bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of user adaptation on prosthetic finger control with an intuitive myoelectric decoder
Authors: Krasoulis Agamemnon , Vijayakumar Sethu , Nazarpour Kianoush
Published: March 2019

Machine learning-based myoelectric control is regarded as an intuitive paradigm, because of the mapping it creates between muscle co-activation patterns and prosthesis movements that aims to simulate the physiological pathways found in the human arm. Despite that, there has been evidence that closed-loop interaction with a classification-based interface results in user adaptation, which leads to performance improvement with experience. Recently, there has been a focus shift towards continuous prosthesis control, yet little is known about whether and how user adaptation affects myoelectric control performance in dexterous, intuitive tasks. We investigate the effect of short-term adaptation with independent finger position control by conducting real-time experiments with 10 able-bodied and two transradial amputee subjects. We demonstrate that despite using an intuitive decoder, experience leads to significant improvements in performance. We argue that this is due to the lack of an utterly natural control scheme, which is mainly caused by differences in the anatomy of human and artificial hands, movement intent decoding inaccuracies, and lack of proprioception. Finally, we extend previous work in classification-based and wrist continuous control by verifying that offline analyses cannot reliably predict real-time performance, thereby reiterating the importance of validating myoelectric control algorithms with real-time experiments.
Error, noise and bias in de novo transcriptome assemblies
Authors: Freedman Adam H. , Clamp Michele , Sackton Timothy B.
Published: March 2019

De novo transcriptome assembly is a powerful tool, widely used over the last decade for making evolutionary inferences. However, it relies on two implicit assumptions: that the assembled transcriptome is an unbiased representation of the underlying expressed transcriptome, and that expression estimates from the assembly are good, if noisy approximations of the relative abundance of expressed transcripts. Using publicly available data for model organisms, we demonstrate that, across assembly algorithms and data sets, these assumptions are consistently violated. Bias exists at the nucleotide level, with genotyping error rates ranging from 30-83%. As a result, diversity is underestimated in transcriptome assemblies, with consistent under-estimation of heterozygosity in all but the most inbred samples. Even at the gene level, expression estimates show wide deviations from map-to-reference estimates, and positive bias at lower expression levels. Standard filtering of transcriptome assemblies improves the robustness of gene expression estimates but leads to the loss of a meaningful number of protein-coding genes, including many that are highly expressed. We demonstrate a computational method, length-rescaled CPM, to partly alleviate noise and bias in expression estimates. Researchers should consider ways to minimize the impact of bias in transcriptome assemblies.
The shift from life in water to life on land advantaged planning in visually-guided behavior
Authors: Mugan Ugurcan , MacIver Malcolm A.
Published: March 2019

Other than formerly land-based mammals such as whales and dolphins that have returned to an aquatic existence, it is uncontroversial that land animals have developed more elaborated cognitive abilities than aquatic animals. Yet there is no apparenta-priorireason for this to be the case. A key cognitive faculty is the ability to plan. Here we provide evidence that in a dynamic visually-guided behavior of crucial evolutionary importance, prey evading a predator, planning provides a significant advantage over habit-based action selection, but only on land. This advantage is dependent on the massive increase in visual range and spatial complexity that greeted the first vertebrates to view the world above the waterline 380 million years ago. Our results have implications for understanding the evolutionary basis of the limited ability of animals, including humans, to think ahead to meet slowly looming and distant threats, toward a neuroscience of sustainability.
OMGS: Optical Map-based Genome Scaffolding
Authors: Pan Weihua , Jiang Tao , Lonardi Stefano
Published: March 2019

Due to the current limitations of sequencing technologies,de novogenome assembly is typically carried out in two stages, namely contig (sequence) assembly and scaffolding. While scaffolding is computationally easier than sequence assembly, the scaffolding problem can be challenging due to the high repetitive content of eukaryotic genomes, possible mis-joins in assembled contigs and inaccuracies in the linkage information. Genome scaffolding tools either use paired-end/mate-pair/linked/Hi-C reads or genome-wide maps (optical, physical or genetic) as linkage information. Optical maps (in particular Bionano Genomics maps) have been extensively used in many recent large-scale genome assembly projects (e.g., goat, apple, barley, maize, quinoa, sea bass, among others). However, the most commonly used scaffolding tools have a serious limitation: they can only deal with one optical map at a time, forcing users to alternate or iterate over multiple maps. In this paper, we introduce a novel scaffolding algorithm called OMGS that for the first time can take advantages of multiple optical maps. OMGS solves several optimization problems to generate scaffolds with optimal contiguity and correctness. Extensive experimental results demonstrate that our tool outperforms existing methods when multiple optical maps are available, and produces comparable scaffolds using a single optical map. OMGS can be obtained fromhttps://github.com/ucrbioinfo/OMGS
TM3’seq: a tagmentation-mediated 3’ sequencing approach for improving scalability of RNA-seq experiments
Authors: Pallares Luisa F. , Picard Serge , Ayroles Julien F.
Published: March 2019

RNA-seq has become the standard tool for collecting genome-wide expression data in diverse fields, from quantitative genetics and medical genomics to ecology and developmental biology. However, RNA-seq library preparation is still prohibitive for many laboratories. Recently, the field of single-cell transcriptomics has reduced costs and increased throughput by adopting early barcoding and pooling of individual samples —producing a single final library containing all samples. In contrast, RNA-seq protocols where each sample is processed individually are significantly more expensive and lower throughput than single-cell approaches. Yet, many projects depend on individual library generation to preserve important samples or for follow-up re-sequencing experiments. Improving on currently available RNA-seq methods we have developed TM3’seq, a 3’-enriched library preparation protocol that uses Tn5 transposase and preserves sample identity at each step. TM3’seq is designed for high-throughput processing of individual samples (96 samples in 6h, with only 3h hands-on time) at a fraction of the cost of commercial kits ($1.5 per sample). The protocol was tested in a range of human andDrosophila melanogasterRNA samples, recovering transcriptomes of the same quality and reliability than the commercial NEBNext®kit. We expect that the cost-and time-efficient features of TM3’seq make large-scale RNA-seq experiments more permissive for the entire scientific community.
Circular RNA profiling in the oocyte and cumulus cells reveals thatcircARMC4is essential for porcine oocyte maturation
Authors: Cao Zubing , Gao Di , Xu Tengteng , Zhang Ling , Tong Xu , Zhang Dandan , Wang Yiqing , Ning Wei , Qi Xin , Ma Yangyang , Ji Kaiyuan , Yu Tong , Li Yunsheng , Zhang Yunhai
Published: March 2019

Thousands of circular RNAs (circRNAs) have been recently discovered in cumulus cells and oocytes from several species. However, the expression and function of circRNA during porcine oocyte meiotic maturation have been never examined. Here, we separately identified 7,067 and 637 circRNAs in both the cumulus cell and the oocyte via deep sequencing and bioinformatic analysis. Further analysis revealed that a faction of circRNAs is differentially expressed (DE) in a developmental stage-specific manner. The host genes of DE circRNAs are markedly enriched to multiple signaling pathways associated with cumulus cell function and oocyte maturation. Additionally, most DE circRNAs harbor several miRNA targets, suggesting that these DE circRNAs potentially act as miRNA sponge. Importantly, we found that maternalcircARMC4knockdown by siRNA microinjection caused a severely impaired chromosome alignment, and significantly inhibited first polar body extrusion and early embryo development. Taken together, these results demonstrate for the first time that circRNAs are abundantly and dynamically expressed in a developmental stage-specific manner in cumulus cells and oocytes, and maternally expressedcircARMC4is essential for porcine oocyte meiotic maturation and early embryo development.
Neurodegeneration caused by LRRK2-G2019S requires Rab10 in select dopaminergic neurons
Authors: Petridi Stavroula , Middleton C. Adam , Fellgett Alison , Covill Laura , Stewart Amy , Munns Jack , Kohrs Friederike Elisabeth , Hiesinger P. Robin , Wilson Laurence , Chawla Sangeeta , Elliott Christopher J. H.
Published: March 2019

Inherited mutations in the LRRK2 protein are the commonest known cause of Parkinson’s, but the molecular link from increased kinase activity to pathological neurodegeneration remains to be determined.In vitro(biochemical and cell culture) assays led to the hypothesis that several Rab GTPases might be LRRK2 substrates. Here we show that Rab10 potently modifiesLRRK2-G2019Smediated electrophysiological responses in anin vivoscreen, in which each Rab was overexpressed inDrosophiladopaminergic neurons. We therefore tested the effect ofRab10loss of function on threeLRRK2-G2019Sphenotypes (vision, movement and sleep) that rely on dopaminergic circuits in both flies and mammals. The knock-out of Rab10in vivofully rescues the reduced responses induced by dopaminergicLRRK2-G2019Sin visual and motor (reaching, proboscis extension) assays, but the sleep phenotype is unaffected. We show that Rab10 is expressed in dopaminergic (tyrosine hydroxylase positive) neurons controlling vision and proboscis movement, but undetectable in those controlling sleep, indicating that anatomical and physiological patterns of Rab10 are related. Our results support the idea that LRRK2 phosphorylates separate targets in distinct neurons and confirm that one degenerative pathway starts with Rab10. Although Rab3 is another putative substrate of LRRK2, it shows no synergy with G2019S and localises to a different subset of dopaminergic neurons from Rab10. We propose that variations inRabexpression may contribute to differences in the rate of neurodegeneration seen in different dopaminergic nuclei in Parkinson’s.

Significance Statement

A key question in Parkinson’s is why dopamine neurons die particularly fast in some parts of thesubstantia nigra. We focused on the commonest Parkinson’s-related mutation, LRRK2-G2019S.In vitroassays suggested that neurodegeneration may start by LRRK2-G2019S increasing phosphorylation of Rab10. We found Rab10 in fly dopamine neurons in visual and motor pathways, but not in the sleep system. Rab10 knock-out rescues G2019S-induced visual and movement degeneration, leaving sleep dysfunction unaffected. Thus, LRRK2 activates at least two pathways, one Rab10-dependent, leading to neurodegenerationin vivo. Rab3 is found in a different subset of dopaminergic neurons and shows no synergy withLRRK2-G2019S. We propose that variations inRabexpression contribute to differences in neurodegeneration seen in Parkinson’s.
Enhanced 4Pi single-molecule localization microscopy with coherent pupil based localization and light sheet illumination
Authors: Liu Sheng , Huang Fang
Published: March 2019

Over the last decades, super-resolution techniques have revolutionized the field of fluorescence microscopy. Among them, interferometric or 4Pi microscopy methods exhibit supreme resolving power in the axial dimension. Combining with single-molecule detection/localization and adaptive optics, iPALM/4PiSMS/W-4PiSMSN allowed 10-15 nm isotropic 3D resolution throughout the whole cell. However, further improving the achieved 3D resolution poses significantly challenges which, in part, is blocked by the complexity of single-molecule emission pattern generated by these systems rendering a large portion of information carrying photons unusable. Here we introduce a localization algorithm that achieves the theoretical information limit for 4Pi based single-molecule switching nanoscopy (4Pi-SMSN), and demonstrates improvements in resolution, accuracy as well as applicability comparing with the state of art 4Pi-SMSN methods. Further, with a novel 4Pi-compatible light-sheet illumination reducing the fluorescence background by >5-fold, we demonstrated the new system enables further improvement in the achievable resolution of 4Pi/interferometric single-molecule imaging systems.
Cryo-EM Map of the CFA/I Pilus Rod
Authors: Zheng Weili , Andersson Magnus , Mortezaei Narges , Bullitt Esther , Egelman Edward H.
Published: March 2019

EnterotoxigenicEscherichia coli(ETEC) are common agents of diarrhea for travelers and a major cause of mortality in children in developing countries. To attach to intestinal cells ETEC express colonization factors, among them CFA/I, which are the most prevalent factors and are the archetypical representative of Class 5 pili. Due to their helical quaternary structure that can be unwound and works as a damper of force, CFA/I pili help ETEC bacteria withstand intestinal fluid motion. We report in this work the CFA/I pilus structure at 4.0 Å resolution and report details of the donor strand complementation. The density map allows us to identify the buried surface area between subunits, and these regions are correlated to quaternary structural stability in class 5 and Chaperone-Usher pili. In addition, from the EM map we also predicted that residue 13 (proline) of the N-terminal β-strand could have a major impact on the filament’s structural stability. Therefore, we used optical tweezers to measure and compare the stability of the quaternary structure of wild type CFA/I and point-mutated CFA/I in which proline 13 was changed to a non-polar residue, phenylalanine. We found that pili with this mutated CFA/I require a lower force to unwind, supporting our hypothesis that Pro 13 is important for structural stability. The high-resolution CFA/I pilus structure presented in this work and the analysis of structural stability will be useful for the development of novel antimicrobial drugs that target the pilus structure to reduce its damping properties, which are needed for initial attachment and sustained adhesion of ETEC.

Synopsis

The structure of a common virulence factor expressed on the surface of diarrheacausing bacteria, CFA/I pili, has been determined at 4.0 Å resolution. The role of proline 13 in stabilizing the pilus structure has been confirmed using force-measuring optical tweezers on wild type and point-mutated pili.
Coolpup.py:versatile pile-up analysis of Hi-C data
Authors: Flyamer Ilya M. , Illingworth Robert S. , Bickmore Wendy A.
Published: March 2019

Motivation

Hi-C is currently the method of choice to investigate the global 3D organisation of the genome. A major limitation of Hi-C is the sequencing depth required to robustly detect loops in the data. A popular approach used to mitigate this issue, even in single-cell Hi-C data, is genome-wide averaging (piling-up) of peaks, or other features, annotated in high-resolution datasets, to measure their prominence in less deeply sequenced data. However current tools do not provide a computationally efficient and versatile implementation of this approach.

Results

Here we describecoolpup.py– a versatile tool to perform pile-up analysis on Hi-C data. We demonstrate its utility by replicating previously published findings regarding the role of cohesin and CTCF in 3D genome organization, as well as discovering novel details of Polycomb-driven interactions. We also present a novel variation of the pile-up approach that can aid the in statistical analysis of looping interactions. We anticipate thatcoolpup.pywill aid in Hi-C data analysis by allowing easy to use, versatile and efficient generation of pileups.

Availability and implementation

Coolpup.pyis cross-platform, open-source and free (MIT licensed) software. Source code is available fromhttps://github.com/Phlya/coolpuppyand it can be installed from the Python Packaging Index.

Contact

Ilya.Flyamer@igmm.ed.ac.uk
Spatial structure governs the mode of tumour evolution
Authors: Noble Robert , Burri Dominik , Kather Jakob Nikolas , Beerenwinkel Niko
Published: March 2019

Characterizing the mode – the way, manner, or pattern – of evolution in tumours is important for clinical forecasting and optimizing cancer treatment. DNA sequencing studies have inferred various modes, including branching, punctuated and neutral evolution, but it is unclear why a particular pattern predominates in any given tumour.1, 2Here we propose that differences in tumour architecture alone can explain the variety of observed patterns. We examine this hypothesis using spatially explicit population genetic models and demonstrate that, within biologically relevant parameter ranges, human tumours are expected to exhibit four distinct onco-evolutionary modes (oncoevotypes): rapid clonal expansion (predicted in leukaemia); progressive diversification (in colorectal adenomas and early-stage colorectal carcinomas); branching evolution (in invasive glandular tumours); and effectively almost neutral evolution (in certain non-glandular and poorly differentiated solid tumours). We thus provide a simple, mechanistic explanation for a wide range of empirical observations. Oncoevotypes are governed by the mode of cell dispersal and the range of cell-cell interaction, which we show are essential factors in accurately characterizing, forecasting and controlling tumour evolution.
3D microenvironment stiffness regulates tumor spheroid growth and mechanics via p21 and ROCK
Authors: Taubenberger Anna V. , Girardo Salvatore , Träber Nicole , Fischer-Friedrich Elisabeth , Kräter Martin , Wagner Katrin , Kurth Thomas , Richter Isabel , Haller Barbara , Binner Marcus , Hahn Dominik , Freudenberg Uwe , Werner Carsten , Guck Jochen
Published: March 2019

Mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using two-dimensional (2D) substrates, much less is known about it in a physiologically more relevant 3D context. Here we demonstrate that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa built up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy (AFM) revealed that tumor spheroid stiffness increased with hydrogel stiffness. Also, constituent cell stiffness increased in a ROCK- and F-actin-dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition reversed not only cell stiffening upon culture in stiff hydrogels but also increased tumor spheroid growth. Taken together, we reveal here a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment. Thus, our findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments and provide the basis for a more in-depth exploration of the underlying mechanosensory response.
The polyploid genome of the mitotic parthenogenetic root-knot nematodeMeloidogyne enterolobii
Authors: Koutsovoulos Georgios D. , Poullet Marine , Ashry Abdelnaser El , Kozlowski Djampa K. , Sallet Erika , Rocha Martine Da , Martin-Jimenez Cristina , Perfus-Barbeoch Laetitia , Frey Juerg-Ernst , Ahrens Christian , Kiewnick Sebastian , Danchin Etienne G.J.
Published: March 2019

Root-knot nematodes (genusMeloidogyne) are plant parasitic species that cause huge economic loss in the agricultural industry and affect the prosperity of communities in developing countries. Control methods against these plant pests are sparse and the current preferred method is deployment of plant cultivars bearing resistance genes againstMeloidogynespecies. However, some species such asM. enterolobiiare not controlled by the resistance genes deployed in the most important crop plants cultivated in Europe. The recent identification of this species in Europe is thus a major concern. Like the other most damaging Meloidogyne species (e.g.M. incognita,M. arenariaandM. javanica),M. enterolobiireproduces by obligatory mitotic parthenogenesis. Genomic singularities such as a duplicated genome structure and a relatively high proportion of transposable elements have previously been described in the above mentioned mitotic parthenogenetic Meloidogyne.

To gain a better understanding of the genomic and evolutionary background we sequenced the genome ofM. enterolobiiusing high coverage short and long read technologies. The information contained in the long reads helped produce a highly contiguous genome assembly ofM. enterolobii, thus enabling us to perform high quality annotations of coding and non-coding genes, and transposable elements.

The genome assembly and annotation reveals a genome structure similar to the ones described in the other mitotic parthenogenetic Meloidogyne, described as recent hybrids. Most of the genome is present in 3 different copies that show high divergence. Because most of the genes belong to these duplicated regions only few gene losses took place, which suggest a recent polyploidization. The most likely hypothesis to reconcile high divergence between genome copies despite few gene losses and translocations is also a recent hybrid origin. Consistent with this hypothesis, we found an abundance of transposable elements at least as high as the one observed in the mitotic parthenogenetic nematodesM. incognitaandM. javanica.
FSHR-1/GPCR activates the mitochondrial unfolded protein response in Caenorhabditis elegans
Authors: Kim Sungjin , Sieburth Derek
Published: March 2019

The mitochondrial unfolded protein response (UPRmt) is an evolutionarily conserved adaptive response that functions to maintain mitochondrial homeostasis following mitochondrial damage. In C. elegans, the nervous system plays a central role in responding to mitochondrial stress by releasing endocrine signals that act upon distal tissues to activate the UPRmt. The mechanisms by which mitochondrial stress is sensed by neurons and transmitted to distal tissues is not fully understood. Here, we identify a role for the conserved follicle-stimulating hormone G protein coupled receptor (GPCR), FSHR-1, in promoting UPRmt activation. Genetic deficiency of fshr-1 severely attenuates UPRmt activation and organism-wide survival in response to mitochondrial stress. FSHR-1 functions in a common genetic pathway with SPHK-1/sphingosine kinase to promote UPRmt activation, and FSHR-1 regulates the mitochondrial association of SPHK-1 in the intestine. Through tissue-specific rescue assays, we show that FSHR-1 functions in neurons to activate the UPRmt, to promote mitochondrial association of SPHK-1 in the intestine, and to promote organism-wide survival in response to mitochondrial stress. We propose that FSHR-1 functions cell non-autonomously in neurons to activate UPRmt upstream of SPHK-1 signaling in the intestine.
eIF5B gates the transition from translation initiation to elongation
Authors: Wang Jinfan , Johnson Alex G. , Lapointe Christopher P. , Choi Junhong , Prabhakar Arjun , Chen Dong-Hua , Petrov Alexey N. , Puglisi Joseph D.
Published: March 2019

Translation initiation determines both the quantity and identity of the protein product by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors (eIFs) prepare ribosomes for polypeptide elongation, yet the underlying dynamics of this process remain enigmatic1–4. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here, we applied in vitro single-molecule fluorescence microscopy approaches to monitor directly in real time the pathways of late translation initiation and the transition to elongation using a purified yeast Saccharomyces cerevisiae translation system. This transition was remarkably slower in our eukaryotic system than that reported for Escherichia coli5–7. The slow entry to elongation was defined by a long residence time of eIF5B on the 80S ribosome after joining of individual ribosomal subunits, which is catalyzed by this universally conserved initiation factor. Inhibition of eIF5B GTPase activity following subunit joining prevented eIF5B dissociation from the 80S complex, thereby preventing elongation. Our findings illustrate how eIF5B dissociation serves as a kinetic checkpoint for the transition from initiation to elongation, and its release may be governed by a conformation of the ribosome complex that triggers GTP hydrolysis.
DNA-scaffolded biomaterials enable modular and tunable control of cell-based cancer immunotherapies
Authors: Huang Xiao , Williams Jasper Z. , Chang Ryan , Li Zhongbo , Gai Eric , Patterson David M. , Wei Yu , Lim Wendell A. , Desai Tejal A.
Published: March 2019

Advanced biomaterials provide versatile ways to spatially and temporally control immune cell activity, potentially enhancing their therapeutic potency and safety. Precise cell modulation demands multi-modal display of functional proteins with controlled densities on biomaterials. Here, we develop an artificial immune cell engager (AICE) platform – biodegradable particles onto which multiple proteins are densely loaded with ratiometric control via short nucleic acid tethers. We demonstrate the impact of AICE with varying ratios of anti-CD3 and anti-CD28 antibodies onex vivoexpansion of human primary T cells. We also show that AICE can be used to control the activity of engineered T cellsin vivo. AICE injected intratumorally can provide a local priming signal for systemically administered AND-gate chimeric antigen receptor T cells, driving local tumor clearance while sparing uninjected tumors that model potentially cross-reactive healthy tissues. This modularly functionalized biomaterial thus provides a flexible platform to achieve sophisticated control over cell-based immunotherapies.
Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP Kinases to SPEECHLESS
Authors: Putarjunan Aarthi , Ruble Jim , Srivastava Ashutosh , Zhao Chunzhao , Rychel Amanda L. , Hofstetter Alex K. , Tang Xiaobo , Zhu Jian-Kang , Tama Florence , Zheng Ning , Torii Keiko U.
Published: March 2019

Cell-fate in eukaryotes is regulated by MAP Kinases (MAPKs) that translate external cues to cellular responses. In plants, two MAPKs, MPK3/6, regulate diverse processes of development, environmental response, and immunity. Yet, the mechanism bridging these shared signaling components with a specific target remains unresolved. Focusing on the development of stomata, epidermal valves for gas exchange and transpiration, we report here that the bHLH protein SCREAM functions as a scaffold by recruiting MPK3/6 to downregulate SPEECHLESS, a transcription factor initiating stomatal cell lineages. SCREAM directly binds with MPK3/6 through an evolutionarily-conserved yet unconventional bipartite motif. Mutations in this motif abrogate association, phosphorylation and degradation of SCREAM, unmask hidden non-redundancies between MPK3 and MPK6, and result in uncontrolled stomatal differentiation. Structural analyses of MPK6 at the 2.75Å resolution unraveled bipartite binding of SCREAM with MPK6, that is distinct from an upstream MAPKK. Our findings elucidate, at the atomic resolution, the mechanism directly linking extrinsic signals to transcriptional reprogramming during the establishment of stomatal cell-fate, and highlight a unique substrate-binding mode adopted by plant MAPKs.
Constitutive expression of a fluorescent protein reports the size of live human cells
Authors: Berenson Daniel F. , Zatulovskiy Evgeny , Xie Shicong , Skotheim Jan M.
Published: March 2019

Cell size is intimately related to cell physiology because it sets the geometric scale of organelles and biosynthesis. A number of methods exist to measure different aspects of size of individual cells, but each has significant drawbacks. Here, we present an alternative method to measure the size of single human cells using a nuclear localized fluorescent protein expressed from a constitutive promoter. We validate this method by comparing it to several established cell size measurement strategies, including flow cytometry optical scatter, total protein dyes, and quantitative phase microscopy. We directly compare our fluorescent protein measurement to the commonly used measurement of nuclear volume and show that our measurements are more robust and less dependent on image segmentation. We apply our method to examine how cell size impacts the cell division cycle, which reaffirms the importance of G1/S size control. Importantly, combining our size reporter with fluorescent labeling of a different protein in a different color channel allows measurement of concentration dynamics using simple widefield fluorescence imaging. Thus, we expect our method will be of use to other researchers interested in the topics of cell size control and, more broadly, how dynamically changing protein concentrations control cell fates.
Intelligent Design of 14-3-3 Docking Proteins Utilizing Synthetic Evolution Artificial Intelligence (SYN-AI)
Authors: Davis Leroy K.
Published: March 2019

The ability to write DNA code from scratch will allow for the discovery of new and interesting chemistries as well as allowing the rewiring of cell signal pathways. Herein, we have utilized synthetic evolution artificial intelligence (SYN-AI) to intelligently design a set of 14-3-3 docking genes. SYN-AI engineers synthetic genes utilizing a parental gene as an evolution template. Wherein, evolution is fast forwarded by transforming template gene sequences to DNA secondary and tertiary codes based upon gene hierarchical structural levels. The DNA secondary code allows identification of genomic building blocks across an orthologous sequence space comprising multiple genomes. Where, the DNA tertiary code allows engineering of super secondary structures. SYN-AI constructed a library of 10 million genes that was reduced to three structurally functional 14-3-3 docking genes by applying natural selection protocols. Synthetic protein identity was verified utilizing Clustal Omega sequence alignments and Phylogeny.fr phylogenetic analysis. Wherein, we were able to confirm three-dimensional structure utilizing I-TASSER and protein ligand interactions utilizing COACH and Cofactor. Conservation of allosteric communications were confirmed utilizing elastic and anisotropic network models. Whereby, we utilized elNemo and ANM2.1 to confirm conservation of the 14-3-3 ζ amphipathic groove. Notably, to the best of our knowledge, we report the first 14-3-3 docking genes to be written from scratch.
Impact of Genome Architecture Upon the Functional Activation and Repression of Hox Regulatory Landscapes
Authors: Rodríguez-Carballo Eddie , Lopez-Delisle Lucille , Yakushiji-Kaminatsui Nayuta , Ullate-Agote Asier , Duboule Denis
Published: March 2019

Background

The spatial organization of the mammalian genome relies upon the formation of chromatin domains of various scales. At the level of gene regulation in cis, collections of enhancer sequences define large regulatory landscapes that usually match with the presence of topologically associating domains (TADs). These domains are largely determined by bound CTCF molecules and often contain ranges of enhancers displaying similar or related tissue specificity, suggesting that in some cases such domains may act as coherent regulatory units, with a global on or off state.

Results

By using the HoxD gene cluster as a paradigm, we investigated the effect of large genomic rearrangements affecting the two TADs flanking this locus, including their fusion into a single chromatin domain. We show that, within a single hybrid TAD, the activation of both proximal and distal limb enhancers initially positioned in either TADs globally occurred as when both TADs are intact. We also show that the timely implementation of distal limb enhancers depends on whether or not target genes had previously responded to proximal enhancers, due to the presence or absence of H3K27me3 marks.

Conclusions

From this work, we conclude that antagonistic limb proximal and distal enhancers can exert their specificities when positioned into the same TAD and in the absence of their genuine target genes. We also conclude that removing these target genes reduced the coverage of a regulatory landscape by chromatin marks associated with silencing and thus prolonged its activity in time. Since Polycomb group proteins are mainly recruited at the Hox gene cluster, our results suggest that Polycomb Repressive Complex 2 (PRC2) can extend its coverage to far-cis regulatory sequences as long as confined to the neighboring TAD structure.
High-throughput Multimodal Automated Phenotyping (MAP) with Application to PheWAS
Authors: Liao Katherine P. , Sun Jiehuan , Cai Tianrun A. , Link Nicholas , Hong Chuan , Huang Jie , Huffman Jennifer E. , Gronsbell Jessica , Zhang Yichi , Ho Yuk-Lam , Castro Victor , Gainer Vivian , Murphy Shawn N. , O’Donnell Christopher J. , Gaziano J. Michael , Cho Kelly , Szolovits Peter , Kohane Isaac S. , Yu Sheng , Cai Tianxi ,   with the VA Million Veteran Program
Published: March 2019

Objective

Electronic health records (EHR) linked with biorepositories are a powerful platform for translational studies. A major bottleneck exists in the ability to phenotype patients accurately and efficiently. The objective of this study was to develop an automated high-throughput phenotyping method integrating International Classification of Diseases (ICD) codes and narrative data extracted using natural language processing (NLP).

Method

We developed a mapping method for automatically identifying relevant ICD and NLP concepts for a specific phenotype leveraging the UMLS. Aggregated ICD and NLP counts along with healthcare utilization were jointly analyzed by fitting an ensemble of latent mixture models. The MAP algorithm yields a predicted probability of phenotype for each patient and a threshold for classifying subjects with phenotype yes/no. The algorithm was validated using labeled data for 16 phenotypes from a biorepository and further tested in an independent cohort PheWAS for two SNPs with known associations.

Results

The MAP algorithm achieved higher or similar AUC and F-scores compared to the ICD code across all 16 phenotypes. The features assembled via the automated approach had comparable accuracy to those assembled via manual curation (AUCMAP 0.943, AUCmanual 0.941). The PheWAS results suggest that the MAP approach detected previously validated associations with higher power when compared to the standard PheWAS method based on ICD codes.

Conclusion

The MAP approach increased the accuracy of phenotype definition while maintaining scalability, facilitating use in studies requiring large scale phenotyping, such as PheWAS.
Single-shot autofocus microscopy using deep learning
Authors: Pinkard Henry , Phillips Zachary , Babakhani Arman , Fletcher Daniel A. , Waller Laura
Published: March 2019

Maintaining an in-focus image over long time scales is an essential and non-trivial task for a variety of microscopic imaging applications. Here, we present an autofocusing method that is inexpensive, fast, and robust. It requires only the addition of one or a few off-axis LEDs to a conventional transmitted light microscope. Defocus distance can be estimated and corrected based on a single image under this LED illumination using a neural network that is small enough to be trained on a desktop CPU in a few hours. In this work, we detail the procedure for generating data and training such a network, explore practical limits, and describe relevant design principles governing the illumination source and network architecture.
Lipid membranes trigger misfolding and self-assembly of amyloid β 42 protein into aggregates
Authors: Banerjee Siddhartha , Hashemi Mohtadin , Zagorski Karen , Lyubchenko Yuri L.
Published: March 2019

The assembly of polypeptides and proteins into nanoscale aggregates is a phenomenon observed in a vast majority of proteins. Importantly, aggregation of amyloid β (Aβ) proteins is considered as a major cause for the development of Alzheimer’s disease. The process depends on various conditions and typical test-tube experiments require high protein concentration that complicates the translation of results obtained in vitro to understanding the aggregation process in vivo. Here we demonstrate that Aβ42 monomers at the membrane bilayer are capable of self-assembling into aggregates at physiologically low concentrations, and the membrane in this aggregation process plays a role of a catalyst. We applied all-atom molecular dynamics to demonstrate that the interaction with the membrane surface dramatically changes the conformation of Aβ42 protein. As a result, the misfolded Aβ42 rapidly assembles into dimers, trimers and tetramers, so the on-surface aggregation is the mechanism by which amyloid oligomers are produced and spread.
Human DNA-PK activates a STING-independent DNA sensing pathway
Authors: Burleigh Katelyn , Maltbaek Joanna H. , Cambier Stephanie , Green Richard , Gale Michael , James Richard C. , Stetson Daniel B.
Published: March 2019

Detection of intracellular DNA by the cGAS-STING pathway activates a type I interferon-mediated innate immune response that protects from virus infection and can be harnessed to promote anti-tumor immunity. Whether there are additional DNA sensing pathways, and how such pathways might function, remains controversial. We show here that humans – but not mice – have a second, potent, STING-independent DNA sensing pathway that is blocked by the E1A viral oncogene of human adenovirus 5. We identify human DNA-PK as the sensor of this pathway and demonstrate that DNA-PK kinase activity drives a robust and broad antiviral response. We discover that the heat shock protein HSPA8/HSC70 is a unique target of DNA-PK. Finally, we demonstrate that detection of foreign DNA and DNA damage trigger distinct modalities of DNA-PK activity. These findings reveal the existence, sensor, unique target, and viral antagonists of a STING-independent DNA sensing pathway (SIDSP) in human cells.
Class A GPCRs use the membrane potential to increase their sensitivity and selectivity
Authors: Shalaeva Daria N. , Cherepanov Dmitry A. , Galperin Michael Y. , Vriend Gert , Mulkidjanian Armen Y.
Published: March 2019

The human genome contains about 700 genes of G protein-coupled receptors (GPCRs) of class A; these seven-helical membrane proteins are the targets of almost half of all known drugs. In the middle of the helix bundle, crystal structures revealed a highly conserved sodium-binding site, which is connected with the extracellular side by a water-filled tunnel. Sodium ions are observed in GPCRs crystallized in their inactive conformations, but not in GPCRs that were trapped in agonist-bound active conformations. The escape route of the sodium ion upon the inactive-to-active transition and its very direction, either into the cytoplasm or back outside the cell, hitherto remained obscure. We modeled sodium-binding GPCRs as electrogenic carriers of sodium ions. In this model the sodium gradient over the cell membrane would increase the sensitivity of GPCRs if their activation is thermodynamically coupled to the translocation of the sodium ion into the cytoplasm, while decreasing it if the sodium ion retreats into the extracellular space upon receptor activation. The model quantitatively describes the available data on both activation and suppression of distinct GPCRs by membrane voltage. The model also predicts selective amplification of the signal from (endogenous) agonists if only they, but not their (partial) analogs, could induce sodium translocation. Comparative structure and sequence analyses of sodium-binding GPCRs indicate a key role for the conserved leucine residue in the second transmembrane helix (Leu2.46) in coupling sodium translocation to receptor activation. Hence, class A GPCRs appear to utilize the energy of the transmembrane sodium potential to increase their sensitivity and selectivity.
Human Foveal Cone Photoreceptor Topography and its Dependence on Eye Length
Authors: Wang Yiyi , Bensaid Nicolas , Tiruveedhula Pavan , Ma Jianqiang , Ravikumar Sowmya , Roorda Austin
Published: March 2019

We provide the first measures of foveal cone density as a function of axial length in living eyes and discuss the physical and visual implications of our findings. We used a new generation Adaptive Optics Scanning Laser Ophthalmoscope to image cones at and near the fovea in 28 eyes of 16 subjects. Cone density and other metrics were computed in units of visual angle and linear retinal units. The foveal cone mosaic in longer eyes is expanded at the fovea, but not in proportion to eye length. Despite retinal stretching (decrease in cones/mm2), myopes generally have a higher angular sampling density (increase in cones/deg2) in and around the fovea compared to emmetropes, offering the potential for better visual acuity. Reports of deficits in best-corrected foveal vision in myopes compared to emmetropes cannot be explained by increased spacing between photoreceptors caused by retinal stretching during myopic progression.
Omics of the early molecular dialogue between Frankia alni and Alnus glutinosa and the cellulase synton
Authors: Pujic Petar , Alloisio Nicole , Fournier Pascale , Roche David , Sghaier Haitham , Miotello Guylaine , Armengaud Jean , Berry Alison M. , Normand Philippe
Published: March 2019

The early Frankia‐Alnus symbiotic molecular exchanges were analyzed in detail by protein and RNA omics. For this, Frankia cells were placed in the presence of Alnus roots but separated by a dialysis membrane for 64 h. The bacterial cells were then harvested and analyzed by high‐throughput proteomics and transcriptomics (RNA‐seq). The most upregulated gene clusters were found to be the potassium transporter operon kdp and an ABC transporter operon of uncharacterized function. The most upregulated proteins were found to be acyl dehydrogenases and the potassium transporter Kdp. These suggest a preadaptation to the impending stresses linked to the penetration into isotonic host tissues and a possible rearrangement of the membrane. Another cluster among the 60 most upregulated ones that comprised two cellulases and a cellulose synthase was conserved among the Frankia and other actinobacteria such as Streptomyces. Cellulase activity was detected on CMC all along the length of the root but not away from it. Frankia alni ACN14a was found to be unable to respire or grow on glucose as sole carbon source. The cellulose synthase was found active at the tip of hyphae in response to Alnus root exudates, resulting in a calcofluor stained tip.
The field of graduate recruitment: leading financial and consultancy firms and elite class formation
Authors: Donnelly Michael , Gamsu Sol
Published: March 2019

The symbolic value of being recruited by a high status multinational company likely represents an important marker of distinction. For the first time, a unique Destinations of Leavers in Higher Education (DLHE) data‐set is used here to model entry to elite multinational company in finance, accountancy and consultancy sectors among graduates of different social origins, universities, degree subjects and with different degree classifications. From a sample of 11,755 graduates working across these three sectors, we examine what predicts entry to 31 leading firms and then examine pay hierarchies among the 3,260 graduates working for these companies using random‐effects models. At first glance, significantly, we find that elite recruits come from a much broader range of universities than might be imagined. However, a closer look at the highest paid graduates within these firms reveals more familiar patterns of social and institutional stratification. We argue that these patterns likely reflect the nature of work undertaken by graduates in these elite firms, with institutional and social origins of graduates differing according to the particular track taken in what are likely to be highly differentiated graduate recruitment schemes.
An r package and online resource for macroevolutionary studies using the ray‐finned fish tree of life
Authors: Chang Jonathan , Rabosky Daniel L. , Smith Stephen A. , Alfaro Michael E.
Published: March 2019

Comprehensive, time‐scaled phylogenies provide a critical resource for many questions in ecology, evolution and biodiversity. Methodological advances have increased the breadth of taxonomic coverage in phylogenetic data; however, accessing and reusing these data remain challenging.

We introduce the Fish Tree of Life website and associated r package fishtree to provide convenient access to sequences, phylogenies, fossil calibrations and diversification rate estimates for the most diverse group of vertebrate organisms, the ray‐finned fishes. The Fish Tree of Life website presents subsets and visual summaries of phylogenetic and comparative data, and is complemented by the r package, which provides flexible programmatic access to the same underlying data source for advanced users wishing to extend or reanalyse the data.

We demonstrate functionality with an overview of the website, and show three examples of advanced usage through the r package. First, we test for the presence of long branch attraction artefacts across the fish tree of life. The second example examines the effects of habitat on diversification rate in the pufferfishes. The final example demonstrates how a community phylogenetic analysis could be conducted with the package.

This resource makes a large comparative vertebrate dataset easily accessible via the website, while the r package enables the rapid reuse and reproducibility of research results via its ability to easily integrate with other r packages and software for molecular biology and comparative methods.