses. Life cycle modeling systems, including minigenome systems and transcription- and replication-competent virus-like particle (trVLP) systems, allow modeling of the virus life cycle under BSL2 conditions; however, all current systems model only certain aspects of the virus life cycle, rely on plasmid-based viral protein expression, and have been used to model only single infectious cycles. We have developed a novel life cycle modeling system allowing continuous passaging of infectious trVLPs containing a tetracistronic minigenome that encodes a reporter and the viral proteins VP40, VP24, and GP1,2. This system is ideally suited for studying morphogenesis, budding, and entry, in addition to genome replication and transcription. Importantly, the specific infectivity of trVLPs in this system was ∼ 500-fold higher than that in previous systems. Using this system for functional studies of VP24, we showed that, contrary to previous reports, VP24 only very modestly inhibits genome replication and transcription when expressed in a regulated fashion, which we confirmed using infectious Ebola viruses. Interestingly, we also discovered a genome length-dependent effect of VP24 on particle infectivity, which was previously undetected due to the short length of monocistronic minigenomes and which is due at least partially to a previously unknown function of VP24 in RNA packaging. Based on our findings, we propose a model for the function of VP24 that reconciles all currently available data regarding the role of VP24 in nucleocapsid assembly as well as genome replication and transcription. Ebola viruses cause severe hemorrhagic fevers in humans, with no countermeasures currently being available, and must be studied in maximum-containment laboratories. Only a few of these laboratories exist worldwide, limiting our ability to study Ebola viruses and develop countermeasures. Here we report the development of a novel reverse genetics-based system that allows the study of Ebola viruses without maximum-containment laboratories. We used this system to investigate the Ebola virus protein VP24, showing that, contrary to previous reports, it only modestly inhibits virus genome replication and transcription but is important for packaging of genomes into virus particles, which constitutes a previously unknown function of VP24 and a potential antiviral target. We further propose a comprehensive model for the function of VP24 in nucleocapsid assembly. Importantly, on the basis of this approach, it should easily be possible to develop similar experimental systems for other viruses that are currently restricted to maximum-containment laboratories. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Analyses of Coronavirus Assembly Interactions with Interspecies Membrane and Nucleocapsid Protein Chimeras

PubMed Central

Kuo, Lili; Hurst-Hess, Kelley R.; Koetzner, Cheri A.

2016-01-01

ABSTRACT The coronavirus membrane (M) protein is the central actor in virion morphogenesis. M organizes the components of the viral membrane, and interactions of M with itself and with the nucleocapsid (N) protein drive virus assembly and budding. In order to further define M-M and M-N interactions, we constructed mutants of the model coronavirus mouse hepatitis virus (MHV) in which all or part of the M protein was replaced by its phylogenetically divergent counterpart from severe acute respiratory syndrome coronavirus (SARS-CoV). We were able to obtain viable chimeras containing the entire SARS-CoV M protein as well as mutants with intramolecular substitutions that partitioned M protein at the boundaries between the ectodomain, transmembrane domains, or endodomain. Our results show that the carboxy-terminal domain of N protein, N3, is necessary and sufficient for interaction with M protein. However, despite some previous genetic and biochemical evidence that mapped interactions with N to the carboxy terminus of M, it was not possible to define a short linear region of M protein sufficient for assembly with N. Thus, interactions with N protein likely involve multiple linearly discontiguous regions of the M endodomain. The SARS-CoV M chimera exhibited a conditional growth defect that was partially suppressed by mutations in the envelope (E) protein. Moreover, virions of the M chimera were markedly deficient in spike (S) protein incorporation. These findings suggest that the interactions of M protein with both E and S protein are more complex than previously thought. IMPORTANCE The assembly of coronavirus virions entails concerted interactions among the viral structural proteins and the RNA genome. One strategy to study this process is through construction of interspecies chimeras that preserve or disrupt particular inter- or intramolecular associations. In this work, we replaced the membrane (M) protein of the model coronavirus mouse hepatitis virus with its counterpart from a heterologous coronavirus. The results clarify our understanding of the interaction between the coronavirus M protein and the nucleocapsid protein. At the same time, they reveal unanticipated complexities in the interactions of M with the viral spike and envelope proteins. PMID:26889024

MYBPH inhibits NM IIA assembly via direct interaction with NMHC IIA and reduces cell motility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hosono, Yasuyuki; Usukura, Jiro; Yamaguchi, Tomoya

2012-11-09

Highlights: Black-Right-Pointing-Pointer MYBPH inhibits NMHC IIA assembly and cell motility. Black-Right-Pointing-Pointer MYBPH interacts to assembly-competent NM IIA. Black-Right-Pointing-Pointer MYBPH inhibits RLC and NMHC IIA, independent components of NM IIA. -- Abstract: Actomyosin filament assembly is a critical step in tumor cell migration. We previously found that myosin binding protein H (MYBPH) is directly transactivated by the TTF-1 lineage-survival oncogene in lung adenocarcinomas and inhibits phosphorylation of the myosin regulatory light chain (RLC) of non-muscle myosin IIA (NM IIA) via direct interaction with Rho kinase 1 (ROCK1). Here, we report that MYBPH also directly interacts with an additional molecule, non-muscle myosinmore » heavy chain IIA (NMHC IIA), which was found to occur between MYBPH and the rod portion of NMHC IIA. MYBPH inhibited NMHC IIA assembly and reduced cell motility. Conversely, siMYBPH-induced increased motility was partially, yet significantly, suppressed by blebbistatin, a non-muscle myosin II inhibitor, while more profound effects were attained by combined treatment with siROCK1 and blebbistatin. Electron microscopy observations showed well-ordered paracrystals of NMHC IIA reflecting an assembled state, which were significantly less frequently observed in the presence of MYBPH. Furthermore, an in vitro sedimentation assay showed that a greater amount of NMHC IIA was in an unassembled state in the presence of MYBPH. Interestingly, treatment with a ROCK inhibitor that impairs transition of NM IIA from an assembly-incompetent to assembly-competent state reduced the interaction between MYBPH and NMHC IIA, suggesting that MYBPH has higher affinity to assembly-competent NM IIA. These results suggest that MYBPH inhibits RLC and NMHC IIA, independent components of NM IIA, and negatively regulates actomyosin organization at 2 distinct steps, resulting in firm inhibition of NM IIA assembly.« less

Simple tools for assembling and searching high-density picolitre pyrophosphate sequence data.

PubMed

Parker, Nicolas J; Parker, Andrew G

2008-04-18

The advent of pyrophosphate sequencing makes large volumes of sequencing data available at a lower cost than previously possible. However, the short read lengths are difficult to assemble and the large dataset is difficult to handle. During the sequencing of a virus from the tsetse fly, Glossina pallidipes, we found the need for tools to search quickly a set of reads for near exact text matches. A set of tools is provided to search a large data set of pyrophosphate sequence reads under a "live" CD version of Linux on a standard PC that can be used by anyone without prior knowledge of Linux and without having to install a Linux setup on the computer. The tools permit short lengths of de novo assembly, checking of existing assembled sequences, selection and display of reads from the data set and gathering counts of sequences in the reads. Demonstrations are given of the use of the tools to help with checking an assembly against the fragment data set; investigating homopolymer lengths, repeat regions and polymorphisms; and resolving inserted bases caused by incomplete chain extension. The additional information contained in a pyrophosphate sequencing data set beyond a basic assembly is difficult to access due to a lack of tools. The set of simple tools presented here would allow anyone with basic computer skills and a standard PC to access this information.

TCGA-assembler 2: software pipeline for retrieval and processing of TCGA/CPTAC data.

PubMed

Wei, Lin; Jin, Zhilin; Yang, Shengjie; Xu, Yanxun; Zhu, Yitan; Ji, Yuan

2018-05-01

The Cancer Genome Atlas (TCGA) program has produced huge amounts of cancer genomics data providing unprecedented opportunities for research. In 2014, we developed TCGA-Assembler, a software pipeline for retrieval and processing of public TCGA data. In 2016, TCGA data were transferred from the TCGA data portal to the Genomic Data Commons (GDCs), which is supported by a different set of data storage and retrieval mechanisms. In addition, new proteomics data of TCGA samples have been generated by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) program, which were not available for downloading through TCGA-Assembler. It is desirable to acquire and integrate data from both GDC and CPTAC. We develop TCGA-assembler 2 (TA2) to automatically download and integrate data from GDC and CPTAC. We make substantial improvement on the functionality of TA2 to enhance user experience and software performance. TA2 together with its previous version have helped more than 2000 researchers from 64 countries to access and utilize TCGA and CPTAC data in their research. Availability of TA2 will continue to allow existing and new users to conduct reproducible research based on TCGA and CPTAC data. http://www.compgenome.org/TCGA-Assembler/ or https://github.com/compgenome365/TCGA-Assembler-2. zhuyitan@gmail.com or koaeraser@gmail.com. Supplementary data are available at Bioinformatics online.

CO2 Compressor Requirements for Integration of Space Station Carbon Dioxide Removal and Carbon Dioxide Reduction Assemblies

NASA Technical Reports Server (NTRS)

Jeng, Frank F.; Lewis, John F.; Graf, John; LaFuse, Sharon; Nicholson, Leonard S. (Technical Monitor)

1999-01-01

This paper describes the analysis on integration requirements, CO2 compressor in particular, for integration of Carbon Dioxide Removal Assembly (CDRA) and CO2 Reduction Assembly (CRA) as a part of the Node 3 project previously conducted at JSC/NASA. A system analysis on the volume and operation pressure range of the CO2 accumulator was conducted. The hardware and operational configurations of the CO2 compressor were developed. The performance and interface requirements of the compressor were specified. An existing Four-Bed Molecular Sieve CO2 removal computer model was modified into a CDRA model and used in analyzing the requirements of the CDRA CO2 compressor. This CDRA model was also used in analyzing CDRA operation parameters that dictate CO2 pump sizing. Strategy for the pump activation was also analyzed.

Solid state electro-optic color filter and iris

NASA Technical Reports Server (NTRS)

1975-01-01

A pair of solid state electro-optic filters (SSEF) in a binocular holder were designed and fabricated for evaluation of field sequential stereo TV applications. The electronic circuitry for use with the stereo goggles was designed and fabricated, requiring only an external video input. A polarizing screen suitable for attachment to various size TV monitors for use in conjunction with the stereo goggles was designed and fabricated. An improved engineering model 2 filter was fabricated using the bonded holder technique developed previously and integrated to a GCTA color TV camera. An engineering model color filter was fabricated and assembled using PLZT control elements. In addition, a ruggedized holder assembly was designed, fabricated and tested. This assembly provides electrical contacts, high voltage protection, and support for the fragile PLZT disk, and also permits mounting and optical alignment of the associated polarizers.

Thermomechanical Response of Self-Assembled Nanoparticle Membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yifan; Chan, Henry; Narayanan, Badri

2017-07-21

Monolayers composed of colloidal nanoparticles, with a thickness of less than 10 nm, have remarkable mechanical moduli and can suspend over micrometer-sized holes to form free-standing membranes. In this paper, we discuss experiment's and coarse-grained molecular dynamics simulations characterizing the thermomechanical properties of these self-assembled nanoparticle membranes. These membranes remain strong and resilient up to temperatures much higher than previous simulation predictions and exhibit an unexpected hysteretic behavior during the first heating cooling cycle. We show this hysteretic behavior can be explained by an asymmetric ligand configuration from the self assembly process and can be controlled by changing the ligandmore » coverage or cross-linking the ligand molecules. Finally, we show the screening effect of water molecules on the ligand interactions can strongly affect the moduli and thermomechanical behavior.« less

Self-assembly of an electronically conductive network through microporous scaffolds.

PubMed

Sebastian, H Bri; Bryant, Steven L

2017-06-15

Electron transfer spanning significant distances through a microporous structure was established via the self-assembly of an electronically conductive iridium oxide nanowire matrix enveloping the pore walls. Microporous formations were simulated using two scaffold materials of varying physical and chemical properties; paraffin wax beads, and agar gel. Following infiltration into the micropores, iridium nanoparticles self-assembled at the pore wall/ethanol interface. Subsequently, cyclic voltammetry was employed to electrochemically crosslink the metal, erecting an interconnected, and electronically conductive metal oxide nanowire matrix. Electrochemical and spectral characterization techniques confirmed the formation of oxide nanowire matrices encompassing lengths of at least 1.6mm, 400× distances previously achieved using iridium nanoparticles. Nanowire matrices were engaged as biofuel cell anodes, where electrons were donated to the nanowires by a glucose oxidizing enzyme. Copyright © 2017 Elsevier Inc. All rights reserved.

Assembly and Commissioning of Naval Postgraduate School Gas Gun for Impact Studies

DTIC Science & Technology

2009-12-01

MAIN GAS GUN ASSEMBLY............................................................ 12 1. Launcher Mount Assembly...12 Figure 8. Launcher Mount Assembly [After 5].................................................... 13 Figure 9. Breech...5] The main gas gun assembly comprises of eight sub-assemblies. The assemblies are mounted onto the launcher mount assembly, where it acts as a

Lineage-Specific Biology Revealed by a Finished Genome Assembly of the Mouse

PubMed Central

Hillier, LaDeana W.; Zody, Michael C.; Goldstein, Steve; She, Xinwe; Bult, Carol J.; Agarwala, Richa; Cherry, Joshua L.; DiCuccio, Michael; Hlavina, Wratko; Kapustin, Yuri; Meric, Peter; Maglott, Donna; Birtle, Zoë; Marques, Ana C.; Graves, Tina; Zhou, Shiguo; Teague, Brian; Potamousis, Konstantinos; Churas, Christopher; Place, Michael; Herschleb, Jill; Runnheim, Ron; Forrest, Daniel; Amos-Landgraf, James; Schwartz, David C.; Cheng, Ze; Lindblad-Toh, Kerstin; Eichler, Evan E.; Ponting, Chris P.

2009-01-01

The mouse (Mus musculus) is the premier animal model for understanding human disease and development. Here we show that a comprehensive understanding of mouse biology is only possible with the availability of a finished, high-quality genome assembly. The finished clone-based assembly of the mouse strain C57BL/6J reported here has over 175,000 fewer gaps and over 139 Mb more of novel sequence, compared with the earlier MGSCv3 draft genome assembly. In a comprehensive analysis of this revised genome sequence, we are now able to define 20,210 protein-coding genes, over a thousand more than predicted in the human genome (19,042 genes). In addition, we identified 439 long, non–protein-coding RNAs with evidence for transcribed orthologs in human. We analyzed the complex and repetitive landscape of 267 Mb of sequence that was missing or misassembled in the previously published assembly, and we provide insights into the reasons for its resistance to sequencing and assembly by whole-genome shotgun approaches. Duplicated regions within newly assembled sequence tend to be of more recent ancestry than duplicates in the published draft, correcting our initial understanding of recent evolution on the mouse lineage. These duplicates appear to be largely composed of sequence regions containing transposable elements and duplicated protein-coding genes; of these, some may be fixed in the mouse population, but at least 40% of segmentally duplicated sequences are copy number variable even among laboratory mouse strains. Mouse lineage-specific regions contain 3,767 genes drawn mainly from rapidly-changing gene families associated with reproductive functions. The finished mouse genome assembly, therefore, greatly improves our understanding of rodent-specific biology and allows the delineation of ancestral biological functions that are shared with human from derived functions that are not. PMID:19468303

Polymerization properties of the Thermotoga maritima actin MreB: roles of temperature, nucleotides, and ions.

PubMed

Bean, Greg J; Amann, Kurt J

2008-01-15

MreB is a bacterial orthologue of actin that affects cell shape, polarity, and chromosome segregation. Although a significant body of work has explored its cellular functions, we know very little about the biochemical behavior of MreB. We have cloned, overexpressed in Escherichia coli, and purified untagged MreB1 from Thermotoga maritima. We have characterized the conditions that regulate its monomer-to-polymer assembly reaction, the critical concentrations of that reaction, the manner in which MreB uses nucleotides, its stability, and the structure of the assembled polymer. MreB requires a bound purine nucleotide for polymerization and rapidly hydrolyzes it following assembly. MreB assembly contains two distinct components, one that does not require divalent cations and one that does, which may comprise the nucleation and elongation phases of assembly, respectively. MreB assembly is strongly favored by increasing temperature or protein concentration but inhibited differentially by high concentrations of monovalent salts. The polymerization rate increases and the bulk critical concentration decreases with increasing temperature, but in contrast to previous reports, MreB is capable of polymerizing across a broad range of temperatures. MreB polymers are shorter and stiffer and scatter more light than eukaryotic actin filaments. Due to rapid ATP hydrolysis and phosphate release, we suggest that most assembled MreB in cells is in the ADP-bound state. Because of only moderate differences between the ATP and ADP critical concentrations, treadmilling may occur, but we do not predict dynamic instability in cells. Because of the relatively low cellular concentration of MreB and the observed structural properties of the polymer, a single MreB assembly may exist in cells.

Optimizing and benchmarking de novo transcriptome sequencing: from library preparation to assembly evaluation.

PubMed

Hara, Yuichiro; Tatsumi, Kaori; Yoshida, Michio; Kajikawa, Eriko; Kiyonari, Hiroshi; Kuraku, Shigehiro

2015-11-18

RNA-seq enables gene expression profiling in selected spatiotemporal windows and yields massive sequence information with relatively low cost and time investment, even for non-model species. However, there remains a large room for optimizing its workflow, in order to take full advantage of continuously developing sequencing capacity. Transcriptome sequencing for three embryonic stages of Madagascar ground gecko (Paroedura picta) was performed with the Illumina platform. The output reads were assembled de novo for reconstructing transcript sequences. In order to evaluate the completeness of transcriptome assemblies, we prepared a reference gene set consisting of vertebrate one-to-one orthologs. To take advantage of increased read length of >150 nt, we demonstrated shortened RNA fragmentation time, which resulted in a dramatic shift of insert size distribution. To evaluate products of multiple de novo assembly runs incorporating reads with different RNA sources, read lengths, and insert sizes, we introduce a new reference gene set, core vertebrate genes (CVG), consisting of 233 genes that are shared as one-to-one orthologs by all vertebrate genomes examined (29 species)., The completeness assessment performed by the computational pipelines CEGMA and BUSCO referring to CVG, demonstrated higher accuracy and resolution than with the gene set previously established for this purpose. As a result of the assessment with CVG, we have derived the most comprehensive transcript sequence set of the Madagascar ground gecko by means of assembling individual libraries followed by clustering the assembled sequences based on their overall similarities. Our results provide several insights into optimizing de novo RNA-seq workflow, including the coordination between library insert size and read length, which manifested in improved connectivity of assemblies. The approach and assembly assessment with CVG demonstrated here would be applicable to transcriptome analysis of other species as well as whole genome analyses.

Multiligand Metal-Phenolic Assembly from Green Tea Infusions.

PubMed

Rahim, Md Arifur; Björnmalm, Mattias; Bertleff-Zieschang, Nadja; Ju, Yi; Mettu, Srinivas; Leeming, Michael G; Caruso, Frank

2018-03-07

The synthesis of hybrid functional materials using the coordination-driven assembly of metal-phenolic networks (MPNs) is of interest in diverse areas of materials science. To date, MPN assembly has been explored as monoligand systems (i.e., containing a single type of phenolic ligand) where the phenolic components are primarily obtained from natural sources via extraction, isolation, and purification processes. Herein, we demonstrate the fabrication of MPNs from a readily available, crude phenolic source-green tea (GT) infusions. We employ our recently introduced rust-mediated continuous assembly strategy to prepare these GT MPN systems. The resulting hollow MPN capsules contain multiple phenolic ligands and have a shell thickness that can be controlled through the reaction time. These multiligand MPN systems have different properties compared to the analogous MPN systems reported previously. For example, the Young's modulus (as determined using colloidal-probe atomic force microscopy) of the GT MPN system presented herein is less than half that of MPN systems prepared using tannic acid and iron salt solutions, and the disassembly kinetics are faster (∼50%) than other, comparable MPN systems under identical disassembly conditions. Additionally, the use of rust-mediated assembly enables the formation of stable capsules under conditions where the conventional approach (i.e., using iron salt solutions) results in colloidally unstable dispersions. These differences highlight how the choice of phenolic ligand and its source, as well as the assembly protocol (e.g., using solution-based or solid-state iron sources), can be used to tune the properties of MPNs. The strategy presented herein expands the toolbox of MPN assembly while also providing new insights into the nature and robustness of metal-phenolic interfacial assembly when using solution-based or solid-state metal sources.

Protein-Protein Interactions within Late Pre-40S Ribosomes

PubMed Central

Campbell, Melody G.; Karbstein, Katrin

2011-01-01

Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps. PMID:21283762

Protein-protein interactions within late pre-40S ribosomes.

PubMed

Campbell, Melody G; Karbstein, Katrin

2011-01-20

Ribosome assembly in eukaryotic organisms requires more than 200 assembly factors to facilitate and coordinate rRNA transcription, processing, and folding with the binding of the ribosomal proteins. Many of these assembly factors bind and dissociate at defined times giving rise to discrete assembly intermediates, some of which have been partially characterized with regards to their protein and RNA composition. Here, we have analyzed the protein-protein interactions between the seven assembly factors bound to late cytoplasmic pre-40S ribosomes using recombinant proteins in binding assays. Our data show that these factors form two modules: one comprising Enp1 and the export adaptor Ltv1 near the beak structure, and the second comprising the kinase Rio2, the nuclease Nob1, and a regulatory RNA binding protein Dim2/Pno1 on the front of the head. The GTPase-like Tsr1 and the universally conserved methylase Dim1 are also peripherally connected to this second module. Additionally, in an effort to further define the locations for these essential proteins, we have analyzed the interactions between these assembly factors and six ribosomal proteins: Rps0, Rps3, Rps5, Rps14, Rps15 and Rps29. Together, these results and previous RNA-protein crosslinking data allow us to propose a model for the binding sites of these seven assembly factors. Furthermore, our data show that the essential kinase Rio2 is located at the center of the pre-ribosomal particle and interacts, directly or indirectly, with every other assembly factor, as well as three ribosomal proteins required for cytoplasmic 40S maturation. These data suggest that Rio2 could play a central role in regulating cytoplasmic maturation steps.

Interleaved arrays antenna technology development

NASA Technical Reports Server (NTRS)

1986-01-01

Phase one and two of a program to further develop and investigate advanced graphite epoxy waveguides, radiators, and components with application to space antennas are discussed. The objective of the two phases were to demonstrate mechanical integrity of a small panel of radiators and parts procured under a previous contract and to develop alternate designs and applications of the technology. Most of the emphasis was on the assembly and test of a 5 x 5 element module. This effort was supported by evaluation of adhesives and waveguide joint configurations. The evaluation and final assembly considered not only mechanical performance but also producibility in large scale.

Palladium nanoparticles formed on titanium silicate ETS-10.

PubMed

Lin, Christopher C H; Danaie, Mohsen; Mitlin, David; Kuznicki, Steven M

2011-03-01

We report that surface templated and supported palladium nanoparticles self assemble on ETS-10 type molecular sieve surfaces by simple exchange and activation procedures in the absence of a reductant. This procedure is similar to the one previously reported for silver nanoparticle self assembly on ETS-10. We observed a bimodal distribution with particle sizes ranging from 2-5 and 15-30 nm. This simple, economical method generates high concentrations (approximately 12 wt% of total composite) of uniform, metallic palladium nanoparticles that are multiply twinned and thermally stable making them potentially unique for advanced catalytic and electronic applications.

Microfluidic perfusion culture system for multilayer artery tissue models.

PubMed

Yamagishi, Yuka; Masuda, Taisuke; Matsusaki, Michiya; Akashi, Mitsuru; Yokoyama, Utako; Arai, Fumihito

2014-11-01

We described an assembly technique and perfusion culture system for constructing artery tissue models. This technique differed from previous studies in that it does not require a solid biodegradable scaffold; therefore, using sheet-like tissues, this technique allowed the facile fabrication of tubular tissues can be used as model. The fabricated artery tissue models had a multilayer structure. The assembly technique and perfusion culture system were applicable to many different sizes of fabricated arteries. The shape of the fabricated artery tissue models was maintained by the perfusion culture system; furthermore, the system reproduced the in vivo environment and allowed mechanical stimulation of the arteries. The multilayer structure of the artery tissue model was observed using fluorescent dyes. The equivalent Young's modulus was measured by applying internal pressure to the multilayer tubular tissues. The aim of this study was to determine whether fabricated artery tissue models maintained their mechanical properties with developing. We demonstrated both the rapid fabrication of multilayer tubular tissues that can be used as model arteries and the measurement of their equivalent Young's modulus in a suitable perfusion culture environment.

[Advances in the study of the nucleolus].

PubMed

Feng, Jin-Mei; Sun, Jun; Wen, Jian-Fan

2012-12-01

As the most prominent sub-nuclear compartment in the interphase nucleus and the site of ribosome biogenesis, the nucleolus synthesizes and processes rRNA and also assembles ribosomal subunits. Though several lines of research in recent years have indicated that the nucleolus might have additional functions-such as the assembling of signal recognition particles, the processing of mRNA, tRNA and telomerase activities, and regulating the cell cycle-proteomic analyses of the nucleolus in three representative eukaryotic species has shown that a plethora of proteins either have no association with ribosome biogenesis or are of presently unknown function. This phenomenon further indicates that the composition and function of the nucleolus is far more complicated than previously thought. Meanwhile, the available nucleolar proteome databases has provided new approaches and led to remarkable progress in understanding the nucleolus. Here, we have summarized recent advances in the study of the nucleolus, including new discoveries of its structure, function, genomics/proteomics as well as its origin and evolution. Moreover, we highlight several of the important unresolved issues in this field.

QIL1 mutation causes MICOS disassembly and early onset fatal mitochondrial encephalopathy with liver disease

PubMed Central

Guarani, Virginia; Jardel, Claude; Chrétien, Dominique; Lombès, Anne; Bénit, Paule; Labasse, Clémence; Lacène, Emmanuelle; Bourillon, Agnès; Imbard, Apolline; Benoist, Jean-François; Dorboz, Imen; Gilleron, Mylène; Goetzman, Eric S; Gaignard, Pauline; Slama, Abdelhamid; Elmaleh-Bergès, Monique; Romero, Norma B; Rustin, Pierre; Ogier de Baulny, Hélène; Paulo, Joao A; Harper, J Wade; Schiff, Manuel

2016-01-01

Previously, we identified QIL1 as a subunit of mitochondrial contact site (MICOS) complex and demonstrated a role for QIL1 in MICOS assembly, mitochondrial respiration, and cristae formation critical for mitochondrial architecture (Guarani et al., 2015). Here, we identify QIL1 null alleles in two siblings displaying multiple clinical symptoms of early-onset fatal mitochondrial encephalopathy with liver disease, including defects in respiratory chain function in patient muscle. QIL1 absence in patients’ fibroblasts was associated with MICOS disassembly, abnormal cristae, mild cytochrome c oxidase defect, and sensitivity to glucose withdrawal. QIL1 expression rescued cristae defects, and promoted re-accumulation of MICOS subunits to facilitate MICOS assembly. MICOS assembly and cristae morphology were not efficiently rescued by over-expression of other MICOS subunits in patient fibroblasts. Taken together, these data provide the first evidence of altered MICOS assembly linked with a human mitochondrial disease and confirm a central role for QIL1 in stable MICOS complex formation. DOI: http://dx.doi.org/10.7554/eLife.17163.001 PMID:27623147

Loss of SPARC dysregulates basal lamina assembly to disrupt larval fat body homeostasis in Drosophila melanogaster.

PubMed

Shahab, Jaffer; Baratta, Cristina; Scuric, Bianca; Godt, Dorothea; Venken, Koen J T; Ringuette, Maurice J

2015-04-01

SPARC is a collagen-binding glycoprotein whose functions during early development are unknown. We previously reported that SPARC is expressed in Drosophila by hemocytes and the fat body (FB) and enriched in basal laminae (BL) surrounding tissues, including adipocytes. We sought to explore if SPARC is required for proper BL assembly in the FB. SPARC deficiency leads to larval lethality, associated with remodeling of the FB. In the absence of SPARC, FB polygonal adipocytes assume a spherical morphology. Loss-of-function clonal analyses revealed a cell-autonomous accumulation of BL components around mutant cells that include collagen IV (Col lV), Laminin, and Perlecan. Ultrastructural analyses indicate SPARC-deficient adipocytes are surrounded by an aberrant accumulation of a fibrous extracellular matrix. Our data indicate a critical requirement for SPARC for the proper BL assembly in Drosophila FB. Since Col IV within the BL is a prime determinant of cell shape, the rounded appearance of SPARC-deficient adipocytes is due to aberrant assembly of Col IV. © 2014 Wiley Periodicals, Inc.

IHF-independent assembly of the Tn10 strand transfer transpososome: implications for inhibition of disintegration.

PubMed

Stewart, Barry J; Wardle, Simon J; Haniford, David B

2002-08-15

The frequency of DNA transposition in transposition systems that employ a strand transfer step may be significantly affected by the occurrence of a disintegration reaction, a reaction that reverses the strand transfer event. We have asked whether disintegration occurs in the Tn10 transposition system. We show that disintegration substrates (substrates constituting one half of the strand transfer product) are assembled into a transpososome that mimics the strand transfer intermediate. This strand transfer transpososome (STT) does appear to support an intermolecular disintegration reaction, but only at a very low level. Strikingly, assembly of the STT is not dependent on IHF, a host protein that is required for de novo assembly of all previously characterized Tn10 transpososomes. We suggest that disintegration substrates are able to form both transposon end and target type contacts with transposase because of their enhanced conformational flexibility. This probably allows the conformation of DNA within the complex that prevents the destructive disintegration reaction, and is responsible for relaxing the DNA sequence requirements for STT formation relative to other Tn10 transpososomes.

IHF-independent assembly of the Tn10 strand transfer transpososome: implications for inhibition of disintegration

PubMed Central

Stewart, Barry J.; Wardle, Simon J.; Haniford, David B.

2002-01-01

The frequency of DNA transposition in transposition systems that employ a strand transfer step may be significantly affected by the occurrence of a disintegration reaction, a reaction that reverses the strand transfer event. We have asked whether disintegration occurs in the Tn10 transposition system. We show that disintegration substrates (substrates constituting one half of the strand transfer product) are assembled into a transpososome that mimics the strand transfer intermediate. This strand transfer transpososome (STT) does appear to support an intermolecular disintegration reaction, but only at a very low level. Strikingly, assembly of the STT is not dependent on IHF, a host protein that is required for de novo assembly of all previously characterized Tn10 transpososomes. We suggest that disintegration substrates are able to form both transposon end and target type contacts with transposase because of their enhanced conformational flexibility. This probably allows the conformation of DNA within the complex that prevents the destructive disintegration reaction, and is responsible for relaxing the DNA sequence requirements for STT formation relative to other Tn10 transpososomes. PMID:12169640

Self-Powered Forward Error-Correcting Biosensor Based on Integration of Paper-Based Microfluidics and Self-Assembled Quick Response Codes.

PubMed

Yuan, Mingquan; Liu, Keng-Ku; Singamaneni, Srikanth; Chakrabartty, Shantanu

2016-10-01

This paper extends our previous work on silver-enhancement based self-assembling structures for designing reliable, self-powered biosensors with forward error correcting (FEC) capability. At the core of the proposed approach is the integration of paper-based microfluidics with quick response (QR) codes that can be optically scanned using a smart-phone. The scanned information is first decoded to obtain the location of a web-server which further processes the self-assembled QR image to determine the concentration of target analytes. The integration substrate for the proposed FEC biosensor is polyethylene and the patterning of the QR code on the substrate has been achieved using a combination of low-cost ink-jet printing and a regular ballpoint dispensing pen. A paper-based microfluidics channel has been integrated underneath the substrate for acquiring, mixing and flowing the sample to areas on the substrate where different parts of the code can self-assemble in presence of immobilized gold nanorods. In this paper we demonstrate the proof-of-concept detection using prototypes of QR encoded FEC biosensors.

Transfer orbit stage mechanisms thermal vacuum test

NASA Technical Reports Server (NTRS)

Oleary, Scott T.

1990-01-01

A systems level mechanisms test was conducted on the Orbital Sciences Corp.'s Transfer Orbit Stage (TOS). The TOS is a unique partially reusable transfer vehicle which will boost a satellite into its operational orbit from the Space Shuttle's cargo bay. The mechanical cradle and tilt assemblies will return to earth with the Space Shuttle while the Solid Rocket Motor (SRM) and avionics package are expended. A mechanisms test was performed on the forward cradle and aft tilting assemblies of the TOS under thermal vacuum conditions. Actuating these assemblies under a 1 g environment and thermal vacuum conditions proved to be a complex task. Pneumatic test fixturing was used to lift the forward cradle, and tilt the SRM, and avionics package. Clinometers, linear voltage displacement transducers, and load cells were used in the thermal vacuum chamber to measure the performance and characteristics of the TOS mechanism assembly. Incorporation of the instrumentation and pneumatic system into the test setup was not routine since pneumatic actuation of flight hardware had not been previously performed in the facility. The methods used are presented along with the problems experienced during the design, setup and test phases.

Estimating gene gain and loss rates in the presence of error in genome assembly and annotation using CAFE 3.

PubMed

Han, Mira V; Thomas, Gregg W C; Lugo-Martinez, Jose; Hahn, Matthew W

2013-08-01

Current sequencing methods produce large amounts of data, but genome assemblies constructed from these data are often fragmented and incomplete. Incomplete and error-filled assemblies result in many annotation errors, especially in the number of genes present in a genome. This means that methods attempting to estimate rates of gene duplication and loss often will be misled by such errors and that rates of gene family evolution will be consistently overestimated. Here, we present a method that takes these errors into account, allowing one to accurately infer rates of gene gain and loss among genomes even with low assembly and annotation quality. The method is implemented in the newest version of the software package CAFE, along with several other novel features. We demonstrate the accuracy of the method with extensive simulations and reanalyze several previously published data sets. Our results show that errors in genome annotation do lead to higher inferred rates of gene gain and loss but that CAFE 3 sufficiently accounts for these errors to provide accurate estimates of important evolutionary parameters.

Evolution of bird genomes-a transposon's-eye view.

PubMed

Kapusta, Aurélie; Suh, Alexander

2017-02-01

Birds, the most species-rich monophyletic group of land vertebrates, have been subject to some of the most intense sequencing efforts to date, making them an ideal case study for recent developments in genomics research. Here, we review how our understanding of bird genomes has changed with the recent sequencing of more than 75 species from all major avian taxa. We illuminate avian genome evolution from a previously neglected perspective: their repetitive genomic parasites, transposable elements (TEs) and endogenous viral elements (EVEs). We show that (1) birds are unique among vertebrates in terms of their genome organization; (2) information about the diversity of avian TEs and EVEs is changing rapidly; (3) flying birds have smaller genomes yet more TEs than flightless birds; (4) current second-generation genome assemblies fail to capture the variation in avian chromosome number and genome size determined with cytogenetics; (5) the genomic microcosm of bird-TE "arms races" has yet to be explored; and (6) upcoming third-generation genome assemblies suggest that birds exhibit stability in gene-rich regions and instability in TE-rich regions. We emphasize that integration of cytogenetics and single-molecule technologies with repeat-resolved genome assemblies is essential for understanding the evolution of (bird) genomes. © 2016 New York Academy of Sciences.

The Red Spiral Galaxy UGC11680: Clues for the Inside-Out Quenching.

NASA Astrophysics Data System (ADS)

Bárcenas, J.; Sanchez, S. F.

2016-06-01

Broadly, galaxies can be divided in two groups, thanks to the Color-Magnitude Diagram: the lively star formation ones, ``The blue Cloud'' and galaxies which halted their star formation, ``The Red Sequence''. It is a currently accepted that the galaxies start their lifespan as a blue objects, turning red when they stop to assembly more mass and thus more stars. Nevertheless, This change need to be quick (˜ 1 Gyr), due to the dearth of galaxies between this two populations (the so called ``green valley'').Previous works have found two distinct stellar mass assembly modes, they are termed as ``the inside-out'' and ``the outside-in'' growth scenarios in the literature. In the ``inside-out'' scenario, mass assembly is finished in the galactic central region. In some cases, the inflow gas can fuel the central SuperMassive BlackHole. The subsequent AGN feedback will then shut-off the central star formation. One possible case of this scenario is the galaxy UGC11680, an unusual face-on red spiral galaxy with an AGN type 2, at the red sequence belonging to the CALIFA survey. We used the so called fossil method to study its star formation history and try to understand what happened to its stellar populations.

Comparison of taxon-specific versus general locus sets for targeted sequence capture in plant phylogenomics.

PubMed

Chau, John H; Rahfeldt, Wolfgang A; Olmstead, Richard G

2018-03-01

Targeted sequence capture can be used to efficiently gather sequence data for large numbers of loci, such as single-copy nuclear loci. Most published studies in plants have used taxon-specific locus sets developed individually for a clade using multiple genomic and transcriptomic resources. General locus sets can also be developed from loci that have been identified as single-copy and have orthologs in large clades of plants. We identify and compare a taxon-specific locus set and three general locus sets (conserved ortholog set [COSII], shared single-copy nuclear [APVO SSC] genes, and pentatricopeptide repeat [PPR] genes) for targeted sequence capture in Buddleja (Scrophulariaceae) and outgroups. We evaluate their performance in terms of assembly success, sequence variability, and resolution and support of inferred phylogenetic trees. The taxon-specific locus set had the most target loci. Assembly success was high for all locus sets in Buddleja samples. For outgroups, general locus sets had greater assembly success. Taxon-specific and PPR loci had the highest average variability. The taxon-specific data set produced the best-supported tree, but all data sets showed improved resolution over previous non-sequence capture data sets. General locus sets can be a useful source of sequence capture targets, especially if multiple genomic resources are not available for a taxon.

Characterization of two distinct beta2-microglobulin unfolding intermediates that may lead to amyloid fibrils of different morphology.

PubMed

Armen, Roger S; Daggett, Valerie

2005-12-13

The self-assembly of beta(2)-microglobulin into fibrils leads to dialysis-related amyloidosis. pH-mediated partial unfolding is required for the formation of the amyloidogenic intermediate that then self-assembles into amyloid fibrils. Two partially folded intermediates of beta(2)-microglobulin have been identified experimentally and linked to the formation of fibrils of distinct morphology, yet it remains difficult to characterize these partially unfolded states at high resolution using experimental approaches. Consequently, we have performed molecular dynamics simulations at neutral and low pH to determine the structures of these partially unfolded amyloidogenic intermediates. In the low-pH simulations, we observed the formation of alpha-sheet structure, which was first proposed by Pauling and Corey. Multiple simulations were performed, and two distinct intermediate state ensembles were identified that may account for the different fibril morphologies. The predominant early unfolding intermediate was nativelike in structure, in agreement with previous NMR studies. The late unfolding intermediate was significantly disordered, but it maintained an extended elongated structure, with hydrophobic clusters and residual alpha-extended chain strands in specific regions of the sequence that map to amyloidogenic peptides. We propose that the formation of alpha-sheet facilitates self-assembly into partially unfolded prefibrillar amyloidogenic intermediates.

Active Immunization Against hIAPP Oligomers Ameliorates the Diabetes- Associated Phenotype in a Transgenic Mice Model.

PubMed

Bram, Yaron; Peled, Sivan; Brahmachari, Sayanti; Harlev, Michael; Gazit, Ehud

2017-10-25

Type 2 diabetes is characterized by insulin tolerance in target cells followed by a reduction of pancreatic β-cell mass. Islet amyloid polypeptide oligomeric assemblies were shown to contribute to β-cell apoptosis by forming discrete pores that destabilize the cellular membrane. We previously characterized α-helical cytotoxic islet amyloid polypeptide oligomers which interact with cell membranes, following a complete internalization that leads to cellular apoptosis. Moreover, antibodies which bind the oligomers and neutralize the cytotoxicity were exclusively identified in the serum of type 2 diabetes patients. Here, we examined the usage of the newly characterized oligomers as an active immunization agent targeting amyloid self- assembly in a diabetes-associated phenotype transgenic mice model. Immunized transgenic mice showed an increase in hIAPP-antibody serum titer as well as improvement in diabetes-associated parameters. Lower fasting blood glucose levels, higher insulin, and lower islet amyloid polypeptide accumulation were observed. Furthermore, antibodies derived from the immunized mice reduced hIAPP oligomers cytotoxicity towards β-cells in a dose-dependent manner. This study highlights the significance of targeting the early amyloid self-assembly events for potential disease management. Furthermore, it demonstrates that α-helical oligomers conformers are valid epitope for the development of future immunization therapy.

Diversity of anaerobic microbes in spacecraft assembly clean rooms.

PubMed

Probst, Alexander; Vaishampayan, Parag; Osman, Shariff; Moissl-Eichinger, Christine; Andersen, Gary L; Venkateswaran, Kasthuri

2010-05-01

Although the cultivable and noncultivable microbial diversity of spacecraft assembly clean rooms has been previously documented using conventional and state-of-the-art molecular techniques, the occurrence of obligate anaerobes within these clean rooms is still uncertain. Therefore, anaerobic bacterial communities of three clean-room facilities were analyzed during assembly of the Mars Science Laboratory rover. Anaerobic bacteria were cultured on several media, and DNA was extracted from suitable anaerobic enrichments and examined with conventional 16S rRNA gene clone library, as well as high-density phylogenetic 16S rRNA gene microarray (PhyloChip) technologies. The culture-dependent analyses predominantly showed the presence of clostridial and propionibacterial strains. The 16S rRNA gene sequences retrieved from clone libraries revealed distinct microbial populations associated with each clean-room facility, clustered exclusively within gram-positive organisms. PhyloChip analysis detected a greater microbial diversity, spanning many phyla of bacteria, and provided a deeper insight into the microbial community structure of the clean-room facilities. This study presents an integrated approach for assessing the anaerobic microbial population within clean-room facilities, using both molecular and cultivation-based analyses. The results reveal that highly diverse anaerobic bacterial populations persist in the clean rooms even after the imposition of rigorous maintenance programs and will pose a challenge to planetary protection implementation activities.

Physiological spacecraft environment data documentation

NASA Technical Reports Server (NTRS)

1977-01-01

The physiological limits of exposure to environmental parameters encountered during space flight was documented. The environmental limits which have been previously established were described in terms of acceptable physiological changes. The process of coordinating data and assembling the completed data book is described in this report.

A transdisciplinary approach to developing an eastern broccoli industry

USDA-ARS?s Scientific Manuscript database

Current economic, social and scientific situations have made it attractive to establish a substantial and profitable broccoli industry in the eastern US, where several obstacles have prevented that from happening previously. Because many barriers must be overcome simultaneously, we assembled a tran...

Laser-directed 3D assembly of carbon nanotubes using two-photon polymerization (Conference Presentation)

NASA Astrophysics Data System (ADS)

Liu, Ying; Xiong, Wei; Jiang, Li Jia; Zhou, Yunshen; Li, Dawei; Jiang, Lan; Silvain, Jean-Francois; Lu, Yongfeng

2017-02-01

Precise assembly of carbon nanotubes (CNTs) in arbitrary 3D space with proper alignment is critically important and desirable for CNT applications but still remains as a long-standing challenge. Using the two-photon polymerization (TPP) technique, it is possible to fabricate 3D micro/nanoscale CNT/polymer architectures with proper CNT alignments in desired directions, which is expected to enable a broad range of applications of CNTs in functional devices. To unleash the full potential of CNTs, it is strategically important to develop TPP-compatible resins with high CNT concentrations for precise assembly of CNTs into 3D micro/nanostructures for functional device applications. We investigated a thiol grafting method in functionalizing multiwalled carbon nanotubes (MWNTs) to develop TPP-compatible MWNT-thiol-acrylate (MTA) composite resins. The composite resins developed had high MWNT concentrations up to 0.2 wt%, over one order of magnitude higher than previously published work. Significantly enhanced electrical and mechanical properties of the 3D micro/nanostructures were achieved. Precisely controlled MWNT assembly and strong anisotropic effects were confirmed. Microelectronic devices made of the MTA composite polymer were demonstrated. The nanofabrication method can achieve controlled assembly of MWNTs in 3D micro/nanostructures, enabling a broad range of CNT applications, including 3D electronics, integrated photonics, and micro/nanoelectromechanical systems (MEMS/NEMS).

Imbalance in Fatty-Acid-Chain Length of Gangliosides Triggers Alzheimer Amyloid Deposition in the Precuneus

PubMed Central

Oikawa, Naoto; Matsubara, Teruhiko; Fukuda, Ryoto; Yasumori, Hanaki; Hatsuta, Hiroyuki; Murayama, Shigeo; Sato, Toshinori; Suzuki, Akemi; Yanagisawa, Katsuhiko

2015-01-01

Amyloid deposition, a crucial event of Alzheimer’s disease (AD), emerges in distinct brain regions. A key question is what triggers the assembly of the monomeric amyloid ß-protein (Aß) into fibrils in the regions. On the basis of our previous findings that gangliosides facilitate the initiation of Aß assembly at presynaptic neuritic terminals, we investigated how lipids, including gangliosides, cholesterol and sphingomyelin, extracted from synaptic plasma membranes (SPMs) isolated from autopsy brains were involved in the Aß assembly. We focused on two regions of the cerebral cortex; precuneus and calcarine cortex, one of the most vulnerable and one of the most resistant regions to amyloid deposition, respectively. Here, we show that lipids extracted from SPMs isolated from the amyloid-bearing precuneus, but neither the amyloid-free precuneus nor the calcarine cortex, markedly accelerate the Aß assembly in vitro. Through liquid chromatography-mass spectrometry of the lipids, we identified an increase in the ratio of the level of GD1b-ganglioside containing C20:0 fatty acid to that containing C18:0 as a cause of the enhanced Aß assembly in the precuneus. Our results suggest that the local glycolipid environment play a critical role in the initiation of Alzheimer amyloid deposition. PMID:25798597

A systematic comparison of error correction enzymes by next-generation sequencing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lubock, Nathan B.; Zhang, Di; Sidore, Angus M.

Gene synthesis, the process of assembling genelength fragments from shorter groups of oligonucleotides (oligos), is becoming an increasingly important tool in molecular and synthetic biology. The length, quality and cost of gene synthesis are limited by errors produced during oligo synthesis and subsequent assembly. Enzymatic error correction methods are cost-effective means to ameliorate errors in gene synthesis. Previous analyses of these methods relied on cloning and Sanger sequencing to evaluate their efficiencies, limiting quantitative assessment. Here, we develop a method to quantify errors in synthetic DNA by next-generation sequencing. We analyzed errors in model gene assemblies and systematically compared sixmore » different error correction enzymes across 11 conditions. We find that ErrASE and T7 Endonuclease I are the most effective at decreasing average error rates (up to 5.8-fold relative to the input), whereas MutS is the best for increasing the number of perfect assemblies (up to 25.2-fold). We are able to quantify differential specificities such as ErrASE preferentially corrects C/G transversions whereas T7 Endonuclease I preferentially corrects A/T transversions. More generally, this experimental and computational pipeline is a fast, scalable and extensible way to analyze errors in gene assemblies, to profile error correction methods, and to benchmark DNA synthesis methods.« less

Active Glutaminase C Self-assembles into a Supratetrameric Oligomer That Can Be Disrupted by an Allosteric Inhibitor*

PubMed Central

Ferreira, Amanda Petrina Scotá; Cassago, Alexandre; Gonçalves, Kaliandra de Almeida; Dias, Marília Meira; Adamoski, Douglas; Ascenção, Carolline Fernanda Rodrigues; Honorato, Rodrigo Vargas; de Oliveira, Juliana Ferreira; Ferreira, Igor Monteze; Fornezari, Camila; Bettini, Jefferson; Oliveira, Paulo Sérgio Lopes; Paes Leme, Adriana Franco; Portugal, Rodrigo Villares; Ambrosio, Andre Luis Berteli; Dias, Sandra Martha Gomes

2013-01-01

The phosphate-dependent transition between enzymatically inert dimers into catalytically capable tetramers has long been the accepted mechanism for the glutaminase activation. Here, we demonstrate that activated glutaminase C (GAC) self-assembles into a helical, fiber-like double-stranded oligomer and propose a molecular model consisting of seven tetramer copies per turn per strand interacting via the N-terminal domains. The loop 321LRFNKL326 is projected as the major regulating element for self-assembly and enzyme activation. Furthermore, the previously identified in vivo lysine acetylation (Lys311 in humans, Lys316 in mouse) is here proposed as an important down-regulator of superoligomer assembly and protein activation. Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, a known glutaminase inhibitor, completely disrupted the higher order oligomer, explaining its allosteric mechanism of inhibition via tetramer stabilization. A direct correlation between the tendency to self-assemble and the activity levels of the three mammalian glutaminase isozymes was established, with GAC being the most active enzyme while forming the longest structures. Lastly, the ectopic expression of a fiber-prone superactive GAC mutant in MDA-MB 231 cancer cells provided considerable proliferative advantages to transformed cells. These findings yield unique implications for the development of GAC-oriented therapeutics targeting tumor metabolism. PMID:23935106

Failure of Harmonic Gears During Verification of a Two-Axis Gimbal for the Mars Reconnaissance Orbiter Spacecraft

NASA Technical Reports Server (NTRS)

Johnson, Michael R.; Gehling, Russ; Head, Ray

2006-01-01

The Mars Reconnaissance Orbiter (MRO) spacecraft has three two-axis gimbal assemblies that support and move the High Gain Antenna and two solar array wings. The gimbal assemblies are required to move almost continuously throughout the mission's seven-year lifetime, requiring a large number of output revolutions for each actuator in the gimbal assemblies. The actuator for each of the six axes consists of a two-phase brushless dc motor with a direct drive to the wave generator of a size-32 cup-type harmonic gear. During life testing of an actuator assembly, the harmonic gear teeth failed completely, leaving the size-32 harmonic gear with a maximum output torque capability less than 10% of its design capability. The investigation that followed the failure revealed limitations of the heritage material choices that were made for the harmonic gear components that had passed similar life requirements on several previous programs. Additionally, the methods used to increase the stiffness of a standard harmonic gear component set, while accepted practice for harmonic gears, is limited in its range. The stiffness of harmonic gear assemblies can be increased up to a maximum stiffness point that, if exceeded, compromises the reliability of the gear components for long life applications.

A systematic comparison of error correction enzymes by next-generation sequencing

DOE PAGES

Lubock, Nathan B.; Zhang, Di; Sidore, Angus M.; ...

2017-08-01

Gene synthesis, the process of assembling genelength fragments from shorter groups of oligonucleotides (oligos), is becoming an increasingly important tool in molecular and synthetic biology. The length, quality and cost of gene synthesis are limited by errors produced during oligo synthesis and subsequent assembly. Enzymatic error correction methods are cost-effective means to ameliorate errors in gene synthesis. Previous analyses of these methods relied on cloning and Sanger sequencing to evaluate their efficiencies, limiting quantitative assessment. Here, we develop a method to quantify errors in synthetic DNA by next-generation sequencing. We analyzed errors in model gene assemblies and systematically compared sixmore » different error correction enzymes across 11 conditions. We find that ErrASE and T7 Endonuclease I are the most effective at decreasing average error rates (up to 5.8-fold relative to the input), whereas MutS is the best for increasing the number of perfect assemblies (up to 25.2-fold). We are able to quantify differential specificities such as ErrASE preferentially corrects C/G transversions whereas T7 Endonuclease I preferentially corrects A/T transversions. More generally, this experimental and computational pipeline is a fast, scalable and extensible way to analyze errors in gene assemblies, to profile error correction methods, and to benchmark DNA synthesis methods.« less

mTORC1 and CK2 coordinate ternary and eIF4F complex assembly

PubMed Central

Gandin, Valentina; Masvidal, Laia; Cargnello, Marie; Gyenis, Laszlo; McLaughlan, Shannon; Cai, Yutian; Tenkerian, Clara; Morita, Masahiro; Balanathan, Preetika; Jean-Jean, Olivier; Stambolic, Vuk; Trost, Matthias; Furic, Luc; Larose, Louise; Koromilas, Antonis E.; Asano, Katsura; Litchfield, David; Larsson, Ola; Topisirovic, Ivan

2016-01-01

Ternary complex (TC) and eIF4F complex assembly are the two major rate-limiting steps in translation initiation regulated by eIF2α phosphorylation and the mTOR/4E-BP pathway, respectively. How TC and eIF4F assembly are coordinated, however, remains largely unknown. We show that mTOR suppresses translation of mRNAs activated under short-term stress wherein TC recycling is attenuated by eIF2α phosphorylation. During acute nutrient or growth factor stimulation, mTORC1 induces eIF2β phosphorylation and recruitment of NCK1 to eIF2, decreases eIF2α phosphorylation and bolsters TC recycling. Accordingly, eIF2β mediates the effect of mTORC1 on protein synthesis and proliferation. In addition, we demonstrate a formerly undocumented role for CK2 in regulation of translation initiation, whereby CK2 stimulates phosphorylation of eIF2β and simultaneously bolsters eIF4F complex assembly via the mTORC1/4E-BP pathway. These findings imply a previously unrecognized mode of translation regulation, whereby mTORC1 and CK2 coordinate TC and eIF4F complex assembly to stimulate cell proliferation. PMID:27040916

Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

PubMed Central

Faundez, Victor; Koval, Michael; Mattheyses, Alexa L.; Kowalczyk, Andrew P.

2014-01-01

Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV. PMID:24498201

Annotation of a hybrid partial genome of the coffee rust (Hemileia vastatrix) contributes to the gene repertoire catalog of the Pucciniales

PubMed Central

Cristancho, Marco A.; Botero-Rozo, David Octavio; Giraldo, William; Tabima, Javier; Riaño-Pachón, Diego Mauricio; Escobar, Carolina; Rozo, Yomara; Rivera, Luis F.; Durán, Andrés; Restrepo, Silvia; Eilam, Tamar; Anikster, Yehoshua; Gaitán, Alvaro L.

2014-01-01

Coffee leaf rust caused by the fungus Hemileia vastatrix is the most damaging disease to coffee worldwide. The pathogen has recently appeared in multiple outbreaks in coffee producing countries resulting in significant yield losses and increases in costs related to its control. New races/isolates are constantly emerging as evidenced by the presence of the fungus in plants that were previously resistant. Genomic studies are opening new avenues for the study of the evolution of pathogens, the detailed description of plant-pathogen interactions and the development of molecular techniques for the identification of individual isolates. For this purpose we sequenced 8 different H. vastatrix isolates using NGS technologies and gathered partial genome assemblies due to the large repetitive content in the coffee rust hybrid genome; 74.4% of the assembled contigs harbor repetitive sequences. A hybrid assembly of 333 Mb was built based on the 8 isolates; this assembly was used for subsequent analyses. Analysis of the conserved gene space showed that the hybrid H. vastatrix genome, though highly fragmented, had a satisfactory level of completion with 91.94% of core protein-coding orthologous genes present. RNA-Seq from urediniospores was used to guide the de novo annotation of the H. vastatrix gene complement. In total, 14,445 genes organized in 3921 families were uncovered; a considerable proportion of the predicted proteins (73.8%) were homologous to other Pucciniales species genomes. Several gene families related to the fungal lifestyle were identified, particularly 483 predicted secreted proteins that represent candidate effector genes and will provide interesting hints to decipher virulence in the coffee rust fungus. The genome sequence of Hva will serve as a template to understand the molecular mechanisms used by this fungus to attack the coffee plant, to study the diversity of this species and for the development of molecular markers to distinguish races/isolates. PMID:25400655

Ordering pathway of block copolymers under dynamic thermal gradients studied by in situ GISAXS

DOE PAGES

Samant, Saumil; Strzalka, Joseph; Yager, Kevin G.; ...

2016-10-31

Dynamic thermal gradient-based processes for directed self-assembly of block copolymer (BCP) thin films such as cold zone annealing (CZA) have demonstrated much potential for rapidly fabricating highly ordered patterns of BCP domains with facile orientation control. As a demonstration, hexagonally packed predominantly vertical cylindrical morphology, technologically relevant for applications such as membranes and lithography, was achieved in 1 μm thick cylinder-forming PS-b-PMMA (cBCP) films by applying sharp thermal gradients (CZA-Sharp) at optimum sample sweep rates. A thorough understanding of the molecular level mechanisms and pathways of the BCP ordering that occur during this CZA-S process is presented, useful to fullymore » exploit the potential of CZA-S for large-scale BCP-based device fabrication. To that end, we developed a customized CZA-S assembly to probe the dynamic structure evolution and ordering of the PS-b-PMMA cBCP film in situ as it undergoes the CZA-S process using the grazing incidence small-angle X-ray scattering (GISAXS) technique. Four distinct regimes of BCP ordering were observed within the gradient that include microphase separation from an “as cast” unordered state (Regime I), evolution of vertical cylinders under a thermally imposed strain gradient (Regime II), reorientation of a fraction of cylinders due to preferential substrate interactions (Regime III), and finally grain-coarsening on the cooling edge (Regime IV). The ordering pathway in the different regimes is further described within the framework of an energy landscape. A novel aspect of this study is the identification of a grain-coarsening regime on the cooling edge of the gradient, previously obscure in zone annealing studies of BCPs. Furthermore, such insights into the development of highly ordered BCP nanostructures under template-free thermal gradient fields can potentially have important ramifications in the field of BCP-directed self-assembly and self-assembling polymer systems more broadly.« less

Directional RNA-seq reveals highly complex condition-dependent transcriptomes in E. coli K12 through accurate full-length transcripts assembling.

PubMed

Li, Shan; Dong, Xia; Su, Zhengchang

2013-07-30

Although prokaryotic gene transcription has been studied over decades, many aspects of the process remain poorly understood. Particularly, recent studies have revealed that transcriptomes in many prokaryotes are far more complex than previously thought. Genes in an operon are often alternatively and dynamically transcribed under different conditions, and a large portion of genes and intergenic regions have antisense RNA (asRNA) and non-coding RNA (ncRNA) transcripts, respectively. Ironically, similar studies have not been conducted in the model bacterium E coli K12, thus it is unknown whether or not the bacterium possesses similar complex transcriptomes. Furthermore, although RNA-seq becomes the major method for analyzing the complexity of prokaryotic transcriptome, it is still a challenging task to accurately assemble full length transcripts using short RNA-seq reads. To fill these gaps, we have profiled the transcriptomes of E. coli K12 under different culture conditions and growth phases using a highly specific directional RNA-seq technique that can capture various types of transcripts in the bacterial cells, combined with a highly accurate and robust algorithm and tool TruHMM (http://bioinfolab.uncc.edu/TruHmm_package/) for assembling full length transcripts. We found that 46.9 ~ 63.4% of expressed operons were utilized in their putative alternative forms, 72.23 ~ 89.54% genes had putative asRNA transcripts and 51.37 ~ 72.74% intergenic regions had putative ncRNA transcripts under different culture conditions and growth phases. As has been demonstrated in many other prokaryotes, E. coli K12 also has a highly complex and dynamic transcriptomes under different culture conditions and growth phases. Such complex and dynamic transcriptomes might play important roles in the physiology of the bacterium. TruHMM is a highly accurate and robust algorithm for assembling full-length transcripts in prokaryotes using directional RNA-seq short reads.

Directional RNA-seq reveals highly complex condition-dependent transcriptomes in E. coli K12 through accurate full-length transcripts assembling

PubMed Central

2013-01-01

Background Although prokaryotic gene transcription has been studied over decades, many aspects of the process remain poorly understood. Particularly, recent studies have revealed that transcriptomes in many prokaryotes are far more complex than previously thought. Genes in an operon are often alternatively and dynamically transcribed under different conditions, and a large portion of genes and intergenic regions have antisense RNA (asRNA) and non-coding RNA (ncRNA) transcripts, respectively. Ironically, similar studies have not been conducted in the model bacterium E coli K12, thus it is unknown whether or not the bacterium possesses similar complex transcriptomes. Furthermore, although RNA-seq becomes the major method for analyzing the complexity of prokaryotic transcriptome, it is still a challenging task to accurately assemble full length transcripts using short RNA-seq reads. Results To fill these gaps, we have profiled the transcriptomes of E. coli K12 under different culture conditions and growth phases using a highly specific directional RNA-seq technique that can capture various types of transcripts in the bacterial cells, combined with a highly accurate and robust algorithm and tool TruHMM (http://bioinfolab.uncc.edu/TruHmm_package/) for assembling full length transcripts. We found that 46.9 ~ 63.4% of expressed operons were utilized in their putative alternative forms, 72.23 ~ 89.54% genes had putative asRNA transcripts and 51.37 ~ 72.74% intergenic regions had putative ncRNA transcripts under different culture conditions and growth phases. Conclusions As has been demonstrated in many other prokaryotes, E. coli K12 also has a highly complex and dynamic transcriptomes under different culture conditions and growth phases. Such complex and dynamic transcriptomes might play important roles in the physiology of the bacterium. TruHMM is a highly accurate and robust algorithm for assembling full-length transcripts in prokaryotes using directional RNA-seq short reads. PMID:23899370

Do supercontinents introvert or extrovert?: Sm-Nd isotope evidence

NASA Astrophysics Data System (ADS)

Brendan Murphy, J.; Damian Nance, R.

2003-10-01

In recent years, two end-member models for the formation of supercontinents have emerged. In the classical Wilson cycle, oceanic crust generated during supercontinent breakup (the interior ocean) is consumed during subsequent amalgamation so that the supercontinent turns “inside in” (introversion). Alternatively, following supercontinent breakup, the exterior margins of the dispersing continental fragments collide during reassembly so that the supercontinent turns “outside in” (extroversion). These end-member models can be distinguished by comparing the Sm-Nd crust-formation ages of accreted mafic complexes (e.g., ophiolites) in the collisional orogens formed during supercontinent assembly with the breakup age of the previous supercontinent. For supercontinents generated by introversion, these crust-formation ages postdate rifting of the previous supercontinent. For supercontinents generated by extroversion, the oceanic lithosphere consumed during reassembly predates breakup of the previous supercontinent, so that crust-formation ages of accreted mafic complexes are older than the age of rifting. In the Paleozoic Appalachian-Caledonide-Variscan orogen, a key collisional orogen in the assembly of Pangea, crust-formation ages of accretionary mafic complexes postdate the formation of the Iapetus Ocean (i.e., are younger than ca. 0.6 Ga), suggesting supercontinent reassembly by introversion. By contrast, the Neoproterozoic East African and Brasiliano orogens, which formed during the amalgamation of Gondwana, are characterized by mafic complexes with crust-formation ages (ca. 0.75 1.2 Ga) that predate the ca. 750 Ma breakup of Rodinia. Hence, these complexes must have formed from lithosphere in the exterior ocean that surrounded Rodinia, implying that this ocean was consumed during the amalgamation of Gondwana. These data indicate that Pangea and Gondwana were formed by introversion and extroversion, respectively, implying that supercontinents can be assembled by fundamentally distinct geodynamic processes.

Exact algorithms for haplotype assembly from whole-genome sequence data.

PubMed

Chen, Zhi-Zhong; Deng, Fei; Wang, Lusheng

2013-08-15

Haplotypes play a crucial role in genetic analysis and have many applications such as gene disease diagnoses, association studies, ancestry inference and so forth. The development of DNA sequencing technologies makes it possible to obtain haplotypes from a set of aligned reads originated from both copies of a chromosome of a single individual. This approach is often known as haplotype assembly. Exact algorithms that can give optimal solutions to the haplotype assembly problem are highly demanded. Unfortunately, previous algorithms for this problem either fail to output optimal solutions or take too long time even executed on a PC cluster. We develop an approach to finding optimal solutions for the haplotype assembly problem under the minimum-error-correction (MEC) model. Most of the previous approaches assume that the columns in the input matrix correspond to (putative) heterozygous sites. This all-heterozygous assumption is correct for most columns, but it may be incorrect for a small number of columns. In this article, we consider the MEC model with or without the all-heterozygous assumption. In our approach, we first use new methods to decompose the input read matrix into small independent blocks and then model the problem for each block as an integer linear programming problem, which is then solved by an integer linear programming solver. We have tested our program on a single PC [a Linux (x64) desktop PC with i7-3960X CPU], using the filtered HuRef and the NA 12878 datasets (after applying some variant calling methods). With the all-heterozygous assumption, our approach can optimally solve the whole HuRef data set within a total time of 31 h (26 h for the most difficult block of the 15th chromosome and only 5 h for the other blocks). To our knowledge, this is the first time that MEC optimal solutions are completely obtained for the filtered HuRef dataset. Moreover, in the general case (without the all-heterozygous assumption), for the HuRef dataset our approach can optimally solve all the chromosomes except the most difficult block in chromosome 15 within a total time of 12 days. For both of the HuRef and NA12878 datasets, the optimal costs in the general case are sometimes much smaller than those in the all-heterozygous case. This implies that some columns in the input matrix (after applying certain variant calling methods) still correspond to false-heterozygous sites. Our program, the optimal solutions found for the HuRef dataset available at http://rnc.r.dendai.ac.jp/hapAssembly.html.

Arenavirus Coinfections Are Common in Snakes with Boid Inclusion Body Disease.

PubMed

Hepojoki, J; Salmenperä, P; Sironen, T; Hetzel, U; Korzyukov, Y; Kipar, A; Vapalahti, O

2015-08-01

Recently, novel arenaviruses were found in snakes with boid inclusion body disease (BIBD); these form the new genus Reptarenavirus within the family Arenaviridae. We used next-generation sequencing and de novo sequence assembly to investigate reptarenavirus isolates from our previous study. Four of the six isolates and all of the samples from snakes with BIBD contained at least two reptarenavirus species. The viruses sequenced comprise four novel reptarenavirus species and a representative of a new arenavirus genus. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Consolidated fuel reprocessing program

NASA Astrophysics Data System (ADS)

1985-04-01

A survey of electrochemical methods applications in fuel reprocessing was completed. A dummy fuel assembly shroud was cut using the remotely operated laser disassembly equipment. Operations and engineering efforts have continued to correct equipment operating, software, and procedural problems experienced during the previous uranium compaigns. Fuel cycle options were examined for the liquid metal reactor fuel cycle. In high temperature gas cooled reactor spent fuel studies, preconceptual designs were completed for the concrete storage cask and open field drywell storage concept. These and other tasks operating under the consolidated fuel reprocessing program are examined.

Concept definition for space station technology development experiments. Experiment definition, task 2

NASA Technical Reports Server (NTRS)

1986-01-01

The second task of a study with the overall objective of providing a conceptual definition of the Technology Development Mission Experiments proposed by LaRC on space station is discussed. During this task, the information (goals, objectives, and experiment functional description) assembled on a previous task was translated into the actual experiment definition. Although still of a preliminary nature, aspects such as: environment, sensors, data acquisition, communications, handling, control telemetry requirements, crew activities, etc., were addressed. Sketches, diagrams, block diagrams, and timeline analyses of crew activities are included where appropriate.

Metagenomic Sequencing of an Echovirus 30 Genome From Cerebrospinal Fluid of a Patient With Aseptic Meningitis and Orchitis.

PubMed

Piantadosi, Anne; Mukerji, Shibani S; Chitneni, Pooja; Cho, Tracey A; Cosimi, Lisa A; Hung, Deborah T; Goldberg, Marcia B; Sabeti, Pardis C; Kuritzkes, Daniel R; Grad, Yonatan H

2017-01-01

Enteroviruses cause a wide spectrum of clinical disease. In this study, we describe the case of a young man with orchitis and aseptic meningitis who was diagnosed with enterovirus infection. Using unbiased "metagenomic" massively parallel sequencing, we assembled a near-complete viral genome, the first use of this method for full-genome viral sequencing from cerebrospinal fluid. We found that the genome belonged to the subgroup echovirus 30, which is a common cause of aseptic meningitis but has not been previously reported to cause orchitis.

An efficient and scalable graph modeling approach for capturing information at different levels in next generation sequencing reads

PubMed Central

2013-01-01

Background Next generation sequencing technologies have greatly advanced many research areas of the biomedical sciences through their capability to generate massive amounts of genetic information at unprecedented rates. The advent of next generation sequencing has led to the development of numerous computational tools to analyze and assemble the millions to billions of short sequencing reads produced by these technologies. While these tools filled an important gap, current approaches for storing, processing, and analyzing short read datasets generally have remained simple and lack the complexity needed to efficiently model the produced reads and assemble them correctly. Results Previously, we presented an overlap graph coarsening scheme for modeling read overlap relationships on multiple levels. Most current read assembly and analysis approaches use a single graph or set of clusters to represent the relationships among a read dataset. Instead, we use a series of graphs to represent the reads and their overlap relationships across a spectrum of information granularity. At each information level our algorithm is capable of generating clusters of reads from the reduced graph, forming an integrated graph modeling and clustering approach for read analysis and assembly. Previously we applied our algorithm to simulated and real 454 datasets to assess its ability to efficiently model and cluster next generation sequencing data. In this paper we extend our algorithm to large simulated and real Illumina datasets to demonstrate that our algorithm is practical for both sequencing technologies. Conclusions Our overlap graph theoretic algorithm is able to model next generation sequencing reads at various levels of granularity through the process of graph coarsening. Additionally, our model allows for efficient representation of the read overlap relationships, is scalable for large datasets, and is practical for both Illumina and 454 sequencing technologies. PMID:24564333

Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters.

PubMed

Dallery, Jean-Félix; Lapalu, Nicolas; Zampounis, Antonios; Pigné, Sandrine; Luyten, Isabelle; Amselem, Joëlle; Wittenberg, Alexander H J; Zhou, Shiguo; de Queiroz, Marisa V; Robin, Guillaume P; Auger, Annie; Hainaut, Matthieu; Henrissat, Bernard; Kim, Ki-Tae; Lee, Yong-Hwan; Lespinet, Olivier; Schwartz, David C; Thon, Michael R; O'Connell, Richard J

2017-08-29

The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications. The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.

Neuronal growth on L- and D-cysteine self-assembled monolayers reveals neuronal chiral sensitivity.

PubMed

Baranes, Koby; Moshe, Hagay; Alon, Noa; Schwartz, Shmulik; Shefi, Orit

2014-05-21

Studying the interaction between neuronal cells and chiral molecules is fundamental for the design of novel biomaterials and drugs. Chirality influences all biological processes that involve intermolecular interaction. One common method used to study cellular interactions with different enantiomeric targets is the use of chiral surfaces. Based on previous studies that demonstrated the importance of cysteine in the nervous system, we studied the effect of L- and D-cysteine on single neuronal growth. L-Cysteine, which normally functions as a neuromodulator or a neuroprotective antioxidant, causes damage at elevated levels, which may occur post trauma. In this study, we grew adult neurons in culture enriched with L- and D-cysteine as free compounds or as self-assembled monolayers of chiral surfaces and examined the effect on the neuronal morphology and adhesion. Notably, we have found that exposure to the L-cysteine enantiomer inhibited, and even prevented, neuronal attachment more severely than exposure to the D-cysteine enantiomer. Atop the L-cysteine surfaces, neuronal growth was reduced and degenerated. Since the cysteine molecules were attached to the surface via the thiol groups, the neuronal membrane was exposed to the molecular chiral site. Thus, our results have demonstrated high neuronal chiral sensitivity, revealing chiral surfaces as indirect regulators of neuronal cells and providing a reference for studying chiral drugs.

A definition study of the on-orbit assembly operations for the outboard photovoltaic power modules for Space Station Freedom. M.S. Thesis - Toledo Univ.

NASA Technical Reports Server (NTRS)

Sours, Thomas J.

1989-01-01

A concept is described for the assembly of the outboard PV modules for Space Station Freedom. Analysis of the on-orbit assembly operations was performed using CADAM design graphics software. A scenario for assembly using the various assembly equipment, as currently defined, is described in words, tables and illustrations. This work is part of ongoing studies in the area of space station assembly. The outboard PV module and the assembly equipment programs are all in definition and preliminary design phases. An input is provided to the design process of assembly equipment programs. It is established that the outboard PV module assembly operations can be performed using the assembly equipment currently planned in the Space Station Freedom Program.

Evaluation of nine popular de novo assemblers in microbial genome assembly.

PubMed

Forouzan, Esmaeil; Maleki, Masoumeh Sadat Mousavi; Karkhane, Ali Asghar; Yakhchali, Bagher

2017-12-01

Next generation sequencing (NGS) technologies are revolutionizing biology, with Illumina being the most popular NGS platform. Short read assembly is a critical part of most genome studies using NGS. Hence, in this study, the performance of nine well-known assemblers was evaluated in the assembly of seven different microbial genomes. Effect of different read coverage and k-mer parameters on the quality of the assembly were also evaluated on both simulated and actual read datasets. Our results show that the performance of assemblers on real and simulated datasets could be significantly different, mainly because of coverage bias. According to outputs on actual read datasets, for all studied read coverages (of 7×, 25× and 100×), SPAdes and IDBA-UD clearly outperformed other assemblers based on NGA50 and accuracy metrics. Velvet is the most conservative assembler with the lowest NGA50 and error rate. Copyright © 2017. Published by Elsevier B.V.

Preburner of Staged Combustion Rocket Engine

NASA Technical Reports Server (NTRS)

Yost, M. C.

1978-01-01

A regeneratively cooled LOX/hydrogen staged combustion assembly system with a 400:1 expansion area ratio nozzle utilizing an 89,000 Newton (20,000 pound) thrust regeneratively cooled thrust chamber and 175:1 tubular nozzle was analyzed, assembled, and tested. The components for this assembly include two spark/torch oxygen-hydrogen igniters, two servo-controlled LOX valves, a preburner injector, a preburner combustor, a main propellant injector, a regeneratively cooled combustion chamber, a regeneratively cooled tubular nozzle with an expansion area ratio of 175:1, an uncooled heavy-wall steel nozzle with an expansion area ratio of 400:1, and interconnecting ducting. The analytical effort was performed to optimize the thermal and structural characteristics of each of the new components and the ducting, and to reverify the capabilities of the previously fabricated components. The testing effort provided a demonstration of the preburner/combustor chamber operation, chamber combustion efficiency and stability, and chamber and nozzle heat transfer.

Computational design of a self-assembling symmetrical β-propeller protein.

PubMed

Voet, Arnout R D; Noguchi, Hiroki; Addy, Christine; Simoncini, David; Terada, Daiki; Unzai, Satoru; Park, Sam-Yong; Zhang, Kam Y J; Tame, Jeremy R H

2014-10-21

The modular structure of many protein families, such as β-propeller proteins, strongly implies that duplication played an important role in their evolution, leading to highly symmetrical intermediate forms. Previous attempts to create perfectly symmetrical propeller proteins have failed, however. We have therefore developed a new and rapid computational approach to design such proteins. As a test case, we have created a sixfold symmetrical β-propeller protein and experimentally validated the structure using X-ray crystallography. Each blade consists of 42 residues. Proteins carrying 2-10 identical blades were also expressed and purified. Two or three tandem blades assemble to recreate the highly stable sixfold symmetrical architecture, consistent with the duplication and fusion theory. The other proteins produce different monodisperse complexes, up to 42 blades (180 kDa) in size, which self-assemble according to simple symmetry rules. Our procedure is suitable for creating nano-building blocks from different protein templates of desired symmetry.

Description of the PMAD DC test bed architecture and integration sequence

NASA Technical Reports Server (NTRS)

Beach, R. F.; Trash, L.; Fong, D.; Bolerjack, B.

1991-01-01

NASA-LEWIS is responsible for the development, fabrication, and assembly of the electric power system (EPS) for the Space Station Freedom (SSF). The SSF power system is radically different from previous spacecraft power systems in both the size and complexity of the system. Unlike past spacecraft power systems, the SSF EPS will grow and be maintained on orbit and must be flexible to meet challenging user power needs. The SSF power system is also unique in comparison with terrestrial power systems because it is dominated by power electronic converters which regulate and control the power. A description is provided of the Power Management and Distribution DC Testbed which was assembled to support the design and early evaluation of the SSF EPS. A description of the integration process used in the assembly sequence is also given along with a description of the support facility.

Engineering the entropy-driven free-energy landscape of a dynamic nanoporous protein assembly.

PubMed

Alberstein, Robert; Suzuki, Yuta; Paesani, Francesco; Tezcan, F Akif

2018-04-30

De novo design and construction of stimuli-responsive protein assemblies that predictably switch between discrete conformational states remains an essential but highly challenging goal in biomolecular design. We previously reported synthetic, two-dimensional protein lattices self-assembled via disulfide bonding interactions, which endows them with a unique capacity to undergo coherent conformational changes without losing crystalline order. Here, we carried out all-atom molecular dynamics simulations to map the free-energy landscape of these lattices, validated this landscape through extensive structural characterization by electron microscopy and established that it is predominantly governed by solvent reorganization entropy. Subsequent redesign of the protein surface with conditionally repulsive electrostatic interactions enabled us to predictably perturb the free-energy landscape and obtain a new protein lattice whose conformational dynamics can be chemically and mechanically toggled between three different states with varying porosities and molecular densities.

Structure and assembly of a paramyxovirus matrix protein

PubMed Central

Battisti, Anthony J.; Meng, Geng; Winkler, Dennis C.; McGinnes, Lori W.; Plevka, Pavel; Steven, Alasdair C.; Morrison, Trudy G.; Rossmann, Michael G.

2012-01-01

Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host’s cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly. PMID:22891297

Structure and assembly of a paramyxovirus matrix protein.

PubMed

Battisti, Anthony J; Meng, Geng; Winkler, Dennis C; McGinnes, Lori W; Plevka, Pavel; Steven, Alasdair C; Morrison, Trudy G; Rossmann, Michael G

2012-08-28

Many pleomorphic, lipid-enveloped viruses encode matrix proteins that direct their assembly and budding, but the mechanism of this process is unclear. We have combined X-ray crystallography and cryoelectron tomography to show that the matrix protein of Newcastle disease virus, a paramyxovirus and relative of measles virus, forms dimers that assemble into pseudotetrameric arrays that generate the membrane curvature necessary for virus budding. We show that the glycoproteins are anchored in the gaps between the matrix proteins and that the helical nucleocapsids are associated in register with the matrix arrays. About 90% of virions lack matrix arrays, suggesting that, in agreement with previous biological observations, the matrix protein needs to dissociate from the viral membrane during maturation, as is required for fusion and release of the nucleocapsid into the host's cytoplasm. Structure and sequence conservation imply that other paramyxovirus matrix proteins function similarly.

Self-assembled nanoparticle of common food constituents that carries a sparingly soluble small molecule.

PubMed

Bhopatkar, Deepak; Feng, Tao; Chen, Feng; Zhang, Genyi; Carignano, Marcelo; Park, Sung Hyun; Zhuang, Haining; Campanella, Osvaldo H; Hamaker, Bruce R

2015-05-06

A previously reported nanoparticle formed through the self-assembly of common food constituents (amylose, protein, and fatty acids) was shown to have the capacity to carry a sparingly soluble small molecule (1-naphthol) in a dispersed system. Potentiometric titration showed that 1-naphthol locates in the lumen of the amylose helix of the nanoparticle. This finding was further supported by calorimetric measurements, showing higher enthalpies of dissociation and reassociation in the presence of 1-naphthol. Visually, the 1-naphthol-loaded nanoparticle appeared to be well-dispersed in aqueous solution. Molecular dynamics simulation showed that the self-assembly was favorable, and at 500 ns, the 1-naphthol molecule resided in the helix of the amylose lumen in proximity to the hydrophobic tail of the fatty acid. Thus, sparingly soluble small molecules, such as some nutraceuticals or drugs, could be incorporated and delivered by this soft nanoparticle carrier.

Reprogrammable Assembly of Molecular Motor on Solid Surfaces via Dynamic Bonds.

PubMed

Yu, Li; Sun, Jian; Wang, Qian; Guan, Yan; Zhou, Le; Zhang, Jingxuan; Zhang, Lanying; Yang, Huai

2017-06-01

Controllable assembly of molecular motors on solid surfaces is a fundamental issue for providing them to perform physical tasks. However, it can hardly be achieved by most previous methods due to their inherent limitations. Here, a general strategy is designed for the reprogrammable assembly of molecular motors on solid surfaces based on dynamic bonds. In this method, molecular motors with disulfide bonds can be remotely, reversibly, and precisely attached to solid surfaces with disulfide bonds, regardless of their chemical composition and microstructure. More importantly, it not only allows encoding geometric information referring to a pattern of molecular motors, but also enables erasing and re-encoding of geometric information via hemolytic photocleavage and recombination of disulfide bonds. Thus, solid surfaces can be regarded as "computer hardware", where molecular motors can be reformatted and reprogramed as geometric information. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase.

PubMed

Takahata, Tatsuro; Takeda, Eri; Tobiume, Minoru; Tokunaga, Kenzo; Yokoyama, Masaru; Huang, Yu-Lun; Hasegawa, Atsuhiko; Shioda, Tatsuo; Sato, Hironori; Kannagi, Mari; Masuda, Takao

2017-01-01

Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied in trans revealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form. Inhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINIs affect the nonenzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its nonenzymatic function. Our results provide a novel mechanistic insight into the nonenzymatic function of HIV-1 IN and its prevention. Copyright © 2016 American Society for Microbiology.

Development of termination and utilization concepts for flat conductor cables. Volume 3: Cost study comparison, flat versus round conductor cable

NASA Technical Reports Server (NTRS)

1972-01-01

A cost study comparing flat conductor cable (FCC) with small-gage wire (SGW) and conventional round conductor cable (RCC) is presented. This study was based on a vehicle wiring system consisting of 110,000 ft of conventional RCC equally divided between AWG sizes 20,22, and 24 using MIL-W-81044-type wire and MIL-C-26500 circular connectors. Basic cost data were developed on a similar-sized commercial jet airplane wiring system on a previous company R&D program in which advanced wiring techniques were carried through equivalent installations on an airplane mockup; and on data developed on typical average bundles during this program. Various cost elements included were engineering labor, operations (manufacturing) labor, material costs, and cost impact on payload. Engineering labor includes design, wiring system integration, wiring diagrams and cable assembly drawings, wire installations, and other related supporting functions such as the electronic data processing for the wiring. Operations labor includes mockup, tooling and production planning, fabrication, assembly, installation, and quality control cost impact on payload is the conversion of wiring system weight variations through use of different wiring concepts to program payload benefits in terms of dollars.

UV-Resistant Non-Spore-Forming Bacteria From Spacecraft-Assembly Facilities

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri

2008-01-01

Four species of non-spore-forming bacteria collected from clean-room surfaces in spacecraft-assembly facilities could survive doses of ultraviolet (UV) radiation that would suffice to kill most known cultivable bacterial species. In a previous study, high UV resistance was found in spores of the SAFR-032 strain of Bacillus pumilus, as reported in "Ultraviolet- Resistant Bacterial Spores," NASA Tech Briefs, Vol. 31, No. 9 (September 2007), page 94. These studies are parts of a continuing effort to understand the survival of hardy species of bacteria under harsh conditions, and develop means of sterilizing spacecraft to prevent biocontamination of Mars that could in turn interfere with future life detection missions. The four species investigated were Arthrobacter sp. KSC_Ak2i, Microbacterium schleiferi LMA_AkK1, Brevundimonas diminuta KSC_Ak3a, and Sphingomonas trueperi JSC_Ak7-3. In the study, cells of these species were mixed into Atacama Desert soil (to elucidate the shadowing effect of soil particles) and the resulting mixtures were tested both in solution and in a desiccated state under simulated Martian atmospheric and UV conditions. The UV-survival indices of Arthrobacter sp. and Microbacterium schleiferi were found to be comparable to those of Bacillus pumilus spores.

The canonical WNT2 pathway and FSH interact to regulate gap junction assembly in mouse granulosa cells.

PubMed

Wang, Hong-Xing; Gillio-Meina, Carolina; Chen, Shuli; Gong, Xiang-Qun; Li, Tony Y; Bai, Donglin; Kidder, Gerald M

2013-08-01

WNTs are extracellular signaling molecules that exert their actions through receptors of the frizzled (FZD) family. Previous work indicated that WNT2 regulates cell proliferation in mouse granulosa cells acting through CTNNB1 (beta-catenin), a key component in canonical WNT signaling. In other cells, WNT signaling has been shown to regulate expression of connexin43 (CX43), a gap junction protein, as well as gap junction assembly. Since previous work demonstrated that CX43 is also essential in ovarian follicle development, the objective of this study was to determine if WNT2 regulates CX43 expression and/or gap-junctional intercellular communication (GJIC) in granulosa cells. WNT2 knockdown via siRNA markedly reduced CX43 expression and GJIC. CX43 expression, the extent of CX43-containing gap junction membrane, and GJIC were also reduced by CTNNB1 transient knockdown. CTNNB1 is mainly localized to the membranes between granulosa cells but disappeared from this location after WNT2 knockdown. Furthermore, CTNNB1 knockdown interfered with the ability of follicle-stimulating hormone (FSH) to promote the mobilization of CX43 into gap junctions. We propose that the WNT2/CTNNB1 pathway regulates CX43 expression and GJIC in granulosa cells by modulating CTNNB1 stability and localization in adherens junctions, and that this is essential for FSH stimulation of GJIC.

Injection tube differentiation in gun cells of a haptoglossa species which infects nematodes

PubMed

Beakes; Glockling

1998-06-01

The gun cells which develop from germinating cysts in Haptoglossa produce a specialized infection apparatus, the injection tube. Upon eversion this tube fires a missile-like projectile which penetrates the host cuticle and then forms an infective sporidium within the body cavity of the nematode host. The temporal assembly of this complex cell organelle has been determined by serial-section reconstructions of maturing gun cells in a previously undescribed Haptoglossa species. The differentiation of the partially walled inverted injection tube is an unusual example of internal tube growth, in which membrane and wall assembly are temporally separated. There is no evidence that the shape of this inverted tube, which coils around the nucleus until it doubles back on itself, is dictated by the disposition of cytoplasmic microtubules. However, actin-like material was associated with the delimiting membrane of the differentiating tube, particularly in the regions of extension. From these studies it seems likely that the "head and buttress" structures previously depicted as the barbed tip of the "harpoon-like" penetration missile are part of a separate, structurally complex system which we suggest locks the "missile" into position in the invaginated injection tube. From this detailed account of cell architecture, models for the likely mechanism of infection cell firing are discussed, and unresolved questions relating to the cell biology and biochemistry of these complex organelles are highlighted. Copyright 1998 Academic Press.

75 FR 12441 - Airworthiness Directives; MD Helicopters, Inc. Model MD-900 Helicopters

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-16

..., DOT. ACTION: Final rule. SUMMARY: This amendment supersedes an existing airworthiness directive (AD... numbers to certain parts, increasing the life limit for various parts, maintaining a previously established life limit for a certain vertical stabilizer control system (VSCS) bellcrank assembly and...

Individualized FAC on bottom tab subassemblies to minimize adhesive gap between emitter and optics

NASA Astrophysics Data System (ADS)

Sauer, Sebastian; Müller, Tobias; Haag, Sebastian; Beleke, Andreas; Zontar, Daniel; Baum, Christoph; Brecher, Christian

2017-02-01

High Power Diode Laser (HPDL) systems with short focal length fast-axis collimators (FAC) require submicron assembly precision. Conventional FAC-Lens assembly processes require adhesive gaps of 50 microns or more in order to compensate for component tolerances (e.g. deviation of back focal length) and previous assembly steps. In order to control volumetric shrinkage of fast-curing UV-adhesives shrinkage compensation is mandatory. The novel approach described in this paper aims to minimize the impact of volumetric shrinkage due to the adhesive gap between HPDL edge emitters and FAC-Lens. Firstly, the FAC is actively aligned to the edge emitter without adhesives or bottom tab. The relative position and orientation of FAC to emitter are measured and stored. Consecutively, an individual subassembly of FAC and bottom tab is assembled on Fraunhofer IPT's mounting station with a precision of +/-1 micron. Translational and lateral offsets can be compensated, so that a narrow and uniform glue gap for the consecutive bonding process of bottom tab to heatsink applies (Figure 4). Accordingly, FAC and bottom tab are mounted to the heatsink without major shrinkage compensation. Fraunhofer IPT's department assembly of optical systems and automation has made several publications regarding active alignment of FAC lenses [SPIE LASE 8241-12], volumetric shrinkage compensation [SPIE LASE 9730-28] and FAC on bottom tab assembly [SPIE LASE 9727-31] in automated production environments. The approach described in this paper combines these and is the logical continuation of that work towards higher quality of HPDLs.

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways.

PubMed

Wingler, Laura M; Cornish, Virginia W

2011-09-13

The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop "Reiterative Recombination," a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10(4) biosynthetic pathways. We anticipate that our system's simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

PubMed Central

Wingler, Laura M.; Cornish, Virginia W.

2011-01-01

The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop “Reiterative Recombination,” a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 104 biosynthetic pathways. We anticipate that our system’s simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo. PMID:21876185

Revisiting the Fundamentals in the Design and Control of Nanoparticulate Colloids in the Frame of Soft Chemistry.

PubMed

Uskoković, Vuk

2013-10-01

This review presents thoughts on some of the fundamental features of conceptual models applied in the design of fine particles in the frames of colloid and soft chemistry. A special emphasis is placed on the limitations of these models, an acknowledgment of which is vital in improving their intricacy and effectiveness in predicting the outcomes of the corresponding experimental settings. Thermodynamics of self-assembly phenomena illustrated on the examples of protein assembly and micellization is analyzed in relation to the previously elaborated thesis that each self-assembly in reality presents a co-assembly, since it implies a mutual reorganization of the assembling system and its immediate environment. Parameters used in the design of fine particles by precipitation are discussed while referring to solubility product, various measures of supersaturation levels, induction time, nucleation and crystal growth rates, interfacial energies, and the Ostwald-Lussac law of phases. Again, the main drawbacks and inadequacies of using the aforementioned parameters in tailoring the materials properties in a soft and colloidal chemical setting were particularly emphasized. The basic and practical limitations of zeta-potential analyses, routinely used to stabilize colloidal dispersions and initiate specific interactions between soft chemical entities, were also outlined. The final section of the paper reiterates the unavoidable presence of practical qualitative models in the design and control of nanoparticulate colloids, which is supported by the overwhelming complexity of quantitative relationships that govern the processes of their formation and assembly.

Revisiting the Fundamentals in the Design and Control of Nanoparticulate Colloids in the Frame of Soft Chemistry1

PubMed Central

Uskoković, Vuk

2013-01-01

This review presents thoughts on some of the fundamental features of conceptual models applied in the design of fine particles in the frames of colloid and soft chemistry. A special emphasis is placed on the limitations of these models, an acknowledgment of which is vital in improving their intricacy and effectiveness in predicting the outcomes of the corresponding experimental settings. Thermodynamics of self-assembly phenomena illustrated on the examples of protein assembly and micellization is analyzed in relation to the previously elaborated thesis that each self-assembly in reality presents a co-assembly, since it implies a mutual reorganization of the assembling system and its immediate environment. Parameters used in the design of fine particles by precipitation are discussed while referring to solubility product, various measures of supersaturation levels, induction time, nucleation and crystal growth rates, interfacial energies, and the Ostwald–Lussac law of phases. Again, the main drawbacks and inadequacies of using the aforementioned parameters in tailoring the materials properties in a soft and colloidal chemical setting were particularly emphasized. The basic and practical limitations of zeta-potential analyses, routinely used to stabilize colloidal dispersions and initiate specific interactions between soft chemical entities, were also outlined. The final section of the paper reiterates the unavoidable presence of practical qualitative models in the design and control of nanoparticulate colloids, which is supported by the overwhelming complexity of quantitative relationships that govern the processes of their formation and assembly. PMID:24490052

Draft genome of the leopard gecko, Eublepharis macularius.

PubMed

Xiong, Zijun; Li, Fang; Li, Qiye; Zhou, Long; Gamble, Tony; Zheng, Jiao; Kui, Ling; Li, Cai; Li, Shengbin; Yang, Huanming; Zhang, Guojie

2016-10-26

Geckos are among the most species-rich reptile groups and the sister clade to all other lizards and snakes. Geckos possess a suite of distinctive characteristics, including adhesive digits, nocturnal activity, hard, calcareous eggshells, and a lack of eyelids. However, one gecko clade, the Eublepharidae, appears to be the exception to most of these 'rules' and lacks adhesive toe pads, has eyelids, and lays eggs with soft, leathery eggshells. These differences make eublepharids an important component of any investigation into the underlying genomic innovations contributing to the distinctive phenotypes in 'typical' geckos. We report high-depth genome sequencing, assembly, and annotation for a male leopard gecko, Eublepharis macularius (Eublepharidae). Illumina sequence data were generated from seven insert libraries (ranging from 170 to 20 kb), representing a raw sequencing depth of 136X from 303 Gb of data, reduced to 84X and 187 Gb after filtering. The assembled genome of 2.02 Gb was close to the 2.23 Gb estimated by k-mer analysis. Scaffold and contig N50 sizes of 664 and 20 kb, respectively, were comparable to the previously published Gekko japonicus genome. Repetitive elements accounted for 42 % of the genome. Gene annotation yielded 24,755 protein-coding genes, of which 93 % were functionally annotated. CEGMA and BUSCO assessment showed that our assembly captured 91 % (225 of 248) of the core eukaryotic genes, and 76 % of vertebrate universal single-copy orthologs. Assembly of the leopard gecko genome provides a valuable resource for future comparative genomic studies of geckos and other squamate reptiles.

Human frataxin activates Fe-S cluster biosynthesis by facilitating sulfur transfer chemistry.

PubMed

Bridwell-Rabb, Jennifer; Fox, Nicholas G; Tsai, Chi-Lin; Winn, Andrew M; Barondeau, David P

2014-08-05

Iron-sulfur clusters are ubiquitous protein cofactors with critical cellular functions. The mitochondrial Fe-S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe-S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and electrons into Fe-S clusters. The physiological function of FXN has received a tremendous amount of attention since the discovery that its loss is directly linked to the neurodegenerative disease Friedreich's ataxia. Previous in vitro results revealed a role for human FXN in activating the cysteine desulfurase and Fe-S cluster biosynthesis activities of the Fe-S assembly complex. Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe-S clusters. Additional mutagenesis, enzyme kinetic, UV-visible, and circular dichroism spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN activation, whereas C35, C61, and C104 are all essential for Fe-S cluster formation on the assembly complex. These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe-S cluster biosynthesis, and further support an allosteric regulator role for FXN. Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe-S cluster biosynthesis.

Human Frataxin Activates Fe–S Cluster Biosynthesis by Facilitating Sulfur Transfer Chemistry

PubMed Central

2015-01-01

Iron–sulfur clusters are ubiquitous protein cofactors with critical cellular functions. The mitochondrial Fe–S assembly complex, which consists of the cysteine desulfurase NFS1 and its accessory protein (ISD11), the Fe–S assembly protein (ISCU2), and frataxin (FXN), converts substrates l-cysteine, ferrous iron, and electrons into Fe–S clusters. The physiological function of FXN has received a tremendous amount of attention since the discovery that its loss is directly linked to the neurodegenerative disease Friedreich’s ataxia. Previous in vitro results revealed a role for human FXN in activating the cysteine desulfurase and Fe–S cluster biosynthesis activities of the Fe–S assembly complex. Here we present radiolabeling experiments that indicate FXN accelerates the accumulation of sulfur on ISCU2 and that the resulting persulfide species is viable in the subsequent synthesis of Fe–S clusters. Additional mutagenesis, enzyme kinetic, UV–visible, and circular dichroism spectroscopic studies suggest conserved ISCU2 residue C104 is critical for FXN activation, whereas C35, C61, and C104 are all essential for Fe–S cluster formation on the assembly complex. These results cannot be fully explained by the hypothesis that FXN functions as an iron donor for Fe–S cluster biosynthesis, and further support an allosteric regulator role for FXN. Together, these results lead to an activation model in which FXN accelerates persulfide formation on NFS1 and favors a helix-to-coil interconversion on ISCU2 that facilitates the transfer of sulfur from NFS1 to ISCU2 as an initial step in Fe–S cluster biosynthesis. PMID:24971490

Bipolarization and Poleward Flux Correlate during Xenopus Extract Spindle AssemblyV⃞

PubMed Central

Mitchison, T.J.; Maddox, P.; Groen, A.; Cameron, L.; Perlman, Z.; Ohi, R.; Desai, A.; Salmon, E.D.; Kapoor, T.M.

2004-01-01

We investigated the mechanism by which meiotic spindles become bipolar and the correlation between bipolarity and poleward flux, using Xenopus egg extracts. By speckle microscopy and computational alignment, we find that monopolar sperm asters do not show evidence for flux, partially contradicting previous work. We account for the discrepancy by describing spontaneous bipolarization of sperm asters that was missed previously. During spontaneous bipolarization, onset of flux correlated with onset of bipolarity, implying that antiparallel microtubule organization may be required for flux. Using a probe for TPX2 in addition to tubulin, we describe two pathways that lead to spontaneous bipolarization, new pole assembly near chromatin, and pole splitting. By inhibiting the Ran pathway with excess importin-alpha, we establish a role for chromatin-derived, antiparallel overlap bundles in generating the sliding force for flux, and we examine these bundles by electron microscopy. Our results highlight the importance of two processes, chromatin-initiated microtubule nucleation, and sliding forces generated between antiparallel microtubules, in self-organization of spindle bipolarity and poleward flux. PMID:15385629

Computational complexity of algorithms for sequence comparison, short-read assembly and genome alignment.

PubMed

Baichoo, Shakuntala; Ouzounis, Christos A

A multitude of algorithms for sequence comparison, short-read assembly and whole-genome alignment have been developed in the general context of molecular biology, to support technology development for high-throughput sequencing, numerous applications in genome biology and fundamental research on comparative genomics. The computational complexity of these algorithms has been previously reported in original research papers, yet this often neglected property has not been reviewed previously in a systematic manner and for a wider audience. We provide a review of space and time complexity of key sequence analysis algorithms and highlight their properties in a comprehensive manner, in order to identify potential opportunities for further research in algorithm or data structure optimization. The complexity aspect is poised to become pivotal as we will be facing challenges related to the continuous increase of genomic data on unprecedented scales and complexity in the foreseeable future, when robust biological simulation at the cell level and above becomes a reality. Copyright © 2017 Elsevier B.V. All rights reserved.

An accelerated photo-magnetic imaging reconstruction algorithm based on an analytical forward solution and a fast Jacobian assembly method

NASA Astrophysics Data System (ADS)

Nouizi, F.; Erkol, H.; Luk, A.; Marks, M.; Unlu, M. B.; Gulsen, G.

2016-10-01

We previously introduced photo-magnetic imaging (PMI), an imaging technique that illuminates the medium under investigation with near-infrared light and measures the induced temperature increase using magnetic resonance thermometry (MRT). Using a multiphysics solver combining photon migration and heat diffusion, PMI models the spatiotemporal distribution of temperature variation and recovers high resolution optical absorption images using these temperature maps. In this paper, we present a new fast non-iterative reconstruction algorithm for PMI. This new algorithm uses analytic methods during the resolution of the forward problem and the assembly of the sensitivity matrix. We validate our new analytic-based algorithm with the first generation finite element method (FEM) based reconstruction algorithm previously developed by our team. The validation is performed using, first synthetic data and afterwards, real MRT measured temperature maps. Our new method accelerates the reconstruction process 30-fold when compared to a single iteration of the FEM-based algorithm.

Structural Basis for Catalytic Activation of a Serine Recombinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Keenholtz, Ross A.; Rowland, Sally-J.; Boocock, Martin R.

2014-10-02

Sin resolvase is a site-specific serine recombinase that is normally controlled by a complex regulatory mechanism. A single mutation, Q115R, allows the enzyme to bypass the entire regulatory apparatus, such that no accessory proteins or DNA sites are required. Here, we present a 1.86 {angstrom} crystal structure of the Sin Q115R catalytic domain, in a tetrameric arrangement stabilized by an interaction between Arg115 residues on neighboring subunits. The subunits have undergone significant conformational changes from the inactive dimeric state previously reported. The structure provides a new high-resolution view of a serine recombinase active site that is apparently fully assembled, suggestingmore » roles for the conserved active site residues. The structure also suggests how the dimer-tetramer transition is coupled to assembly of the active site. The tetramer is captured in a different rotational substate than that seen in previous hyperactive serine recombinase structures, and unbroken crossover site DNA can be readily modeled into its active sites.« less

Space assembly fixtures and aids

NASA Technical Reports Server (NTRS)

Bloom, K. A.; Lillenas, A. N.

1980-01-01

Concepts and requirements for assembly fixtures and aids necessary for the assembly and maintenance of spare platforms were studied. Emphasis was placed on erectable and deployable type structures with the shuttle orbiter as the assembly base. Both single and multiple orbiter flight cases for the platform assembly were considered. Applicable space platform assembly studies were reviewed to provide a data base for establishing the assembly fixture and aids design requirements, assembly constraints, and the development of representative design concepts. Conclusions indicated that fixture requirements will vary with platform size. Larger platforms will require translation relative to the orbiter RMS working volume. The installation of platform payloads and subsystems (e.g., utility distribution) must also be considered in the specification of assembly fixtures and aids.

Development of Scaffolds for Light Harvesting and Photocatalysis from the Coat Protein of Tobacco Mosaic Virus

NASA Astrophysics Data System (ADS)

Dedeo, Michel Toussaint

The utility of a previously developed TMV-based light harvesting system has been dramatically expanded through the introduction of reactive handles for the site-specific modification of the interior and exterior surfaces. Further experiments to reengineer the coat protein have produced structures with unique, unexpected, and useful assembly properties that complement the newly available surface modifications. Energy transfer from chromophores in the RNA channel of self-assembled TMV structures to the exterior was made possible by conjugation of acceptor dyes and porphyrins to the N-terminus. By repositioning the N-terminus to the pore through circular permutation, this process was repeated to create structures that mimic the light harvesting 1 complex of photosynthetic bacteria. To study and improve upon natural photosynthesis, closely packed chromophore arrays and gold nanoparticles were tethered to the pore of stabilized TMV disks through introduction of a uniquely reactive lysine. Finally, a dimeric TMV coat protein was produced to control the distribution and arrangement of synthetic groups with synergistic activity.

Free-energy landscape of protein oligomerization from atomistic simulations

PubMed Central

Barducci, Alessandro; Bonomi, Massimiliano; Prakash, Meher K.; Parrinello, Michele

2013-01-01

In the realm of protein–protein interactions, the assembly process of homooligomers plays a fundamental role because the majority of proteins fall into this category. A comprehensive understanding of this multistep process requires the characterization of the driving molecular interactions and the transient intermediate species. The latter are often short-lived and thus remain elusive to most experimental investigations. Molecular simulations provide a unique tool to shed light onto these complex processes complementing experimental data. Here we combine advanced sampling techniques, such as metadynamics and parallel tempering, to characterize the oligomerization landscape of fibritin foldon domain. This system is an evolutionarily optimized trimerization motif that represents an ideal model for experimental and computational mechanistic studies. Our results are fully consistent with previous experimental nuclear magnetic resonance and kinetic data, but they provide a unique insight into fibritin foldon assembly. In particular, our simulations unveil the role of nonspecific interactions and suggest that an interplay between thermodynamic bias toward native structure and residual conformational disorder may provide a kinetic advantage. PMID:24248370

When Genomics Is Not Enough: Experimental Evidence for a Decrease in LINE-1 Activity During the Evolution of Australian Marsupials

PubMed Central

Gallus, Susanne; Lammers, Fritjof

2016-01-01

The autonomous transposable element LINE-1 is a highly abundant element that makes up between 15% and 20% of therian mammal genomes. Since their origin before the divergence of marsupials and placental mammals, LINE-1 elements have contributed actively to the genome landscape. A previous in silico screen of the Tasmanian devil genome revealed a lack of functional coding LINE-1 sequences. In this study we present the results of an in vitro analysis from a partial LINE-1 reverse transcriptase coding sequence in five marsupial species. Our experimental screen supports the in silico findings of the genome-wide degradation of LINE-1 sequences in the Tasmanian devil, and identifies a high frequency of degraded LINE-1 sequences in other Australian marsupials. The comparison between the experimentally obtained LINE-1 sequences and reference genome assemblies suggests that conclusions from in silico analyses of retrotransposition activity can be influenced by incomplete genome assemblies from short reads. PMID:27389686

Cytotoxic 3,4,5-trimethoxychalcones as mitotic arresters and cell migration inhibitors

PubMed Central

Salum, Lívia B.; Altei, Wanessa F.; Chiaradia, Louise D.; Cordeiro, Marlon N.S.; Canevarolo, Rafael R.; Melo, Carolina P.S.; Winter, Evelyn; Mattei, Bruno; Daghestani, Hikmat N.; Santos-Silva, Maria Cláudia; Creczynski-Pasa, Tânia B.; Yunes, Rosendo A.; Yunes, José A.; Andricopulo, Adriano D.; Day, Billy W.; Nunes, Ricardo J.; Vogt, Andreas

2013-01-01

Based on classical colchicine site ligands and a computational model of the colchicine binding site on beta tubulin, two classes of chalcone derivatives were designed, synthesized and evaluated for inhibition of tubulin assembly and toxicity in human cancer cell lines. Docking studies suggested that the chalcone scaffold could fit the colchicine site on tubulin in an orientation similar to that of the natural product. In particular, a 3,4,5-trimethoxyphenyl ring adjacent to the carbonyl group appeared to benefit the ligand-tubulin interaction, occupying the same subcavity as the corresponding moiety in colchicine. Consistent with modeling predictions, several 3,4,5-trimethoxychalcones showed improved cytotoxicity to murine acute lymphoblastic leukemia cells compared with a previously described parent compound, and inhibited tubulin assembly in vitro as potently as colchicine. The most potent chalcones inhibited the growth of human leukemia cell lines at nanomolar concentrations, caused microtubule destabilization and mitotic arrest in human cervical cancer cells, and inhibited human breast cancer cell migration in scratch wound and Boyden chamber assays. PMID:23524161

Non-Covalent Functionalization of Carbon Nanovectors with an Antibody Enables Targeted Drug Delivery

PubMed Central

Berlin, Jacob M.; Pham, Tam T.; Sano, Daisuke; Mohamedali, Khalid A.; Marcano, Daniela C.; Myers, Jeffrey N.; Tour, James M.

2011-01-01

Current chemotherapeutics are characterized by efficient tumor cell-killing and severe side effects mostly derived from off target toxicity. Hence targeted delivery of these drugs to tumor cells is actively sought. We previously demonstrated that poly(ethylene glycol)-functionalized carbon nanovectors are able to sequester paclitaxel, a widely used hydrophobic cancer drug, by simple physisorption and deliver the drug for killing of cancer cells. The cell-killing when these drug-loaded carbon nanoparticles were used was equivalent to when a commercial formulation of paclitaxel was used. Here we show that by further mixing the drug-loaded nanoparticles with Cetuximab, a monoclonal antibody that recognizes the epidermal growth factor receptor (EGFR), paclitaxel is preferentially targeted to EGFR+ tumor cells in vitro. This supports progressing to in vivo studies. Moreover, the construct is unusual in that all three components are assembled through non-covalent interactions. Such non-covalent assembly could enable high-throughput screening of drug/antibody combinations. PMID:21736358

Pronounced Environmental Effects on Injection Currents in EGaIn Tunneling Junctions Comprising Self-Assembled Monolayers.

PubMed

Carlotti, Marco; Degen, Maarten; Zhang, Yanxi; Chiechi, Ryan C

2016-09-15

Large-area tunneling junctions using eutectic Ga-In (EGaIn) as a top contact have proven to be a robust, reproducible, and technologically relevant platform for molecular electronics. Thus far, the majority of studies have focused on saturated molecules with backbones consisting mainly of alkanes in which the frontier orbitals are either highly localized or energetically inaccessible. We show that self-assembled monolayers of wire-like oligophenyleneethynylenes (OPEs), which are fully conjugated, only exhibit length-dependent tunneling behavior in a low-O 2 environment. We attribute this unexpected behavior to the sensitivity of injection current on environment. We conclude that, contrary to previous reports, the self-limiting layer of Ga 2 O 3 strongly influences transport properties and that the effect is related to the wetting behavior of the electrode. This result sheds light on the nature of the electrode-molecule interface and suggests that adhesive forces play a significant role in tunneling charge-transport in large-area molecular junctions.

Self-assembly formation of palm-based esters nano-emulsion: A molecular dynamics study

NASA Astrophysics Data System (ADS)

Abdul Rahman, Mohd. Basyaruddin; Huan, Qiu-Yi; Tejo, Bimo A.; Basri, Mahiran; Salleh, Abu Bakar; Rahman, Raja Noor Zaliha Abdul

2009-10-01

Palm-oil esters (POEs) are unsaturated and non-ionic esters that can be prepared by enzymatic synthesis from palm oil. Their nano-emulsion properties possess great potential to act as drug carrier for transdermal drug delivery system. A ratio of 75:5:20 (water/POEs/Span20) was chosen from homogenous region in the phase diagram of our previous experimental work to undergo molecular dynamics simulation. A 15 ns molecular dynamics simulation of nano-emulsion system (water/POEs/Span20) was carried out using OPLS-AA force field. The aggregations of the oil and surfactant molecules are observed throughout the simulation. After 8 ns of simulation, the molecules start to aggregate to form one spherical micelle where the POEs molecules are surrounded by the non-ionic surfactant (Span20) molecules with an average size of 4.2 ± 0.05 nm. The size of the micelle and the ability of palm-based nano-emulsion to self-assemble suggest that this nano-emulsion can potentially use in transdermal drug delivery system.

Synaptic Vesicle Endocytosis Occurs on Multiple Timescales and Is Mediated by Formin-Dependent Actin Assembly.

PubMed

Soykan, Tolga; Kaempf, Natalie; Sakaba, Takeshi; Vollweiter, Dennis; Goerdeler, Felix; Puchkov, Dmytro; Kononenko, Natalia L; Haucke, Volker

2017-02-22

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Recent data suggest that at physiological temperature SVs are internalized via clathrin-independent ultrafast endocytosis (UFE) within hundreds of milliseconds, while other studies have postulated a key role for clathrin-mediated endocytosis (CME) of SV proteins on a timescale of seconds to tens of seconds. Here we demonstrate using cultured hippocampal neurons as a model that at physiological temperature SV endocytosis occurs on several timescales from less than a second to several seconds, yet, is largely independent of clathrin. Clathrin-independent endocytosis (CIE) of SV membranes is mediated by actin-nucleating formins such as mDia1, which are required for the formation of presynaptic endosome-like vacuoles from which SVs reform. Our results resolve previous discrepancies in the field and suggest that SV membranes are predominantly retrieved via CIE mediated by formin-dependent actin assembly. Copyright © 2017 Elsevier Inc. All rights reserved.

Deeper insight into the structure of the anaerobic digestion microbial community; the biogas microbiome database is expanded with 157 new genomes.

PubMed

Treu, Laura; Kougias, Panagiotis G; Campanaro, Stefano; Bassani, Ilaria; Angelidaki, Irini

2016-09-01

This research aimed to better characterize the biogas microbiome by means of high throughput metagenomic sequencing and to elucidate the core microbial consortium existing in biogas reactors independently from the operational conditions. Assembly of shotgun reads followed by an established binning strategy resulted in the highest, up to now, extraction of microbial genomes involved in biogas producing systems. From the 236 extracted genome bins, it was remarkably found that the vast majority of them could only be characterized at high taxonomic levels. This result confirms that the biogas microbiome is comprised by a consortium of unknown species. A comparative analysis between the genome bins of the current study and those extracted from a previous metagenomic assembly demonstrated a similar phylogenetic distribution of the main taxa. Finally, this analysis led to the identification of a subset of common microbes that could be considered as the core essential group in biogas production. Copyright © 2016 Elsevier Ltd. All rights reserved.