bluuebunny/crossref_metadata_2025_split huggingface dataset:
A high-throughput screen for transcription activation domains reveals their sequence characteristics and permits reliable prediction by deep learning
Authors: Erijman Ariel , Kozlowski Lukasz , Sohrabi-Jahromi Salma , Fishburn James , Warfield Linda , Schreiber Jacob , Noble William S. , Söding Johannes , Hahn Steven 
Published: December 2019

AbstractTranscription activation domains (ADs) are encoded by a wide range of seemingly unrelated amino acid sequences, making it difficult to recognize features that permit their dynamic behavior, fuzzy interactions and target specificity. We screened a large set of random 30-mer peptides for AD function and trained a deep neural network (ADpred) on the AD-positive and negative sequences. ADpred correctly identifies known ADs within protein sequences and accurately predicts the consequences of mutations. We show that functional ADs are (1) located within intrinsically disordered regions with biased amino acid composition, (2) contain clusters of hydrophobic residues near acidic side chains, (3) are enriched or depleted for particular dipeptide sequences, and (4) have higher helical propensity than surrounding regions. Taken together, our findings fit the model of “fuzzy” binding through hydrophobic protein-protein interfaces, where activator-coactivator binding takes place in a dynamic hydrophobic environment rather than through combinations of sequence-specific interactions.
Maternal alum-adjuvanted recombinant HIV Env vaccine does not enhance autologous virus neutralization in HIV-infected pregnant women
Authors: Hompe Eliza D. , Mangold Jesse F. , Eudailey Joshua A. , Giorgi Elena E. , Kumar Amit , McGuire Erin , Haynes Barton F. , Moody M. Anthony , Wright Peter F. , Fouda Genevieve G. , Gao Feng , Permar Sallie R. 
Published: December 2019

AbstractPreventive strategies beyond ART will be required to end the pediatric HIV epidemic. A maternal vaccine capable of boosting neutralizing antibody (nAb) responses against circulating viruses in HIV-infected pregnant women could effectively decrease mother-to-child transmission of HIV. However, it is not known if an HIV envelope (Env) vaccine administered to infected pregnant women can enhance autologous virus neutralization.Here, we assessed autologous virus nAb responses in maternal plasma samples obtained from AIDS Vaccine Evaluation Group (AVEG) Protocols 104 and 102, historical Phase I safety and immunogenicity trials of recombinant HIV Env subunit vaccines in HIV-infected pregnant women (NCT00001041). AVEG 104 participants were randomized to receive 300 µg Env subunit MN recombinant gp120 with alum adjuvant or alum alone. AVEG 102 participants were randomized to receive 640 µg Env subunit recombinant gp160 or placebo. HIV Env-specific maternal plasma binding and neutralizing responses were characterized before and after vaccination in 15 AVEG 104 (n=10 vaccinee, n=5 placebo) and 2 AVEG 102 (n=1 vaccinee, n=1 placebo) participants. Single genome amplification (SGA) was used to obtain HIVenvgene sequences from autologous viruses for pseudovirus production in pre- and post-vaccination plasma of HIV-infected pregnant vaccinees (n=6 gp120, n=1 gp160) and placebo recipients (n=3).We detected an increase in MN gp120-specific IgG binding in the vaccinee group between the first immunization visit and the last visit at delivery (p=0.027, 2-sided Wilcoxon test). However, no difference was observed in the neutralization potency of maternal plasma collected at delivery against autologous viruses isolated from early or late pregnancy. Thus, maternal vaccination with gp120/160 did not boost maternal autologous virus nAb responses. Immunization strategies capable of more potent B cell stimulation will likely be required to effectively boost autologous virus nAb responses in pregnant women and synergize with ART to further reduce infant HIV infections.HighlightsPrior maternal HIV Env vaccine trial did not assess autologous virus neutralizationCirculating viruses isolated from mothers were tested against autologous plasmaMaternal vaccination with HIV Env gp120/160 increased MN gp120-specific IgG bindingMaternal HIV Env vaccine regimen did not boost autologous virus neutralizationMore potent B cell stimulation will be required to elicit autologous nAb responses
Rapid assessment of phytoplankton assemblages using Next Generation Sequencing – Barcode of Life database: a widely applicable toolkit to monitor biodiversity and harmful algal blooms (HABs)
Authors: Ivanova Natalia V. , Watson L. Cynthia , Comte Jérôme , Bessonov Kyrylo , Abrahamyan Arusyak , Davis Timothy W. , Bullerjahn George S. , Watson Susan B. 
Published: December 2019

AbstractHarmful algal blooms have important implications for the health, functioning and services of aquatic ecosystems. Our ability to detect and monitor these events is often challenged by the lack of rapid and cost-effective methods to identify bloom-forming organisms and their potential for toxin production, Here, we developed and applied a combination of DNA barcoding and Next Generation Sequencing (NGS) for the rapid assessment of phytoplankton community composition with focus on two important indicators of ecosystem health: toxigenic bloom-forming cyanobacteria and impaired planktonic biodiversity. To develop this molecular toolset for identification of cyanobacterial and algal species present in HABs (Harmful Algal Blooms), hereafter called HAB-ID, we optimized NGS protocols, applied a newly developed bioinformatics pipeline and constructed a BOLD (Barcode of Life Data System) 16S reference database from cultures of 203 cyanobacterial and algal strains representing 101 species with particular focus on bloom and toxin producing taxa. Using the new reference database of 16S rDNA sequences and constructed mock communities of mixed strains for protocol validation we developed new NGS primer set which can recover 16S from both cyanobacteria and eukaryotic algal chloroplasts. We also developed DNA extraction protocols for cultured algal strains and environmental samples, which match commercial kit performance and offer a cost-efficient solution for large scale ecological assessments of harmful blooms while giving benefits of reproducibility and increased accessibility. Our bioinformatics pipeline was designed to handle low taxonomic resolution for problematic genera of cyanobacteria such as theAnabaena-Aphanizomenon-Dolichospermumspecies complex, two clusters ofAnabaena(I and II),PlanktothrixandMicrocystis. This newly developed HAB-ID toolset was further validated by applying it to assess cyanobacterial and algal composition in field samples from waterbodies with recurrent HABs events.
Structure of biomolecular condensates from dissipative particle dynamics simulations
Authors: Shillcock Julian C. , Brochut Maelick , Chénais Etienne , Ipsen John H. 
Published: December 2019

ABSTRACTPhase separation of immiscible fluids is a common phenomenon in polymer chemistry, and is recognized as an important mechanism by which cells compartmentalize their biochemical reactions. Biomolecular condensates are condensed fluid droplets in cells that form by liquid-liquid phase separation of intrinsically-disordered proteins. They have a wide range of functions and are associated with chronic neurodegenerative diseases in which they become pathologically rigid. Intrinsically-disordered proteins are conformationally flexible and possess multiple, distributed binding sites for each other or for RNA. However, it remains unclear how their material properties depend on the molecular structure of the proteins. Here we use coarse-grained simulations to explore the phase behavior and structure of a model biomolecular condensate composed of semi-flexible polymers with attractive end-caps in a good solvent. Although highly simplified, the model contains the minimal molecular features that are sufficient to observe liquid-liquid phase separation of soluble polymers. The polymers condense into a porous, three-dimensional network in which their end-caps reversibly bind at junctions. The spatial separation of connected junctions scales with the polymer backbone length as a self-avoiding random walk over a wide range of concentration with a weak affinity-dependent prefactor. By contrast, the average number of polymers that meet at the junctions depends strongly on the end-cap affinity but only weakly on the polymer length. The regularity and porosity of the condensed network suggests a mechanism for cells to regulate biomolecular condensates. Interaction sites along a protein may be turned on or off to modulate the condensate’s porosity and tune the diffusion and interaction of additional proteins.
Molecular Investigations into the Unfoldase Action of Severing Enzymes on Microtubules
Authors: Varikoti Rohith Anand , Macke Amanda C. , Speck Virginia , Ross Jennifer L. , Dima Ruxandra I. 
Published: December 2019

AbstractMicrotubules associated proteins regulate the dynamic behavior of microtubules during cellular processes. Microtubule severing enzymes are the associated proteins which destabilize microtubules by removing subunits from the lattice. One model for how severing enzymes remove tubulin dimers from the microtubule lattice is by unfolding its subunits through pulling on the carboxy-terminal tails of tubulin dimers. This model stems from the fact that severing enzymes are AAA+ unfoldases. To test this mechanism, we apply pulling forces on the carboxy-terminal regions of microtubule subunits using coarse grained molecular simulations. In our simulations we used different microtubule lattices and concentrations of severing enzymes. We compare our simulation results with data fromin-vitrosevering assays and find that the experimental data is best fit by a model of cooperative removal of protofilament fragments by severing enzymes which depends on the severing enzyme concentration and placement on the microtubule lattice.
An allosteric switch regulatesMycobacterium tuberculosisClpP1P2 protease function as established by cryo-EM and methyl-TROSY NMR
Authors: Vahidi Siavash , Ripstein Zev A. , Juravsky Jordan B. , Rennella Enrico , Goldberg Alfred L. , Mittermaier Anthony K. , Rubinstein John L. , Kay Lewis E. 
Published: December 2019

AbstractThe 300-kDa ClpP1P2 protease fromMycobacterium tuberculosiscollaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as novel antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucyl-leucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of anti-TB therapeutics that target the Clp system. Here we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Cryo-EM structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key β-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)-based NMR, cryo-EM, and biochemical assays show that upon binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function, and shows extensive allosteric interactions involving both intra- and inter-ring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to targetM. tuberculosis.Significance StatementThe MtClpP1P2 protease is part of the essential protein degradation machinery that helps maintain protein homeostasis inMycobacterium tuberculosis, the causative agent of TB. Antibiotics that selectively kill both dormant and growing drug-resistant populations ofM. tuberculosisby disrupting MtClpP1P2 function have attracted recent attention. Here we characterize a switch that can control MtClpP1P2 activity through binding of small peptides, leading to a concerted conformational change that potentially can be exploited by drug molecules to interfere with MtClpP1P2 function. Overall, this work highlights the power of a combined NMR and cryo-EM approach to provide detailed insights into the structure-dynamics-function relationship of molecular machines critical to human health.
Quantitative method for estimating stain density in electron microscopy of conventionally prepared biological specimens
Authors: Fera Andrea , He Qianping , Zhang Guofeng , Leapman Richard D. 
Published: December 2019

SummaryStain density is an important parameter for optimizing the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including serial block-face electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). Here, we show how some straightforward measurements in the TEM can be used to determine the stain density based on a simple expression that we derive. Numbers of stain atoms per unit volume are determined from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. The method is tested on specimens of embedded blood platelets, brain tissue, and liver tissue.
coil: an R package for cytochrome C oxidase I (COI) DNA barcode data cleaning, translation, and error evaluation
Authors: Nugent Cameron M. , Elliott Tyler A. , Ratnasingham Sujeevan , Adamowicz Sarah J. 
Published: December 2019

AbstractBiological conclusions based on DNA barcoding and metabarcoding analyses can be strongly influenced by the methods utilized for data generation and curation, leading to varying levels of success in the separation of biological variation from experimental error. The five-prime region of cytochrome c oxidase subunit I (COI-5P) is the most common barcode gene for animals, with conserved structure and function that allows for biologically informed error identification. Here, we present coil (https://CRAN.R-project.org/package=coil), an R package for the pre-processing and error assessment of COI-5P animal barcode and metabarcode sequence data. The package contains functions for placement of barcodes into a common reading frame, accurate translation of sequences to amino acids, and highlighting insertion and deletion errors. The analysis of 10,000 barcode sequences of varying quality demonstrated how coil can place barcode sequences in reading frame and distinguish sequences containing indel errors from error-free sequences with greater than 97.5% accuracy. Package limitations were tested through the analysis of COI-5P sequences from the plant and fungal kingdoms as well as the analysis of potential contaminants: nuclear mitochondrial pseudogenes andWolbachiaCOI-5P sequences. Results demonstrated that coil is a strong technical error identification method but is not reliable for detecting all biological contaminants.
Cell-free genetic devices confer autonomic and adaptive properties to hydrogels
Authors: Whitfield Colette J. , Banks Alice M. , Dura Gema , Love John , Fieldsend Jonathan E. , Goodchild Sarah A. , Fulton David A. , Howard Thomas P. 
Published: December 2019

AbstractSmart materials are able to alter one or more of their properties in response to defined stimuli. Our ability to design and create such materials, however, does not match the diversity and specificity of responses seen within the biological domain. We propose that relocation of molecular phenomena from living cells into hydrogels can be used to confer smart functionality to materials. We establish that cell-free protein synthesis can be conducted in agarose hydrogels, that gene expression occurs throughout the material and that co-expression of genes is possible. We demonstrate that gene expression can be controlled transcriptionally (usingin gelgene interactions) and translationally in response to small molecule and nucleic acid triggers. We use this system to design and build a genetic device that can alter the structural property of its chassis material in response to exogenous stimuli. Importantly, we establish that a wide range of hydrogels are appropriate chassis for cell-free synthetic biology, meaning a designer may alter both the genetic and hydrogel components according to the requirements of a given application. We probe the relationship between the physical structure of the gel andin gelprotein synthesis and reveal that the material itself may act as a macromolecular crowder enhancing protein synthesis. Given the extensive range of genetically encoded information processing networks in the living kingdom and the structural and chemical diversity of hydrogels, this work establishes a model by which cell-free synthetic biology can be used to create autonomic and adaptive materials.Significance statementSmart materials have the ability to change one or more of their properties (e.g. structure, shape or function) in response to specific triggers. They have applications ranging from light-sensitive sunglasses and drug delivery systems to shape-memory alloys and self-healing coatings. The ability to programme such materials, however, is basic compared to the ability of a living organism to observe, understand and respond to its environment. Here we demonstrate the relocation of biological information processing systems from cells to materials. We achieved this by operating small, programmable genetic devices outside the confines of a living cell and inside hydrogel matrices. These results establish a method for developing materials functionally enhanced with molecular machinery from biological systems.
Model-driven design allows growth ofMycoplasma pneumoniaeon serum-free media
Authors: Gaspari Erika , Malachowski Antoni , Garcia-Morales Luis , Burgos Raul , Serrano Luis , Santos Vitor A. P. Martins dos , Suarez-Diez Maria 
Published: December 2019

AbstractMycoplasma pneumoniaeis a slow-growing, human pathogen that causes atypical pneumonia. Because it lacks a cell wall, many antibiotics are ineffective, and vaccination is required. Due to its reduced genome and dearth of many biosynthetic pathways, this fastidious bacterium depends on rich, undefined medium for growth, which makes large-scale cultivation for vaccine production challenging and expensive.To understand factors limiting growth, we developed a genome-scale, constraint-based model ofM. pneumoniaecalled iEG158_mpn to describe the metabolic potential of this bacterium. We have put special emphasis on cell membrane formation to identify key lipid components to maximize bacterial growth. We have used this knowledge to predict and validatein vitrotwo serum-free media able to sustain growth.Our findings also show that glycolysis and lipid metabolism are much less efficient under hypoxia; these findings suggest that factors other than metabolism and membrane formation alone affect the growth ofM. pneumoniae.Altogether, our modelling approach enabled us to optimize medium composition, capacitated growth in defined media and streamlined operational requirements, thereby providing the basis for stable, reproducible and less expensive vaccine production.
Selection for rapid uptake of scarce or fluctuating resource explains vulnerability of glycolysis to imbalance
Authors: Janulevicius Albertas , van Doorn G. Sander 
Published: December 2019

AbstractGlycolysis is a conserved central pathway in energy metabolism that converts glucose to pyruvate with net production of two ATP molecules. Because ATP is produced only in the lower part of glycolysis (LG), preceded by an initial investment of ATP in the upper glycolysis (UG), achieving robust start-up of the pathway upon activation presents a challenge: a sudden increase in glucose concentration can throw a cell into a self-sustaining imbalanced state in which UG outpaces LG, glycolytic intermediates accumulate and the cell is unable to maintain high ATP concentration needed to support cellular functions. Such metabolic imbalance can result in “substrate-accelerated death”, a phenomenon observed in prokaryotes and eukaryotes when cells are exposed to an excess of substrate that previously limited growth. Here, we address why evolution has apparently not eliminated such a costly vulnerability and propose that it is a manifestation of an evolutionary trade-off, whereby the glycolysis pathway is adapted to quickly secure scarce or fluctuating resource at the expense of vulnerability in an environment with ample resource. To corroborate this idea, we perform evolutionary simulations of a simplified yeast glycolysis pathway consisting of UG, LG, phosphate transport between a vacuole and a cytosol, and a general ATP demand reaction. The pathway is evolved in constant or fluctuating resource environments by allowing mutations that affect the (maximum) reaction rate constants, reflecting changing expression levels of different glycolytic enzymes. We demonstrate that under limited constant resource, the population evolves to a genotype that is balanced but exhibits strongly imbalanced dynamics under ample resource conditions. Furthermore, when resource availability is fluctuating, the imbalanced phenotype enjoys a fitness advantage over balanced dynamics: when glucose is abundant, imbalanced pathways can quickly accumulate glycolytic intermediate FBP as intracellular storage that is used during periods of starvation to maintain high ATP concentration needed for growth. Our model further predicts that in environments with fluctuating resource, competition for glucose can result in stable coexistence of balanced and imbalanced cells, as well as repeated cycles of population crashes and recoveries that depend on such polymorphism. Overall, we demonstrate the importance of ecological and evolutionary arguments for understanding seemingly maladaptive aspects of cellular metabolism.