bluuebunny/crossref_metadata_2025 huggingface dataset:
Neglected Quaternary legacy on biodiversity in the Mountains of Southwest China
Authors: Wan Tao , Huang Huateng , Oaks Jamie R. , Jiang Xuelong , Knowles L. Lacey
Published: February 2020

Mountains of Southwest China (MSWC) is a biodiversity hotspot with a very unique and highly complex terrain. However, with the majority of studies focusing on the biogeographic consequences of massive mountain building, the Quaternary legacy of biodiversity for the MSWC has long been overlooked. Here, we took a comparative phylogeography approach to examine factors that shaped community-wide diversification. With data from 30 vertebrate species, the results reveal spatially concordant genetic structure, with temporally clustered divergence events during severe glacial cycles, indicating the importance of riverine barriers in the phylogeographic history of the vertebrate community. We conclude that the repeated glacial cycles are associated with temporal synchrony of divergence patterns that are themselves structured by the heterogeneity of the montane landscape has of the MSWC. This orderly process of diversifications has profound implications for conservation by highlighting the relative independence of different geographic areas in which communities have responded similarly to climate changes and calls for further comparative phylogeographic investigations to reveal the extent to which these findings might apply more broadly to other taxa in this biodiversity hotspot.
More than a feeling: central amygdala mediates social transfer of information about proximity of danger
Authors: Andraka K. , Kondrakiewicz K. , Rojek-Sito K. , Ziegart-Sadowska K. , Meyza K. , Nikolaev T. , Hamed A. , Kursa M. , Wójcik M. , Danielewski K. , Wiatrowska M. , Kublik E. , Bekisz M. , Lebitko T. , Duque D. , Jaworski T. , Madej H. , Konopka W. , Boguszewski P. M. , Knapska E.
Published: February 2020

To survive, an animal must adequately respond to challenges presented by the environment. Social animals can learn about danger from fear of conspecifics which allows them to avoid predation and other threats without costly, first-hand experience. However, it remains unclear as to whether animals can transmit specific information that helps an individual avoid harm or whether the transmitted social signals simply increase vigilance in a non-specific manner. Here we demonstrate that animals can select appropriate defensive strategies in a novel environment depending on cues from the conspecific and, using opsins targeted to behaviorally activated neurons, identify distinct neuronal circuits in the central amygdala (CeA) that are crucial for choosing a context-appropriate reaction. The identified circuits differ in molecular markers and patterns of connectivity. Thus, we show that social signals carry explicit information about proximity of danger, necessary for choosing a context-appropriate reaction, and that the choosing process is mediated by the CeA circuits.
Multithreaded two-pass connected components labelling and particle analysis in ImageJ
Authors: Doube Michael
Published: February 2020

Sequential region labelling, also known as connected components labelling, is a standard image segmentation problem that joins contiguous foreground pixels into blobs. Despite its long development history and widespread use across diverse domains such as bone biology, materials science, and geology, connected components labelling can still form a bottleneck in image processing pipelines. Here, I describe a multithreaded implementation of classical two-pass sequential region labelling and introduce an efficient collision resolution step, ‘bucket fountain’. Code was validated on test images and against commercial software (Avizo). It was performance tested on images from 2 MB (161 particles) to 6.5 GB (437,508 particles) to determine whether theoretical linear scaling (O(n)) had been achieved, and on 1 – 40 CPU threads to measure speed improvements due to multithreading. The new implementation achieves linear scaling (b= 0.905 – 1.052,time∝pixelsb;R2 = 0.985 – 0.996), which improves with increasing thread number up to 8-10 threads, suggesting that it is memory bandwidth limited. This new implementation of sequential region labelling reduces the time required from hours to a few tens of seconds for images of several GB, and is limited only by hardware scale. It is available open source and free of charge in BoneJ.
Genome wide association analysis in dilated cardiomyopathy reveals two new key players in systolic heart failure on chromosome 3p25.1 and 22q11.23
Authors: Garnier Sophie , Harakalova Magdalena , Weiss Stefan , Mokry Michal , Regitz-Zagrosek Vera , Hengstenberg Christian , Cappola Thomas P , Isnard Richard , Arbustini Eloisa , Cook Stuart A. , van Setten Jessica , Callis Jörg , Hakonarson Hakon , Morley Michael P , Stark Klaus , Prasad Sanjay K. , Li Jin , O’Regan Declan P , Grasso Maurizia , Müller-Nurasyid Martina , Meitinger Thomas , Empana Jean-Philippe , Strauch Konstantin , Waldenberger Mélanie , Marguiles Kenneth B , Seidman Christine E. , Meder Benjamin , Boutouyrie Pierre , Lacolley Patrick , Jouven Xavier , Erdman Jeanette , Blankenberg Stefan , Wichter Thomas , Ruppert Volker , Tavazzi Luigi , Dubourg Olivier , Roizes Gerard , Dorent Richard , DeGroote Pascal , Fauchier Laurent , Trochu Jean-Noël , Aupetit Jean-François , Germain Marine , Völker Uwe , Daiane Hemerich , Raji Ibticem , Bacq-Daian Delphine , Proust Carole , Lehnert Kristin , Maas Renee , Olaso Robert , Saripella Ganapathivarma , Felix Stephan B. , Ginn Steven Mc , Duboscq-Bidot Laëtitia , van Mil Alain , Besse Céline , Fontaine Vincent , Blanché Hélène , Keating Brendan , Garcia-Pavia Pablo , Curjol Angélique , Boland Anne , Komajda Michel , Cambien François , Deleuze Jean-François , Dörr Marcus , Asselbergs Folkert W , Villard Eric , Trégouët David-Alexandre , Charron Philippe ,   On behalf of GENMED consortium
Published: February 2020

We present the results of the largest genome wide association study (GWAS) performed so far in dilated cardiomyopathy (DCM), a leading cause of systolic heart failure and cardiovascular death, with 2,719 cases and 4,440 controls in the discovery population. We identified and replicated two new DCM-associated loci, one on chromosome 3p25.1 (lead SNP rs62232870, p = 8.7 × 10−11and 7.7 × 10−4in the discovery and replication step, respectively) and the second on chromosome 22q11.23 (lead SNP rs7284877, p = 3.3 × 10−8and 1.4 × 10−3in the discovery and replication step, respectively) while confirming two previously identified DCM loci on chromosome 10 and 1,BAG3andHSPB7. The genetic risk score constructed from the number of lead risk-alleles at these four DCM loci revealed that individuals with 8 risk-alleles were at a 27% increased risk of DCM compared to individuals with 5 risk alleles (median of the referral population). We estimated the genome wide heritability at 31% ± 8%.

In silicoannotation and functional 4C-sequencing analysis on iPSC-derived cardiomyocytes strongly suggestSLC6A6as the most likely DCM gene at the 3p25.1 locus. This gene encodes a taurine and beta-alanine transporter whose involvement in myocardial dysfunction and DCM is supported by recent observations in humans and mice. Although less easy to discriminate the better candidate at the 22q11.23 locus,SMARCB1appears as the strongest one.

This study provides both a better understanding of the genetic architecture of DCM and new knowledge on novel biological pathways underlying heart failure, with the potential for a therapeutic perspective.
Comparative Gene Essentiality across the Bacterial Domain
Authors: Shaw Daniel , Hermoso Antonio , Lluch-Senar Maria , Serrano Luis
Published: February 2020

Comparative genomics among bacteria has been used to gain insight into the minimal number of conserved genes in biological pathways. Essentiality studies have provided information regarding which genes are non-dispensable (essential, E) for cell growth. Here, we integrated studies of gene conservation, essentiality and function, performed in 47 diverse bacterial species. We showed there is a modest positive correlation between genome size and number of essential genes. Interestingly, we observed a clear shift in the functions assigned to these essential genes as genome size increases. For instance, essential genes related to transcription and translation dominate small genomes. In contrast, in large genomes functions of essential genes are related with cellular processing and metabolism. Finally, and most intriguing, we found a group of genes that while being highly conserved are also typically non-essential. This suggests that some housekeeping genes confer a significant survival benefit in nature while being non-essentialin vitro.
Nested oscillations and brain connectivity during sequential stages of feature-based attention
Authors: Pagnotta Mattia F. , Pascucci David , Plomp Gijs
Published: March 2020

Brain mechanisms of visual selective attention involve both local and network-level activity changes at specific oscillatory rhythms, but their interplay remains poorly explored. Here, we investigate anticipatory and reactive effects of feature-based attention using separate fMRI and EEG recordings, while participants attended to one of two spatially overlapping visual features (motion and orientation). We focused on EEG source analysis of local nested oscillations and on graph analysis of connectivity changes in a network of fMRI-defined regions of interest, and characterized a cascade of attentional effects and their interplay at multiple spatial scales. We discuss how the results may reconcile several theories of selective attention, by showing how β rhythms support anticipatory information routing through increased network efficiency and β-γ coupling in functionally specialized regions (V1 for orientation, V5 for motion), while reactive α-band desynchronization patterns and increased α-γ coupling in V1 and V5 mediate stimulus-evoked processing of task-relevant signals.
Host “cleansing zone” at secondary contact: a new pattern in host-parasite population genetics
Authors: Martinů Jana , Štefka Jan , Poosakkannu Anbu , Hypša Václav
Published: March 2020

We introduce a new pattern of population genetic structure in a host-parasite system that can arise after secondary contact (SC) of previously isolated populations. Due to different generation time and therefore different tempo of molecular evolution the host and parasite populations reach different degrees of genetic differentiation during their separation (e.g. in refugia). Consequently, during the SC the host populations are able to re-establish a single panmictic population across the whole recolonized area, while the parasite populations stop their dispersal at the SC zone and create a narrow hybrid zone (HZ). From the host’s perspective, the parasite’s HZ functions on a microevolutionary scale as a “host-cleansing filter”: while passing from area A to area B, the hosts are rid of the area A parasites and acquire the area B parasites. We demonstrate this novel pattern on a model composed ofApodemusmice andPolyplaxlice by comparing maternally inherited markers (complete mitochondrial genomes, and complete genomes of vertically transmitted symbiontLegionella polyplacis) with SNPs derived from the louse genomic data. We discuss circumstances which may lead to this pattern and possible reasons why it has been overlooked in the studies on host-parasite population genetics.
Mass Photometry of Membrane Proteins
Authors: Olerinyova Anna , Sonn Segev Adar , Gault Joseph , Eichmann Cedric , Schimpf Johannes , Kopf Adrian H , Rudden Lucas S.P. , Ashkinadze Dzmitry , Bomba Radoslaw , Greenwald Jason , Degiacomi Matteo T , Killian J Antoinette , Friedrich Thorsten , Riek Roland , Struwe Weston B , Kukura Philipp
Published: February 2020

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid - like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane mimetic systems at the single particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterising the particle size, sample purity and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high - resolution studies of membrane protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene - maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers - in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non - functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.
Curvature domains in V4 of Macaque Monkey
Authors: Hu Jiaming , Song Xue Mei , Wang Qiannan , Roe Anna Wang
Published: March 2020

An important aspect of visual object recognition is the ability to perceive object shape. How the brain encodes fundamental aspects of shape information remains poorly understood. Models of object shape representation describe a multi-stage process that includes encoding of contour orientation and curvature. While modules encoding contour orientation are well established (orientation domains in V1 and V2 visual cortical areas), whether there are modules for curvature is unknown. In this study, we identify a module for curvature representation in area V4 of monkey visual cortex and illustrate a systematic representation of low to high curvature and of curvature orientation, indicative of curvature hypercolumns in V4. We suggest that identifying systematic modular organizations at each stage of the visual cortical hierarchy signifies the key computations performed.

Significance

We use intrinsic signal optical imaging in area V4 of anesthetized macaque monkey to study the functional organization of curvature representation. We find a modular basis for cue-invariant curvature representation in area V4 of monkey visual cortex and illustrate a systematic representation from low to high curvature and of curvature orientation, replete with curvature pinwheels. This is the first report of systematic functional organization for curvature representation in the visual system. The use of optical imaging has revealed at a population level spatial details of cortical responses, something which has not been evident from previous studies of single neurons. These data support a representational architecture underlying a ‘curvature hypercolumn’ in V4.
Exploring the Pharmacological Mechanism of Bezoar on Cerebral Ischemic Stroke using a Network Pharmacology Approach
Authors: Du Xin , Li Changxiang , Ren Beida , Deng Nan , Chen Congai , Wang Xueqian , Cheng Fafeng , Qingguo Wang Min Liu
Published: February 2020

Background

Ischemic stroke is a common clinical disease limited by its time window for treatment. In addition to its high mortality rate, only one treatment currently exists for ischemic stroke. Nonetheless, traditional Chinese medicine is often used as a reatment for the disease. Bezoar is a multi-functional drug that has been demonstrated to be effective for the treatment of ischemic stroke. However, its mechanism of action is yet to be fully elucidated. Based on network pharmacology, we explored the potential mechanism of action of bezoar. Symmap and literature data mining methods were used to obtain the target of bezoar. The mechanism of bezoar for the treatment of ischemic stroke was identified and ischemic stroke-related targets were retrieved using DrugBank, Online Mendelian Inheritance in Man, and Therapeutic Target Database. Protein-protein interaction networks were constructed using the Cytoscape plugin, BisoGenet, and analysed by topological methods. Gene Ontology and Kyoto Encyclopaedia of Genes and Genomes pathway enrichment were carried out via the Database for Annotation, Visualization, and Integrated Discovery server. We obtained 48 potential targets and 3 signalling pathways, including mitogen-activated protein kinase, hypoxia-inducible factor-1, and tumour necrosis factor signalling pathways. The mechanism of action of bezoar on ischemic stroke involves multiple targets and signalling pathways. Our research provides a network pharmacology framework for future Chinese medicinal research.
Altered B cells homeostasis in child-onset immunoglobulin A vasculitis
Authors: Liu Deying , Jiang Yanfang , Wang Jinghua , Liu Jinxiang , Xu Meng , Liu Congcong , Yang Sirui
Published: February 2020

Background

Immunoglobulin A vasculitis (IgAV), also called Henoch–Schönlein purpura, is a systemic small vessels vasculitis with immunoglobulin A1-dominant immune deposits. B-cells are a heterogeneous population with unique subsets distinguished by their phenotypes and cytokine production. Here, we explored the status of B cell subsets in patients with IgAV.

Methods

Thirty IgAV patients and fifteen age- and sex-matched healthy individuals were enrolled in this study. Fresh blood samples were collected from both healthy and IgAV patients. Upon the distinct expressions of CD3, CD19, CD20, CD38, CD27 and IgD, peripheral blood mononuclear cells (PBMCs) were initially categorized into plasmablasts and memory B cells. Subsequently, using surface markers including CD138 and IgM, and intracellular markers containing IgM and IgG, plasmablasts and memory B cells were further divided into distinct subgroups. A total of eleven populations were detected using multiple flow cytometry.

Results

CD3-CD19+IgD+CD27-, CD3-CD19+CD20-CD38+, CD3-CD19+CD20-CD38+IgM+, and CD3-CD19+CD20-CD38+CD138+B cells were larger in patients with IgAV than in the HCs. Only CD3-CD19+IgD-CD27+IgM+B cell counts were reduced in IgAV. The elevated B cell numbers returned to normal after treatment. Plasma and plasmablast B cell numbers correlated with plasma IgA levels. On the contrary, CD3-CD19+IgD-CD27+IgM+B cell numbers were negatively proportional to the plasma IgA levels while naïve B cell numbers correlated with plasma and plasmablast B cell counts.

Conclusions

We hypothesized that immunoglobulin production was abnormally elevated in IgAV and could be explained by altered B-cell subset homeostasis.
Path sampling simulations reveal how the Q61L mutation restricts the dynamics of KRas
Authors: Roet Sander , Hooft Ferry , Bolhuis Peter G. , Swenson David W.H. , Vreede Jocelyne
Published: February 2020

The GTPase KRas is a signaling protein in networks for cell differentiation, growth, and division. KRas mutations can prolong activation of these networks, resulting in tumor formation. When active, KRas tightly binds GTP. Several oncogenic mutations affect the conversion between this rigid state and inactive, more flexible states. Detailed understanding of these transitions may provide valuable insights into how mutations affect KRas. Path sampling simulations, which focus on transitions, show KRas visiting several states, which are the same for wild type and the oncogenic mutant Q61L. Large differences occur when converting between these states, indicating the dramatic effect of the Q61L mutation on KRas dynamics. For Q61L a route to the flexible state is inaccessible, thus shifting the equilibrium to more rigid states. Our methodology presents a novel way to predict dynamical effects of KRas mutations, which may aid in identifying potential therapeutic targets.

Author summary

Cancer cells frequently contain mutations in the protein KRas. However, KRas is a challenging target for anti-cancer drugs, in part because the dynamic behavior of flexible regions in the protein is difficult to characterize experimentally, and occurs on timescales that are too long for straightforward molecular dynamics simulations. We have used path sampling, an advanced simulation technique that overcomes long timescales, to obtain atomistic insight into the dynamics of KRas. Comparing the oncogenic mutant Q61L to the wild type revealed that the mutation closes off one transition channel for deactivating KRas. Our approach opens up the way for predicting the dynamical effects of mutations in KRas, which may aid in identifying potential therapeutic targets.
STIG: Generation and simulated sequencing of synthetic T cell receptor repertoires
Authors: Woodcock Mark G. , Bortone Dante S. , Vincent Benjamin G.
Published: February 2020

T cell receptor repertoire inference from DNA and RNA sequencing experiments is frequently performed to characterize host immune responses to disease states. Existing tools for repertoire inference have been compared across publicly available biological datasets or unpublished simulated sequencing data. Evaluation and comparison of these tools is challenging without common data sets created from a known repertoire with well-defined biological and sequencing characteristics. Here we introduce STIG, a tool to create simulated T cell receptor sequencing data from a customizable virtual T cell repertoire, with clear attribution of individual reads back to locations within their respective T-cell receptor clonotypes. STIG allows for robust performance evaluation of T cell repertoire inference and downstream analysis methods. STIG is implemented in Python 3 and is freely available for download athttps://github.com/Benjamin-Vincent-Lab/stig

Author summary

As part of the acquired immune system, T cells are integral in the host response to microbes, tumors and autoimmune disease. These cells each have a semi-unique T cell receptor that serves to bind a set of antigens that will in turn stimulate that cell to perform its particular pro- (or anti) inflammatory role. This receptor is the product of DNA rearrangement of germline gene segments, similar to B cell receptor loci rearrangement, which provides a wide variety of potential T cell receptors to respond to antigens. At the site of an immune reaction, T cells can increase their number through clonal expansion and methods have been developed to analyze bulk genetic sequencing data to infer the individual receptors and the relative size of their clonal subpopulations present within a sample. To date, these methods and tools have been tested and compared using either biological samples (where the true quantitiy and types of T cells is unknown) or unshared synthetic datasets. In this paper I describe a new tool to generate biologically-inspired T-cell repertoires in-silico and generate simulated sequencing data from them.
SNCAE46KtransgenicDrosophilaModel of Parkinson’s Disease Confirmed the Causative Role of Oxidative Stress
Authors: Jahromi Samaneh Reiszadeh , Ramesh S R , Finkelstein David I , Haddadi Mohammad
Published: February 2020

Parkinson’s disease (PD) is a class of neurodegenerative disorders in which, complex interactions of genetic and environmental agents are involved in the etiology of both sporadic and familial PD cases. α-synuclein-encodingSNCAgene is known as one of the major genetic contributors of this disease.E46Kmutation inSNCAgene has not been investigated as intensive as otherSNCAgene mutations including A30P and A53T. In this study, to induce PD inDrosophilaflies,UAS-hSNCAWTandUAS-hSNCAE46Ktransgenic fly lines were constructed, whereSNCAgene was over-expressed in flies brains using GAL4-UAS genetic system. Western blot analysis of head samples ofSNCA-expressing flies verifiedSNCAexpression at protein level. Light and electron microscopy analysis of ommatidial structures were performed to verify neurodegeneration as a result of α-synuclein gene overexpression inDrosophilatransgenic flies. Confocal microscopy analysis of dopaminergic neuron clusters verified cell loss followingSNCAE46Kexpression in the flies’ brain.E46Kα-synuclein gene over-expression resulted in an evident decline in longevity as well as climbing ability of the flies. Biochemical studies of transgenic flies showed a remarkable decline in antioxidant enzymes activity and a significant increase in oxidative markers level as well as AchE enzyme activity. Oxidative stress has been known as a causal factor in PD pathogenesis, following expression ofE46Kmutant version of humanSNCAgene. ThisDrosophilamodel is able to facilitate comparative studies of both molecular and cellular assays implicated in the assessment of neurotoxicity of different α-synuclein mutations.
Improved reference genome of the arboviral vectorAedes albopictus
Authors: Palatini U. , Masri R.A. , Cosme L.V. , Koren S. , Thibaud-Nissen F. , Biedler J.K. , Krsticevic F. , Johnston J.S. , Halbach R. , Crawford J.E. , Antoshechkin I. , Failloux A. , Pischedda E. , Marconcini M. , Ghurye J. , Rhie A. , Sharma A. , Karagodin D.A. , Jenrette J. , Gamez S. , Miesen P. , Caccone A. , Sharakhova M.V. , Tu Z. , Papathanos P.A. , Van Rij R.P. , Akbari O. S. , Powell J. , Phillippy A. M. , M. Bonizzoni
Published: February 2020

The Asian tiger mosquitoAedes albopictusis globally expanding and has become the main vector for human arboviruses in Europe. Here we present AalbF2, a dramatically improved assembly of theAe. albopictusgenome that has revealed widespread viral insertions, novel microRNAs and piRNA clusters, the sex determining locus, new immunity genes, and has enabled genome-wide studies of geographically diverseAe. albopictuspopulations and analyses of the developmental and stage-dependent network of expression data. Additionally, we built the first physical map for this species with 75% of the assembled genome anchored to the chromosomes. These up-to-date resources of the genome provide a foundation to improve understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures.

One Sentence Summary

Long-read and Hi-C-basedde novoassembly of the arboviral vectorAedes albopictusgenome fosters deeper understanding of its biological features.
Coordination of asparagine uptake and asparagine synthetase expression is required for T cell activation
Authors: Hope Helen Carrasco , Brownlie Rebecca J. , Steele Lynette , Salmond Robert J.
Published: March 2020

T cell receptor triggering by antigen results in metabolic reprogramming that, in turn, facilitates T cells’ exit from quiescence. The increased nutrient requirements of activated lymphocytes are met in part by upregulation of cell surface transporters and enhanced uptake of amino acids, fatty acids and glucose from the environment. However, the role of intracellular pathways of amino acid biosynthesis in T cell activation is relatively unexplored. Asparagine (Asn) is a non-essential amino acid that can be synthesized intracellularly through the glutamine-hydrolyzing enzyme asparagine synthetase (ASNS). We set out to define the requirements for uptake of extracellular Asn and ASNS activity in CD8+T cell activation. At early timepoints of activation, T cells expressed little or no ASNS and, as a consequence, viability and TCR-stimulated growth, activation and metabolic reprogramming were substantially impaired under conditions of Asn deprivation. At later timepoints (>48h of activation), TCR-induced mTOR-dependent signals resulted in upregulation of ASNS, that endowed T cells with the capacity to function independently of extracellular Asn. Thus, we have determined that the coordinated upregulation of ASNS expression and uptake of extracellular Asn is required for optimal T cell effector responses.
Manipulation of a Cation-π Sandwich Reveals Conformational Flexibility in Phenylalanine Hydroxylase
Authors: Arturo Emilia C. , Merkel George W. , Hansen Michael R. , Lisowski Sophia , Almeida Deeanne , Gupta Kushol , Jaffe Eileen K.
Published: February 2020

Phenylalanine hydroxylase (PAH) is an allosteric enzyme responsible for maintaining phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria. Wild type (WT) PAH equilibrates among resting-state (RS-PAH) and activated (A-PAH) conformations, whose equilibrium position depends upon allosteric Phe binding to the A-PAH conformation. The RS-PAH conformation of WT rat PAH (rPAH) contains a cation-π sandwich between Phe80, Arg123, and Arg420, which cannot exist in the A-PAH conformation. Phe80 variants F80A, F80D, F80L, and F80R were prepared; their conformational equilibrium was evaluated using native PAGE, size exclusion chromatography, ion exchange behavior, intrinsic protein fluorescence, enzyme kinetics, and limited proteolysis, each as a function of [Phe]. Like WT rPAH, F80A and F80D show allosteric activation by Phe while F80L and F80R are constitutively active. Maximal activity of all variants suggests relief of a rate-determining conformational change involving Phe80. Limited proteolysis of WT rPAH in the absence of Phe reveals facile cleavage within a C-terminal 4-helix bundle that is buried in the RS-PAH tetramer interface, reflecting dynamic dissociation of the RS-PAH conformation. This cleavage is not seen for the Phe80 variants, which all show proteolytic hypersensitivity in a linker that repositions during the RS-PAH to A-PAH conformational interchange. Hypersensitivity is corrected by addition of Phe such that all Phe80 variants become like WT rPAH and achieve the A-PAH conformation. Thus, manipulation of Phe80 perturbs the conformational space sampled by PAH, increasing the propensity to sample intermediates in the RS-PAH and A-PAH interchange, which are presumed on-pathway because they can readily achieve the A-PAH conformation by addition of Phe.
Transcriptome profiling of the branchial arches reveals cell type composition and a conserved signature of neural crest cell invasion
Authors: Morrison Jason A , McLennan Rebecca , Teddy Jessica M , Scott Allison R , Kasemeier-Kulesa Jennifer C , Gogol Madelaine M , Kulesa Paul M
Published: February 2020

The vertebrate branchial arches that give rise to structures of the head, neck, and heart form with very dynamic tissue growth and well-choreographed neural crest, ectoderm, and mesoderm cell dynamics. Although this morphogenesis has been studied by marker expression and fate-mapping, the mechanisms that control the collective migration and diversity of the neural crest and surrounding tissues remain unclear, in part due to the effects of averaging and need for cell isolation in conventional transcriptome analysis experiments of multiple cell populations. We used label free single cell RNA sequencing on 95,000 individual cells at 2 developmental stages encompassing formation of the first four chick branchial arches to measure the transcriptional states that define the cellular hierarchy and invasion signature of the migrating neural crest. The results confirmed basic features of cell type diversity and led to the discovery of many novel markers that discriminate between axial level and distal-to-proximal cell populations within the branchial arches and neural crest streams. We identified the transcriptional signature of the most invasive neural crest that is conserved within each branchial arch stream and elucidated a set of genes common to other cell invasion signatures in types in cancer, wound healing and development. These data robustly delineate molecularly distinct cell types within the branchial arches and identify important molecular transitions within the migrating neural crest during development.
In-cell architecture of an actively transcribing-translating expressome
Authors: O’Reilly Francis J. , Xue Liang , Graziadei Andrea , Sinn Ludwig , Lenz Swantje , Tegunov Dimitry , Blötz Cedric , Hagen Wim J. H. , Cramer Patrick , Stülke Jörg , Mahamid Julia , Rappsilber Juri
Published: February 2020

Structural biology performed inside cells can capture molecular machines in action within their native context. Here we develop an integrative in-cell structural approach using the genome-reduced human pathogenMycoplasma pneumoniae. We combine whole-cell crosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpoint NusA at the interface between a NusG-bound elongating RNAP and the ribosome, and propose it could mediate transcription-translation coupling. Translation inhibition dissociates the expressome, whereas transcription inhibition stalls and rearranges it, demonstrating that the active expressome architecture requires both translation and transcription elongation within the cell.
GOT1 Inhibition Primes Pancreatic Cancer for Ferroptosis through the Autophagic Release of Labile Iron
Authors: Kremer Daniel M. , Nelson Barbara S. , Lin Lin , Yarosz Emily L. , Halbrook Christopher J. , Kerk Samuel A. , Sajjakulnukit Peter , Myers Amy , Thurston Galloway , Hou Sean W. , Carpenter Eileen S. , Andren Anthony C. , Nwosu Zeribe C. , Cusmano Nicholas , Wisner Stephanie , Ramos Johanna , Gao Tina , Sastra Stephen A. , Palermo Carmine F. , Badgley Michael A. , Zhang Li , Asara John M. , di Magliano Marina Pasca , Shah Yatrik M. , Crawford Howard C. , Olive Kenneth P. , Lyssiotis Costas A.
Published: March 2020

Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest solid malignancies, with a 5-year survival rate at ten percent. PDA have unique metabolic adaptations in response to cell-intrinsic and environmental stressors, and identifying new strategies to target these adaptions is an area of active research. We previously described a dependency on a cytosolic aspartate aminotransaminase (GOT1)-dependent pathway for NADPH generation. Here, we sought to identify metabolic dependencies induced by GOT1 inhibition that could be exploited to selectively kill PDA. Using pharmacological methods, we identified cysteine, glutathione, and lipid antioxidant function as metabolic vulnerabilities following GOT1 withdrawal. Targeting any of these pathways was synthetic lethal in GOT1 knockdown cells and triggered ferroptosis, an oxidative, non-apoptotic, iron-dependent form of cell death. Mechanistically, GOT1 inhibition promoted the activation of autophagy in response to metabolic stress. This enhanced the availability of labile iron through ferritinophagy, the autolysosome-mediated degradation of ferritin. In sum, our study identifies a novel biochemical connection between GOT1, iron regulation, and ferroptosis, and suggests the rewired malate-aspartate shuttle plays a role in protecting PDA from severe oxidative challenge.

Highlights

PDA exhibit varying dependence on GOT1 forin vitroandin vivogrowth.

Exogenous cystine, glutathione synthesis, and lipid antioxidant fidelity are essential under GOT1 suppression.

GOT1 inhibition sensitizes pancreatic cancer cell lines to ferroptosis.

GOT1 inhibition represses anabolic metabolism and promotes the release of iron through autophagy.
MiR-101a loaded Extracellular Nanovesicles as Bioactive Carriers for Cardiac Repair
Authors: Wang Jinli , Lee Christine J. , Deci Michael B. , Jasiewicz Natalie , Canty John M , Nguyen J.
Published: February 2020

Myocardial infarction (MI) remains a major cause of mortality worldwide. Despite significant advances in MI treatment, many who survive the acute event are at high risk of chronic cardiac morbidity. Here we developed a cell-free therapeutic that capitalizes on the antifibrotic effects of micro(mi)RNA-101a and exploits the multi-faceted regenerative activity of mesenchymal stem cell (MSC) extracellular nanovesicles (eNVs). While the majority of MSC eNVs require local delivery via intramyocardial injection to exert therapeutic efficacy, we have developed MSC eNVs that can be administered in a minimally invasive manner, all while remaining therapeutically active. When loaded with miR-101a, MSC eNVs substantially decreased infarct size (12 ± 2.4% vs. 21.4 ± 5.7%) and increased ejection fraction (53.6 ± 7.6% vs. 40.3 ± 6.0%) and fractional shortening (23.6 ± 4.3% vs. 16.6 ± 3.0%) compared to control. These findings are significant as they represent an advance in the development of minimally invasive cardio-therapies.
Changes in perilesional V1 underlie training-induced recovery in cortically-blind patients
Authors: Barbot Antoine , Das Anasuya , Melnick Michael D. , Cavanaugh Matthew R. , Merriam Elisha P. , Heeger David J. , Huxlin Krystel R.
Published: February 2020

Damage to the primary visual cortex (V1) causes profound, homonymous visual-field loss termed cortical blindness (CB). Though long considered intractable, multiple studies now show that perceptual training can recover visual functions in chronic CB. A popular hypothesis is that training recruits intact extrageniculostriate pathways. Alternatively, training may induce plastic changes within spared regions of the damaged V1. Here, we linked changes in luminance detection sensitivity with retinotopic fMRI activity in eleven chronic CB patients, before and after extensive visual discrimination training. Our results show that the strength of spared V1 activity representing perimetrically blind-field locations before training predicts the amount of training-induced recovery of luminance detection sensitivity. Additionally, training caused an enlargement of population receptive fields in perilesional V1 cortex, which increased blind-field coverage. These findings uncover fundamental changes in perilesional V1 cortex underlying training-induced restoration of conscious luminance detection sensitivity in cortically-blind patients.
Picomolar affinity antagonist and sustained signaling agonist peptide ligands for the adrenomedullin and calcitonin gene-related peptide receptors
Authors: Booe Jason M. , Warner Margaret L. , Pioszak Augen A.
Published: February 2020

The calcitonin receptor-like G protein-coupled receptor (CLR) mediates adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) actions including vasodilation, cardioprotection, and nociception. Receptor activity-modifying proteins (RAMP1-3) determine CLR ligand selectivity through an unresolved mechanism. CLR-RAMP complexes are drug targets, but short AM and CGRP plasma half-lives limit their therapeutic utility. We used combinatorial peptide library and rational design approaches to probe selectivity determinants and develop short AM and CGRP variants with ∼1000-fold increased receptor extracellular domain affinities. Binding and structural studies explained the increased affinities and defined roles for AM Lys46 and RAMP modulation of CLR conformation in selectivity. In longer scaffolds that also bind the CLR transmembrane domain the variants generated picomolar affinity antagonists, one with an estimated 12.5 hr CGRP receptor residence time, andsustainedsignaling agonists ss-AM and ss-CGRP. This work clarifies the RAMP-modulated ligand selectivity mechanism and provides AM and CGRP variants with promise as long-acting therapeutics.
Cumulus cell acetyl-CoA metabolism from acetate is associated with maternal age but inconclusively with oocyte maturity
Authors: Anderson Sharon , Xu Peining , Frey Alexander J. , Goodspeed Jason R. , Doan Mary T. , Orris John J. , Clements Nicolle , Glassner Michael J. , Snyder Nathaniel W.
Published: February 2020

Cumulus cell (CC) clumps that associate with oocytes provide the oocytes with growth and signaling factors. Thus, the metabolism of the CCs may influence oocyte function and CC metabolism may be predictive of oocyte competence for in vitro fertilization. CCs are thought to be highly glycolytic but data on other potential carbon substrates are lacking in humans. This was a prospective and blinded cohort study that was designed to examine the substrate utilization of CCs by age and oocyte competence. Individual sets of CC clumps from participants were removed after oocyte retrieval procedure, incubated with stable isotope labeled substrates, and analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for isotopologue enrichment of major metabolic intermediates, including acetyl-CoA. The acyl-chain of acetyl-CoA contains 2 carbons that can be derived from13C-labeled substrates resulting in a M+2 isotopologue that contains 213C atoms. Comparing the fate of three major carbon sources, mean enrichment of M+2 acetyl-CoA (mean, standard deviation) was for glucose (3.6, 7.7), for glutamine (9.4, 6.2), and for acetate (20.7, 13.9). Due to this unexpected high and variable labeling from acetate, we then examined acetyl-CoA mean % enrichment from acetate of in 278 CCs from 21 women ≤34 (49.06, 12.73) decreased with age compared to 124 CCs from 10 women >34 (43.48, 16.20) (p=0.0004, t test). The CCs associated with the immature prophase I oocytes had significantly lower enrichment in M+2 acetyl CoA compared to the CCs associated with the metaphase I and metaphase II oocytes (difference: −6.02, CI: −1.74,-13.79, p=0.013). Acetate metabolism in individual CC clumps was positively correlated with oocyte maturity and decreased with maternal age. These findings indicate that CC metabolism of non-glucose substrates should be investigated relative to oocyte function and age-related fertility.
Tissue-resident CD4+T helper cells assist protective respiratory mucosal B and CD8+T cell memory responses
Authors: Son Young Min , Cheon In Su , Wu Yue , Li Chaofan , Wang Zheng , Chen Yao , Takahashi Yoshimasa , Dent Alexander L. , Kaplan Mark H , Fu Yang-Xin , Taylor Justin J. , Cui Weiguo , Sun Jie
Published: February 2020

The roles of CD4+T helper cells (TH) in shaping localized memory B and CD8+T cell immunity in the mucosal tissues are largely unexplored. Here, we report that lung THcells provide local assistance for the optimal development of tissue-resident memory B (BRM) and CD8+T (TRM) cells following the resolution of primary influenza virus infection. We identify a population of tissue-resident CD4+TH(akaTRH) cells that co-exhibit follicular T helper (TFH) and TRMcell features and mediate local help of CD4+T cells to B and CD8+T cells. Optimal TRHcell formation requires lung B cells and transcription factors involved in TFHor TRMdevelopment. Further, we show that TRHcells deliver local help to B and CD8 T cells through CD40L and IL-21-dependent mechanisms. Our data have uncovered a new tissue-resident THcell population that is specialized in assisting the development of mucosal protective B and CD8+T cell responsesin situ.
Size-dependent patterns of cell proliferation and migration in freely-expanding epithelia
Authors: Heinrich Matthew A. , Alert Ricard , LaChance Julienne M. , Zajdel Tom J. , Košmrlj Andrej , Cohen Daniel J.
Published: February 2020

The coordination of cell proliferation and migration in growing tissues is crucial in development and regeneration but remains poorly understood. Here, we find that, while expanding with an edge speed independent of initial conditions, millimeter-scale epithelial monolayers exhibit internal patterns of proliferation and migration that depend not on the current but on the initial tissue size, indicating memory effects. Specifically, the core of large tissues becomes very dense, almost quiescent, and ceases cell-cycle progression. In contrast, initially-smaller tissues develop a local minimum of cell density and a tissue-spanning vortex. To explain vortex formation, we propose an active polar fluid model with a feedback between cell polarization and tissue flow. Taken together, our findings suggest that expanding epithelia decouple their internal and edge regions, which enables robust expansion dynamics despite the presence of size- and history-dependent patterns in the tissue interior.
Three-dimensional reconstruction of individual helical nano-filament structures from atomic force microscopy topographs
Authors: Lutter Liisa , Serpell Christopher , Tuite Mick , Serpell Louise , Xue Wei-Feng
Published: February 2020

Atomic force microscopy, AFM, is a powerful tool that can produce detailed topographical images of individual nano-structures with a high signal-to-noise ratio without the need for ensemble averaging. However, the application of AFM in structural biology has been hampered by the tip-sample convolution effect, which distorts images of nano-structures, particularly those that are of similar dimensions to the cantilever probe tips used in AFM. Here we show that the tip-sample convolution results in a feature-dependent and non-uniform distribution of image resolution on AFM topographs. We show how this effect can be utilised in structural studies of nano-sized upward convex objects such as spherical or filamentous molecular assemblies deposited on a flat surface, because it causes ‘magnification’ of such objects in AFM topographs. Subsequently, this enhancement effect is harnessed through contact-point based deconvolution of AFM topographs. Here, the application of this approach is demonstrated through the 3D reconstruction of the surface envelope of individual helical amyloid filaments without the need of cross-particle averaging using the contact-deconvoluted AFM topographs. Resolving the structural variations of individual macromolecular assemblies within inherently heterogeneous populations is paramount for mechanistic understanding of many biological phenomena such as amyloid toxicity and prion strains. The approach presented here will also facilitate the use of AFM for high-resolution structural studies and integrative structural biology analysis of single molecular assemblies.
Propylene glycol-based antifreeze as an effective preservative for DNA metabarcoding of benthic arthropods
Authors: Robinson Chloe V. , Porter Teresita M. , Wright Michael T.G. , Hajibabaei Mehrdad
Published: February 2020

Maintaining the integrity of DNA in bulk environmental samples from source to laboratory is crucial for capturing the true range of taxa present within an ecosystem. Preservation consideration of DNA is particularly important if samples are being collected in remote areas and by non-specialist ‘citizen scientists’ in nationwide programs. Traditionally, absolute ethanol is used as the preferred preservative for environmental samples collected for downstream DNA metabarcoding analyses. However, transport, shipping and DNA extraction of samples preserved in ethanol is a lengthy procedure due to safety restrictions and the requirement of full ethanol evaporation prior to extraction. We examined the efficacy of an easily accessible, non-toxic propylene glycol-based antifreeze as an alternative to absolute ethanol for preserving macroinvertebrate DNA from bulk-benthos DNA samples. We tested the differences in both cytochrome oxidase I (COI) exact sequence variants (ESVs) and COI taxonomic orders detected in both ethanol and antifreeze samples using two processing methods (no evaporation of preservative versus full evaporation). In addition, we assessed the detection of families and genera within the Arthropoda phylum for preservative type, site and processing method. Our results suggest that antifreeze is a suitable alternative to ethanol, a greater global ESV richness reported for antifreeze samples. Additionally, a higher proportion of arthropod reads in ESVs were detected in antifreeze (average 69%) compared with ethanol (average 53%). Finally, antifreeze samples produced similar results for the different processing methods, whereas ethanol samples failed to produce similar results without prior evaporation. Although ethanol is currently widely used for DNA preservation, our results demonstrate that by using antifreeze, it is possible to achieve similar taxonomic coverage and community assemblages of bulk-benthos DNA samples for macroinvertebrates, with the added simplicity and shorter laboratory processing time achieved using an easily available, unregulated preservative.