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At least 865 records · Page 48

Sequence-defined structural transitions by calcium-responsive proteins

Biopolymer sequences dictate their functions, and protein-based polymers are a promising platform to establish sequence–function relationships for novel biopolymers. To efficiently explore vast sequence spaces of natural proteins, sequence repetition is a common strategy to tune and amplify specific functions. This strategy is applied to repeats-in-toxin (RTX) proteins with calcium-responsive folding behavior, which stems from tandem repeats of the nonapeptide GGXGXDXUX in which X can be any amino acid and U is a hydrophobic amino acid. To determine the functional range of this nonapeptide, we modified a naturally occurring RTX protein that forms β-roll structures in the presence of calcium. Sequence modifications focused on calcium-binding turns within the repetitive region, including either global substitution of nonconserved residues or complete replacement with tandem repeats of a consensus nonapeptide GGAGXDTLY. Some sequence modifications disrupted the typical transition from intrinsically disordered random coils to folded β rolls, despite conservation of the underlying nonapeptide sequence. Proteins enriched with smaller, hydrophobic amino acids adopted secondary structures in the absence of calcium and underwent structural rearrangements in calcium-rich environments. In contrast, proteins with bulkier, hydrophilic amino acids maintained intrinsic disorder in the absence of calcium. In conclusion, these results indicate a significant role of nonconserved amino acids in calcium-responsive folding, thereby revealing a strategy to leverage sequences in the design of tunable, calcium-responsive biopolymers.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Statistical properties of DNA sequences

We review evidence supporting the idea that the DNA sequence in genes containing non-coding regions is correlated, and that the correlation is remarkably long range--indeed, nucleotides thousands of base pairs distant are correlated. We do not find such a long-range correlation in the coding regions of the gene. We resolve the problem of the "non-stationarity" feature of the sequence of base pairs by applying a new algorithm called detrended fluctuation analysis (DFA). We address the claim of Voss that there is no difference in the statistical properties of coding and non-coding regions of DNA by systematically applying the DFA algorithm, as well as standard FFT analysis, to every DNA sequence (33301 coding and 29453 non-coding) in the entire GenBank database. Finally, we describe briefly some recent work showing that the non-coding sequences have certain statistical features in common with natural and artificial languages. Specifically, we adapt to DNA the Zipf approach to analyzing linguistic texts. These statistical properties of non-coding sequences support the possibility that non-coding regions of DNA may carry biological information.

Non-NASA Center↗

Proteomic studies of bone and skeletal health outcomes

Proteins are an essential part of essentially all biological processes, and there is enormous variation in protein forms and concentrations that is not reflected in DNA or RNA. Recently there have been rapid advances in the ability to measure protein sequence, modification and concentration, particularly with methods based in mass spectrometry. Global measures of proteins in tissues or in the circulation provide a broad assessment of the proteome that can be extremely useful for discovery, and targeted proteomic measures can yield specific and sensitive assessments of specific peptides and proteins. While most proteomic measures are directed at the detection of consensus peptide sequences, mass spectrometry based proteomic methods also allow a detailed examination of the peptide sequence differences that result from genetic variants and that may have important effects on protein function. In evaluating proteomic data, a number of analytical considerations are important, including an understanding of missing data, the challenge of multiple testing and replication, and the use of rapidly evolving methods in systems biology. While proteomics has not yet had a major impact in skeletal research, interesting recent research has used these approaches in the study of bone cell biology and the discovery of biomarkers of skeletal disorders. Proteomics can be expected to have an increasing influence in the study of bone biology and pathophysiology

Proteomic, Bone↗

Global overview and major challenges of host prediction methods for uncultivated phages

Bacterial communities play critical roles across all of Earth’s biomes, affecting human health and global ecosystem functioning. They do so under strong constraints exerted by viruses, i.e., bacteriophages or “phages”. Phages can reshape bacterial communities’ structure, influence long-term evolution of bacterial populations, and alter host cell metabolism during infection. Metagenomics approaches, i.e., shotgun sequencing of environmental DNA or RNA, recently enabled large-scale exploration of phage genomic diversity, yielding several millions of phage genomes now to be further analyzed and characterized. One major challenge however is the lack of direct host information for these phages. Several methods and tools have been proposed to bioinformatically predict the potential host(s) of uncultivated phages based only on genome sequence information. Here we review these different approaches and highlight their distinct strengths and limitations. We also outline complementary experimental assays which are being proposed to validate and refine these bioinformatic predictions.

59 BASIC BIOLOGICAL SCIENCES↗

Designing Peptide Fossils That Model the Evolution of the Bacterial Ferredoxin Fold

Electron transfer coupled to redox chemistry is at the heart of metabolism. The proteins responsible for moving electrons (protein electron carriers) must have emerged at the origin of life. The small iron–sulfur-binding bacterial ferredoxins were likely among these first proteins. Embedded within the ferredoxin sequence and structure is a symmetry that points to an ancient gene duplication event. Little is understood about the nature of ferredoxins prior to this duplication event or what environmental factors may have driven the selection for more complex forms. The deep-time molecular history of ferredoxins goes back billions of years and cannot be reconstructed by phylogenetic analyses based on amino acid sequences. Here, we use structure-guided protein design to model a fossil half-ferredoxin stage in the evolution of this fold, the semidoxins, and their symmetric full-length counterparts, the symdoxins. Semidoxin designs homodimerize, exhibiting structural, thermodynamic, and electrochemical behaviors in most cases identical to cognate symdoxins. However, the semi- and symdoxin fossil stages behave differently when incorporated into an in vivo electron transfer complementation assay. Both can support bacterial growth dependent on protein expression. Growth rates of bacteria expressing the semidoxins are much more sensitive to oxygen than those of bacteria expressing symdoxins. Motivated by the in vivo functionality of designed semidoxins, we identified putative naturally occurring semidoxins in extant anaerobic microorganisms. This is consistent with the observed in vivo oxygen sensitivity of the semidoxin designs. One natural semidoxin is shown to be folded and redox active. However, it exists as a mixture of monomers and dimers, suggesting a potential connection between semidoxins and even simpler single iron–sulfur cluster-binding peptides.

59 BASIC BIOLOGICAL SCIENCES↗

CDL2PLC translator v0.1.0

The CDL-PLC translator aims at translating control sequences for building energy systems from the CDL CXF format to the PLCopen XML format. The CDL CXF developed at LBL within the OpenBuildingControl project, and now being standardized via ASHRAE Standard 231P, enables expressing control sequences developed in the simulation environment Modelica in a JSON format. The PLCopen XML is an existing exchange format standardized in IEC 61131-10 for Programmable Logic Controllers (PLCs) following the IEC 61131 standard as one target system of CDL among others. The translation from the CDL CXF to the PLCopen XML contributes to a seamless workflow from the model-based development of control sequences in simulation environments, which is not building practice today, and their digital implementation on building controllers, which replaces graphical and textual documents used for this purpose today. The translator is at a prototypical stage and enables, as a proof of concept, the translation of very simple control sequences composed of 4 selected function blocks out of 137 function blocks defined in CDL. The translation includes the connection of inputs and outputs of function blocks and the expression of a control function in CDL to the equivalent code in IEC 61131-3.

Walther, Karl↗

Implementing Distributed Operations: A Comparison of Two Deep Space Missions

Two very different deep space exploration missions--Mars Exploration Rover and Cassini--have made use of distributed operations for their science teams. In the case of MER, the distributed operations capability was implemented only after the prime mission was completed, as the rovers continued to operate well in excess of their expected mission lifetimes; Cassini, designed for a mission of more than ten years, had planned for distributed operations from its inception. The rapid command turnaround timeline of MER, as well as many of the operations features implemented to support it, have proven to be conducive to distributed operations. These features include: a single science team leader during the tactical operations timeline, highly integrated science and engineering teams, processes and file structures designed to permit multiple team members to work in parallel to deliver sequencing products, web-based spacecraft status and planning reports for team-wide access, and near-elimination of paper products from the operations process. Additionally, MER has benefited from the initial co-location of its entire operations team, and from having a single Principal Investigator, while Cassini operations have had to reconcile multiple science teams distributed from before launch. Cassini has faced greater challenges in implementing effective distributed operations. Because extensive early planning is required to capture science opportunities on its tour and because sequence development takes significantly longer than sequence execution, multiple teams are contributing to multiple sequences concurrently. The complexity of integrating inputs from multiple teams is exacerbated by spacecraft operability issues and resource contention among the teams, each of which has their own Principal Investigator. Finally, much of the technology that MER has exploited to facilitate distributed operations was not available when the Cassini ground system was designed, although later adoption of web-based and telecommunication tools has been critical to the success of Cassini operations.

Cassini Mission↗

Strategies for Constructing and Operating DNA Origami Linear Actuators

Abstract Linear actuators are ubiquitous components at all scales of engineering. DNA nanotechnology offers a unique opportunity for bottom‐up assembly at the molecular scale, providing nanoscale precision with multiple methods for constructing and operating devices. In this paper, DNA origami linear actuators with up to 200 nm travel, based on a rail threading a topologically locked slider, are demonstrated. Two strategies, one‐ and two‐pot assembly, are demonstrated whereby the two components are folded from one or two DNA scaffold strands, respectively. In order to control the position of the slider on the rail, the rail and the inside of the slider are decorated with single‐stranded oligonucleotides with distinct sequences. Two positioning strategies, based on diffusion and capture of signaling strands, are used to link the slider reversibly to determined positions on the rail with high yield and precision. These machine components provide a basis for applications in molecular machinery and nanoscale manufacture including programmed chemical synthesis.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

Compact quantum dot surface modification to enable emergent behaviors in quantum dot-DNA composites

Quantum dot (QD) biological imaging and sensing applications often require surface modification with single-stranded deoxyribonucleic acid (ssDNA) oligonucleotides. Furthermore, ssDNA conjugation can be leveraged for precision QD templating via higher-order DNA nanostructures to exploit emergent behaviors in photonic applications. Use of ssDNA-QDs across these platforms requires compact, controlled conjugation that engenders QD stability over a wide pH range and in solutions of high ionic strength. However, cur-rent ssDNA-QD conjugation approaches suffer from limitations, such as the requirement for thick coatings, low control over ssDNA labeling density, requirement of large amounts of ssDNA, or low colloidal or photostability, restraining implementation in many applications. Here, we combine thin, multidentate, phytochelatin-3 (PC3) QD passivation techniques with strain-promoted copper-free alkyne-azide click chemistry to yield functional ssDNA-QDs with high stability. This process was broadly applicable across QD sizes (i.e.,λ em = 540, 560, 600 nm), ssDNA lengths (i.e., 10–16 base pairs, bps), and sequences (poly thymine, mixed bps). The resulting compacts sDNA-QDs displayed a fluorescence quenching efficiency of up to 89% by hybridization with complementary ssDNA-AuNPs. Further-more, ssDNA-QDs were successfully incorporated with higher-order DNA origami nanostructure templates. Finally, this approach, combining PC3 passivation with click chemistry, generates ssDNA-PC3-QDs that enable emergent QD properties in DNA-based devices and applications.

77 NANOSCIENCE AND NANOTECHNOLOGY↗

Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform

Continued advancements in sequencing technologies have fueled the development of new sequencing applications and promise to flood current databases with raw data. A number of factors prevent the seamless and easy use of these data, including the breadth of project goals, the wide array of tools that individually perform fractions of any given analysis, the large number of associated software/hardware dependencies, and the detailed expertise required to perform these analyses. To address these issues, we have developed an intuitive web-based environment with a wide assortment of integrated and cutting-edge bioinformatics tools in pre-configured workflows. These workflows, coupled with the ease of use of the environment, provide even novice next-generation sequencing users with the ability to perform many complex analyses with only a few mouse clicks and, within the context of the same environment, to visualize and further interrogate their results. As a result, this bioinformatics platform is an initial attempt at Empowering the Development of Genomics Expertise (EDGE) in a wide range of applications for microbial research.

59 BASIC BIOLOGICAL SCIENCES↗

Listeria monocytogenes in Stone Fruits Linked to a Multistate Outbreak: Enumeration of Cells and Whole-Genome Sequencing

In 2014, the identification of stone fruits contaminated with led to the subsequent identification of a multistate outbreak. Simultaneous detection and enumeration of L. monocytogenes were performed on 105 fruits, each weighing 127 to 145 g, collected from 7 contaminated lots. The results showed that 53.3% of the fruits yielded L. monocytogenes (lower limit of detection, 5 CFU/fruit), and the levels ranged from 5 to 2,850 CFU/fruit, with a geometric mean of 11.3 CFU/fruit (0.1 CFU/g of fruit). Two serotypes, IVb-v1 and 1/2b, were identified by a combination of PCR- and antiserum-based serotyping among isolates from fruits and their packing environment; certain fruits contained a mixture of both serotypes. Single nucleotide polymorphism (SNP)-based whole-genome sequencing (WGS) analysis clustered isolates from two case-patients with the serotype IVb-v1 isolates and distinguished outbreak-associated isolates from pulsed-field gel electrophoresis (PFGE)-matched, but epidemiologically unrelated, clinical isolates. The outbreak-associated isolates differed by up to 42 SNPs. All but one serotype 1/2b isolate formed another WGS cluster and differed by up to 17 SNPs. Fully closed genomes of isolates from the stone fruits were used as references to maximize the resolution and to increase our confidence in prophage analysis. Putative prophages were conserved among isolates of each WGS cluster. All serotype IVb-v1 isolates belonged to singleton sequence type 382 (ST382); all but one serotype 1/2b isolate belonged to clonal complex 5.

59 BASIC BIOLOGICAL SCIENCES↗

Phylogenetic diversity in the genus Bacillus as seen by 16S rRNA sequencing studies

Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major "Bacillus clusters" can be defined: a "Bacillus subtilis cluster" including B. stearothermophilus, a "B. brevis cluster" including B. laterosporus, a "B. alvei cluster" including B. macerans, B. maquariensis and B. polymyxa and a "B. cycloheptanicus branch".

NASA Discipline Number 52-30↗

Predicting antimicrobial resistance in Pseudomonas aeruginosa with machine learning-enabled molecular diagnostics

Limited therapy options due to antibiotic resistance underscore the need for optimization of current diagnostics. In some bacterial species, antimicrobial resistance can be unambiguously predicted based on their genome sequence. In this study, we sequenced the genomes and transcriptomes of 414 drug-resistant clinical Pseudomonas aeruginosa isolates. By training machine learning classifiers on information about the presence or absence of genes, their sequence variation, and expression profiles, we generated predictive models and identified biomarkers of resistance to four commonly administered antimicrobial drugs. Using these data types alone or in combination resulted in high (0.8–0.9) or very high (> 0.9) sensitivity and predictive values. For all drugs except for ciprofloxacin, gene expression information improved diagnostic performance. Our results pave the way for the development of a molecular resistance profiling tool that reliably predicts antimicrobial susceptibility based on genomic and transcriptomic markers. Finally, the implementation of a molecular susceptibility test system in routine microbiology diagnostics holds promise to provide earlier and more detailed information on antibiotic resistance profiles of bacterial pathogens and thus could change how physicians treat bacterial infections.

59 BASIC BIOLOGICAL SCIENCES↗

Universal Human Papillomavirus Typing Assay: Whole-Genome Sequencing following Target Enrichment

We designed a universal human papillomavirus (HPV) typing assay based on target enrichment and whole-genome sequencing (eWGS). The RNA bait included 23,941 probes targeting 191 HPV types and 12 probes targeting beta-globin as a control. We used the Agilent SureSelect XT2 protocol for library preparation, Illumina HiSeq 2500 for sequencing, and CLC Genomics Workbench for sequence analysis. Mapping stringency for type assignment was determined based on 8 (6 HPV-positive and 2 HPV-negative) control samples. Using the optimal mapping conditions, types were assigned to 24 blinded samples. eWGS results were 100% concordant with Linear Array (LA) genotyping results for 9 plasmid samples and fully or partially concordant for 9 of the 15 cervical-vaginal samples, with 95.83% overall type-specific concordance for LA genotyping. eWGS identified 7 HPV types not included in the LA genotyping. Since this method does not involve degenerate primers targeting HPV genomic regions, PCR bias in genotype detection is minimized. With further refinements aimed at reducing cost and increasing throughput, this first application of eWGS for universal HPV typing could be a useful method to elucidate HPV epidemiology.

59 BASIC BIOLOGICAL SCIENCES↗

Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing

With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.

09 BIOMASS FUELS↗

The Frequency-Coded Pulse-Burst Waveform and the Costas Sequence

The process of simultaneously measuring range and radial velocity with radar is accompanied by an inherent uncertainty or ambiguity in the values obtained. The choice of radar waveform allows some control over this uncertainty. This study examines the frequency-coded pulse-burst waveform as it relates to determination of the range and radial velocity of a moving target. The radar ambiguity function and the matched-filter response are presented for a certain class of frequency-coded pulse bursts, in which each subpulse contains exactly an integer number of cycles. The study is specialized further to the examination of a pulse burst based on a special sequence of integers called a Costas sequence. The advantages and disadvantages of this special waveform are described. Results from simulated measurements are included.

45 MILITARY TECHNOLOGY, WEAPONRY, AND NATIONAL DEF↗

Modeling the Activity of Single Genes

The central dogma of molecular biology states that information is stored in DNA, transcribed to messenger RNA (mRNA) and then translated into proteins. This picture is significantly augmentated when we consider the action of certain proteins in regulating transcription. These transcription factors provide a feedback pathway by which genes can regulate one another's expression as mRNA and then as protein. To review: DNA, RNA and proteins have different functions. DNA is the molecular storehouse of genetic information. When cells divide, the DNA is replicated, so that each daughter cell maintains the same genetic information as the mother cell. RNA acts as a go-between from DNA to proteins. Only a single copy of DNA is present, but multiple copies of the same piece of RNA may be present, allowing cells to make huge amounts of protein. In eukaryotes (organisms with a nucleus), DNA is found in the nucleus only. RNA is copied in the nucleus then translocates(moves) outside the nucleus, where it is transcribed into proteins. Along the way, the RNA may be spliced, i.e., may have pieces cut out. RNA then attaches to ribosomes and is translated to proteins. Proteins are the machinery of the cell other than DNA and RNA, all the complex molecules of the cell are proteins. Proteins are specialized machines, each of which fulfills its own task, which may be transporting oxygen, catalyzing reactions, or responding to extracellular signals, just to name a few. One of the more interesting functions a protein may have is binding directly or indirectly to DNA to perform transcriptional regulation, thus forming a closed feedback loop of gene regulation. The structure of DNA and the central dogma were understood in the 50s; in the early 80s it became possible to make arbitrary modifications to DNA and use cellular machinery to transcribe and translate the resulting genes; more recently, genomes (i.e., the complete DNA sequence) of many organisms have been sequenced. This large-scale sequencing began with simple organisms, viruses and bacteria, progressed to eukaryotes such as yeast, and more recently (1998) progressed to a multi-cellular animal, the nematode Caenorhabditis elegans. Sequencers have now moved on to the fruit fly Drosophila melanogaster, whose sequence is slated for completion by the end of 1999. The human genome project is expected to determine the complete sequence of all 3 billion bases of human DNA within the next five years. In the wake of genome-scale sequencing, further instrumentation is being developed to assay gene expression and function on a comparably large scale. Much of the work in computational biology focuses on computational tools used in sequencing, finding genes that are related to a particular gene, finding which parts of the DNA code for proteins and which do not, understanding what proteins will be formed from a given length of DNA, predicting how the proteins will fold from a one-dimensional structure into a three dimensional structure, and so on. Much less computational work has been done regarding the function of proteins. One reason for this is that different proteins function very differently, and so work on protein function is very specific to certain classes of proteins. There are, for example, proteins such enzymes that catalyze various intracellular reactions, receptors that respond to extracellular signals and ion channels that regulate the flow of charged particles into and out of the cell. In this chapter, we will consider a particular class of proteins called transcription factors(TFs), which are responsible for regulating when a certain gene is expressed in a certain cell, which cells it is express in, and how much is expressed. Understanding these processes will involve developing a deeper understanding of transcription, translation, and the cellular processes that control those processes. All of these elements fall under the aegis of gene regulation or more narrowly transcriptional regulation. Some of the key questions in gene regulation are: What genes are expressed in a certain cell at a certain time? How does gene expression differ from cell to cell in a multicellular organism? Which proteins act as transcription factors, i.e., are important in regulating gene expression? From questions like these, we hope to understand which genes are important for various macroscopic processes. Nearly all of the cells of a multicellular organism contain the same DNA. Yet this same genetic information yields a large number of different cell types. The fundamental difference between a neuron and a liver cell, for example, is which genes are expressed. Thus understanding gene regulation is an important step in understanding development. Furthermore, understanding the usual genes that are expressed in cells may give important clues about various diseases. Some diseases, such as sickle cell anemia and cystic fibrosis, are caused by defects in single, non-regulatory genes; others, such as certain cancers, are caused when the cellular control circuitry malfunctions - an understanding of these diseases will involve pathways of multiple interacting gene products. There are numerous challenges in the area of understanding and modeling gene regulation. First and foremost, biologists would like to develop a deeper understanding of the processes involved, including which genes and families of genes are important, how they interact, etc. From a computation point of view, there has been embarrassingly little work done. In this chapter there are many areas in which we can phrase meaningful, non-trivial computational questions, but questions that have not been addressed. Some of these are purely computational (what is a good algorithm for dealing with a model of type X) and others are more mathematical (given a system with certain characteristics, what sort of model can one use? How does one find biochemical parameters from system-level behavior using as few experiments as possible?). In addition to biological and algorithmic problems, there is also the ever-present issue of theoretical biology - what general principles can be derived from these systems, what can one do with models other than just simulate time-courses, what can be deduced about a class of systems without knowing all the details? The fundamental challenge to computationalists and theorists is to add value to the biology - to use models, modeling techniques and algorithms to understand the biology in new ways.

Mjolsness, Eric↗