bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrative inference of transcriptional networks in Arabidopsis yields novel ROS signalling regulators
Authors: De Clercq Inge , Van de Velde Jan , Luo Xiaopeng , Liu Li , Storme Veronique , Van Bel Michiel , Pottie Robin , Vaneechoutte Dries , Breusegem Frank Van , Vandepoele Klaas
Published: August 2020

Gene regulation is a dynamic process in which transcription factors (TFs) play an important role to control spatiotemporal gene expression. While gene regulatory networks describe the interactions between TFs and their target genes, our global knowledge about the complexity of TF control for different genes and biological processes is incomplete. To enhance our global understanding of regulatory interactions inArabidopsis thaliana, different regulatory input networks capturing complementary information about DNA motifs, open chromatin, TF binding and expression-based regulatory interactions, were combined using a supervised learning approach, resulting in an integrated gene regulatory network (iGRN) covering 1,491 TFs and 31,393 target genes (1.7 million interactions). This iGRN outperforms the different input networks to predict known regulatory interactions and has a similar performance to recover functional interactions compared to state-of-the-art experimental methods like yeast one-hybrid and ChIP-seq. The iGRN correctly inferred known functions for 681 TFs and predicted new gene functions for hundreds of unknown TFs. For regulators predicted to be involved in reactive oxygen species stress regulation, we confirmed in total 75% of TFs with a function in ROS and/or physiological stress responses. This includes 13 novel ROS regulators, previously not connected to any ROS or stress function, that were experimentally validated in our ROS-specific phenotypic assays of loss- or gain-of-function lines. In conclusion, the presented iGRN offers a high-quality starting point to enhance our understanding of gene regulation in plants by integrating different experimental data types at the network level.
Highly-parallel microfluidics-based force spectroscopy on single cytoskeletal motors
Authors: Urbanska Marta , Lüdecke Annemarie , Walter Wim J. , van Oijen Antoine M. , Duderstadt Karl E. , Diez Stefan
Published: August 2020

Cytoskeletal motors transform chemical energy into mechanical work to drive essential cellular functions. Optical trapping experiments have provided crucial insights into the operation of these molecular machines under load. However, the throughput of such force spectroscopy experiments is typically limited to one measurement at a time. Here, we introduce a highly-parallel, microfluidics-based method that allows for rapid collection of force-dependent motility parameters of cytoskeletal motors with two orders of magnitude improvement in throughput compared to currently available methods. We apply tunable hydrodynamic forces to stepping kinesin-1 motors via DNA-tethered beads and utilize a large field of view to simultaneously track the velocities, run lengths and interaction times of hundreds of individual kinesin-1 molecules under varying resisting and assisting loads. Importantly, the 16-μm long DNA tethers between the motors and the beads significantly reduces the vertical component of the applied force pulling the motors away from the microtubule. Our approach is readily applicable to other molecular systems and constitutes a new methodology for parallelized single-molecule force studies on cytoskeletal motors.
A flexible Bayesian framework for unbiased estimation of timescales
Authors: Zeraati Roxana , Engel Tatiana A. , Levina Anna
Published: August 2020

Timescales characterize the pace of change for many dynamic processes in nature. Timescales are usually estimated by fitting the exponential decay of data autocorrelation in the time or frequency domain. We show that this standard procedure often fails to recover the correct timescales due to a statistical bias arising from the finite sample size. We develop an alternative approach to estimating timescales by fitting the sample autocorrelation or power spectrum with a generative model based on a mixture of Ornstein-Uhlenbeck processes using adaptive Approximate Bayesian Computations. Our method accounts for finite sample size and noise in data and returns a posterior distribution of timescales that quantifies the estimation uncertainty and can be used for model selection. We demonstrate the accuracy of our method on synthetic data and illustrate its application to recordings from the primate cortex. We provide a customizable Python package implementing our framework with different generative models suitable for diverse applications.
Humans can visually judge grasp quality and refine their judgments through visual and haptic feedback
Authors: Maiello Guido , Schepko Marcel , Klein Lina K. , Paulun Vivian C. , Fleming Roland W.
Published: August 2020

How humans visually select where to grasp objects is determined by the physical object properties (e.g., size, shape, weight), the degrees of freedom of the arm and hand, as well as the task to be performed. We recently demonstrated that human grasps are near-optimal with respect to a weighted combination of different cost functions that make grasps uncomfortable, unstable or impossible e.g., due to unnatural grasp apertures or large torques. Here, we ask whether humans can consciously access these rules. We test if humans can explicitly judge grasp quality derived from rules regarding grasp size, orientation, torque, and visibility. More specifically, we test if grasp quality can be inferred (i) by using motor imagery alone, (ii) from watching grasps executed by others, and (iii) through performing grasps, i.e. receiving visual, proprioceptive and haptic feedback. Stimuli were novel objects made of 10 cubes of brass and wood (side length 2.5 cm) in various configurations. On each object, one near-optimal and one sub-optimal grasp were selected based on one cost function (e.g. torque), while the other constraints (grasp size, orientation, and visibility) were kept approximately constant or counterbalanced. Participants were visually cued to the location of the selected grasps on each object and verbally reported which of the two grasps was best. Across three experiments, participants could either (i) passively view the static objects, (ii) passively view videos of other participants grasping the objects, or (iii) actively grasp the objects themselves. Our results show that participants could already judge grasp optimality from simply viewing the objects, but were significantly better in the video and grasping session. These findings suggest that humans can determine grasp quality even without performing the grasp—perhaps through motor imagery—and can further refine their understanding of how to correctly grasp an object through sensorimotor feedback but also by passively viewing others grasp objects.
The monologue of the double: allocentric reduplication of the own voice alters bodily self perception
Authors: Lesur Marte Roel , Bolt Elena , Lenggenhager Bigna
Published: August 2020

During autoscopic phenomena, people perceive a double of themselves in extrapersonal space. Such clinical allocentric self-experiences often co-occur with auditory hallucinations, yet experimental setups to induce similar illusions in healthy participants have been limited to visual doubles. We investigated whether feeling the presence of an auditory double could be provoked in healthy participants and how it might affect spatial aspects of bodily self-consciousness. We recorded the own versus another person’s voice while walking around in the room using binaural headphones from an egocentric and an allocentric perspective. In comparison to listening to their own moving voice egocentrically, when listening to themselves allocentrically, participants reported a strong feeling of a presence with a similarly high degree of self-identification, suggesting a successful induction of the feeling of an acoustic doppelganger. When pointing to the source of the own voice participants localized it closer to themselves than when listening to another person’s voice, suggesting a change in spatial perception. Interestingly, the opposite pattern was found in participants that had previous hallucinatory experiences. These findings show that listening to one’s own voice allocentrically can manipulate bodily self-consciousness and self-related spatial perception. This paradigm enables the experimental study of the relationship between auditory vocal hallucinations and bodily self-consciousness, bridging important clinical phenomena and experimental knowledge.
Starburst amacrine cells amplify optogenetic visual restoration through gap junctions
Authors: Katada Yusaku , Kunimi Hiromitsu , Serizawa Naho , Lee Deokho , Kobayashi Kenta , Negishi Kazuno , Okano Hideyuki , Tanaka Kenji F. , Tsubota Kazuo , Kurihara Toshihide
Published: August 2020

Ectopic induction of optogenetic actuators, such as channelrhodopsin, is a promising approach to restoring vision in the degenerating retina. However, the cell type-specific response of ectopic photoreception has not been well understood. It is limited to obtain efficient gene expression in a specifically targeted cell population by a transgenic approach. In the present study, we established a murine model with high efficiency of gene induction to retinal ganglion cells (RGCs)- and amacrine cells using an improved tetracycline transactivator-operator bipartite system (KENGE-tet system). To investigate the cell type-specific visual restorative effect, we expressed the channelrhodopsin gene into RGCs and amacrine cells using the KENGE-tet system. As a result, enhancement in the visual restorative effect was observed to RGCs and starburst amacrine cells. In conclusion, a photoresponse from amacrine cells may enhance the maintained response of RGCs and further increase/improve the visual restorative effect.
Four newScopalinafrom Southern California: the first Scopalinida (Porifera: Demospongiae) from the temperate Eastern Pacific
Authors: Turner Thomas L.
Published: August 2020

Sponges (phylum Porifera) are common inhabitants of kelp forest ecosystems in California, but their diversity and ecological importance are poorly characterized in this biome. Here I use freshly collected samples to describe the diversity of the order Scopalinida in California. Though previously unknown in the region, four new species are described here:Scopalina nausicaesp. nov.,S. kuyamusp. nov.,S. goletensissp. nov., andS. jalisp. nov.. These discoveries illustrate the considerable uncharacterized sponge diversity remaining in California kelp forests, and the utility of SCUBA-based collection to improve our understanding of this diversity.
Genomically Complex Human Angiosarcoma and Canine Hemangiosarcoma Establish Convergent Angiogenic Transcriptional Programs Driven by Novel Gene Fusions
Authors: Kim Jong Hyuk , Megquier Kate , Thomas Rachael , Sarver Aaron L. , Song Jung Min , Kim Yoon Tae , Cheng Nuojin , Schulte Ashley J. , Linden Michael A. , Murugan Paari , Oseth LeAnn , Forster Colleen L. , Elvers Ingegerd , Swofford Ross , Turner-Maier Jason , Karlsson Elinor K. , Breen Matthew , Lindblad-Toh Kerstin , Modiano Jaime F.
Published: August 2020

Sporadic angiosarcomas (ASs) are aggressive vascular sarcomas whose rarity and genomic complexity present significant obstacles in deciphering the pathogenic significance of individual genetic alterations. Numerous fusion genes have been identified across multiple types of cancers, but their existence and significance remain unclear in sporadic ASs. In this study, we leveraged RNA sequencing data from thirteen human ASs and 76 spontaneous canine hemangiosarcomas (HSAs) to identify fusion genes associated with spontaneous vascular malignancies. Ten novel protein-coding fusion genes, includingTEX2-PECAM1andATP8A2-FLT1,were identified in seven of the thirteen human tumors, with two tumors showing mutations ofTP53. HRASandNRASmutations were found in ASs without fusions orTP53mutations. We found fifteen novel protein-coding fusion genes includingMYO16-PTK2, GABRA3-FLT1,andAKT3-XPNPEP1in eleven of the 76 canine HSAs; these fusion genes were seen exclusively in tumors of the angiogenic molecular subtype that contained recurrent mutations inTP53, PIK3CA, PIK3R1,andNRAS.In particular, fusion genes and mutations ofTP53co-occurred in tumors with higher frequency than expected by random chance, and they enriched gene signatures predicting activation of angiogenic pathways. Comparative transcriptomic analysis of human ASs and canine HSAs identified shared molecular signatures associated with activation of PI3K/AKT/mTOR pathways. Our data show that, while driver events of malignant vasoformative tumors of humans and dogs include diverse mutations and stochastic rearrangements that create novel fusion genes, convergent transcriptional programs govern the highly conserved morphological organization and biological behavior of these tumors in both species.
Efficacy of Targeting SARS-CoV-2 by CAR-NK Cells
Authors: Ma Minh , Badeti Saiaditya , Geng Ke , Liu Dongfang
Published: August 2020

SARS-CoV-2, which causes COVID-19 disease, is one of greatest global pandemics in history. No effective treatment is currently available for severe COVID-19 disease. One strategy for implementing cell-based immunity involves the use of chimeric antigen receptor (CAR) technology. Unlike CAR T cells, which need to be developed using primary T cells derived from COVID-19 patients with lymphopenia, clinical success of CAR NK cell immunotherapy is possible through the development of allogeneic, universal, and ‘off-the-shelf’ CAR-NK cells from a third party, which will significantly broaden the application and reduce costs. Here, we develop a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2. CAR-NK cells were generated using the scFv domain of CR3022 (henceforth, CR3022-CAR-NK), a broadly neutralizing antibody for SARS-CoV-1 and SARS-CoV-2. CR3022-CAR-NK cells can specifically bind to RBD of SARS-CoV-2 and pseudotyped SARS-CoV-2 S protein, and can be activated by pseudotyped SARS-CoV-2-S viral particlesin vitro.Further, CR3022-CAR-NK cells can specifically kill pseudo-SARS-CoV-2 infected target cells. Thus, ‘off-the-shelf’ CR3022-CAR-NK cells may have the potential to treat patients with severe COVID-19 disease.
Assessment of ethanol-induced toxicity on iPSC-derived human dopaminergic neurons using a novel high-throughput mitochondrial neuronal health (MNH) assay
Authors: Zink Annika , Conrad Josefin , Telugu Narasimha Swami , Diecke Sebastian , Heinz Andreas , Wanker Erich , Priller Josef , Prigione Alessandro
Published: August 2020

Excessive ethanol exposure can cause mitochondrial and cellular toxicity. In order to discover potential counteracting interventions, it is essential to develop assays capable of capturing the consequences of ethanol exposure in human dopaminergic (DA) neurons, which are crucial for the development and maintenance of alcohol use disorders (AUD). Here, we developed a novel high-throughput (HT) assay to quantify mitochondrial and neuronal toxicity in human DA neurons from induced pluripotent stem cells (iPSCs). The assay, dubbedmitochondrialneuronalhealth (MNH) assay, combines live-cell measurement of mitochondrial membrane potential (MMP) with quantification of neuronal branching complexity post-fixation. Using the MNH assay, we demonstrated that chronic ethanol exposure in human iPSC-derived DA neurons decreases MMP and branching complexity in a dose-dependent manner. The toxic effect of ethanol on DA neurons was already detectable after 1 hour of exposure, and occurred similarly in DA neurons derived from healthy individuals and from patients with AUD. We next used the MNH assay to carry out a proof-of-concept compound screening using FDA-approved drugs. We identified potential candidate drugs modulating acute ethanol toxicity in human DA neurons. Among these drugs, flavoxate and disulfiram influenced mitochondrial neuronal health independently from ethanol, leading to amelioration and worsening, respectively. Altogether, we developed an HT assay to probe human mitochondrial neuronal health and used it to assess ethanol neurotoxicity and to identify modulating agents. The MNH assay represents an effective new tool for discovering modulators of mitochondrial neuronal health and toxicity in live human neurons.
Cycle network model of Prostaglandin H Synthase-1
Authors: Goltsov Alexey , Swat Maciej , Peskov Kirill , Kosinsky Yuri
Published: August 2020

The kinetic model of Prostaglandin H Synthase-1 (PGHS-1) was developed to investigate its complex network kinetics and non-steroidal anti-inflammatory drugs (NSAIDs) efficacy in differentin vitroandin vivoconditions. To correctly describe the complex mechanism of PGHS-1 catalysis, we developed a microscopic approach to modelling of intricate network dynamics of 35 intraenzyme reactions among 24 intermediate states of the enzyme. The developed model quantitatively describes interconnection between cyclooxygenase and peroxidase enzyme activities; substrate (arachidonic acid, AA) and reducing cosubstrate competitive consumption; enzyme self-inactivation; autocatalytic role of AA; enzyme activation threshold, and synthesis of intermediate PGG2and final PGH2products under wide experimental conditions. In the paper we provided the detailed description of the enzyme catalytic cycle, model calibration based on a series ofin vitrokinetic data and model validation using experimental data on the regulatory properties of PGHS-1.

The validated model of PGHS-1 with a unified set of kinetic parameters is applicable forin silicoscreening and prediction of the inhibition effects of NSAIDs and their combination on the balance of pro-thrombotic (thromboxane) and anti-thrombotic (prostacyclin) prostaglandin biosynthesis in platelets and endothelial cells expressing PGHS-1.
Mean-field models for EEG/MEG: from oscillations to waves
Authors: Byrne Á. , Ross James , Nicks Rachel , Coombes Stephen
Published: August 2020

Neural mass models have been actively used since the 1970s to model the coarse-grained activity of large populations of neurons. They have proven especially fruitful for understanding brain rhythms. However, although motivated by neurobiological considerations they are phenomeno-logical in nature, and cannot hope to recreate some of the rich repertoire of responses seen in real neuronal tissue. Here we consider a simple spiking neuron network model that has recently been shown to admit to an exact mean-field description for both synaptic and gap-junction interactions. The mean-field model takes a similar form to a standard neural mass model, with an additional dynamical equation to describe the evolution of population synchrony. As well as reviewing the origins of thisnext generationmass model we discuss its extension to describe an idealised spatially extended planar cortex. To emphasise the usefulness of this model for EEG/MEG modelling we show how it can be used to uncover the role of local gap-junction coupling in shaping large scale synaptic waves.
Protein arginine methyltransferase 5 promotes metastasis via enhancing EGFR transcription and modulating AKT1 activation by methylation
Authors: Huang Lei , Zhang Xiao-Ou , Verdejo-Torres Odette , Wigglesworth Kim , Sun Xiaomei , Sallis Benjamin , Moon Daniel , Huang Tingting , Rozen Esteban , Wang Gang , Zhang Lei , Shohet Jason , Lee Mary M. , Wu Qiong
Published: August 2020

Protein arginine methyltransferase 5 (PRMT5) regulates a wide range of physiological processes, including cancer cell proliferation and metastasis, by generating symmetric di-methyl-arginine marks on histones and non-histone proteins. Here, we report that PRMT5 directly regulates epidermal growth factor receptor (EGFR) transcription to control EGF stimulated EGFR signaling. Furthermore, PRMT5 modulates protein kinase B (AKT) activation by methylation of AKT1 Arg 15, which is required for its subsequent phosphorylation at AKT1 Thr 308 and Ser 473. The PRMT5/EGFR/AKT axis converges to regulate transcription factors ZEB1, SNAIL, and TWIST1 to promote the epithelial-mesenchymal transition (EMT), in the manner that EGFR and AKT1 compensate each other to support tumor cell invasion and metastasis. Inhibiting PRMT5 methyltransferase activity with a small molecule inhibitor attenuated primary tumor growth and prevented hepatic metastasis in aggressivein vivotumor models. Collectively, our results support the use of PRMT5 based therapies for metastatic cancer.
Alteration of mucins in the submandibular gland during aging in mice
Authors: Kameyama Akihiko , Thet Tin Wai Wai , Nishijima Risa , Yamakoshi Kimi
Published: August 2020

Objective

Mucins are large glycosylated glycoproteins that are produced in the salivary glands, and their changes may contribute to the development of xerostomia due to aging and the accompanying deterioration of oral hygiene. This study aimed to characterize the changes in the mucins produced in submandibular gland (SMG) during the aging process.

Methods

SMG mucins derived from mice of each age were separated using supported molecular matrix electrophoresis (SMME). Subsequently, the membranes were stained with AB or blotted with MAL-II lectin. The SMME membranes stained with AB were subjected to densitometric analysis and glycan analysis. The detailed structures of O-glycan were investigated by MS/MS spectra.

Results

The SMG of mice secreted three mucins with different glycan profiles: age-specific mucin, youth-specific mucin, and a mucin expressed throughout life, and the expression patterns of these mucins change during aging. Additionally, age-specific mucin began to be detected at about 12 months of age. A mucin expressed throughout life and age-specific mucin had the same mass of major glycans but different structures.

Furthermore, the proportion of mucin glycan species expressed throughout life changed during the aging process, and aging tended to decrease the proportion of fucosylated glycans and increase the proportion of sialoglycans.

Conclusion

There are three secretory mucins with different glycan profiles in the SMG of mice, and their expression patterns change according to the period of the aging process. The proportion of glycan species of mucin expressed throughout life also changes during the aging process.

Highlights

Three secreted mucins with different glycan profiles are detected in SMG using SMME.

Each mucin has its own peak production age during the aging process.

Age-specific mucin begins to be detected at about 12 months of age.

Same mass of major glycans but different structures in mucins expressed throughout life vs. age-specific mucins.

Proportion of mucin glycan species expressed throughout life changes during aging.
Kinesin-1 motility traced by an activity-based precipitating dye
Authors: Angerani Simona , Lindberg Eric , Klena Nikolai , Bleck Christopher K. E. , Aumeier Charlotte , Winssinger Nicolas
Published: August 2020

Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. As tracks for cargo delivery, kinesin-1 uses a subset of microtubules within the dense microtubule network. It is still debated what defines the specific binding of kinesin-1 to a subset of microtubules. Therefore, the ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate for kinesin-1 that yields a precipitating dye. The activity of kinesin-1 thus leaves a fluorescent trail along its walking path and can be visualized without loss of signal due to diffusion. Kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time.
Single cell 3’UTR analysis identifies changes in alternative polyadenylation throughout neuronal differentiation and in autism
Authors: Göpferich Manuel , George Nikhil Oommen , Muelas Ana Domingo , Bizyn Alex , Pascual Rosa , Fijalkowska Daria , Kalamakis Georgios , Müller Ulrike , Krijgsveld Jeroen , Mendez Raul , Fariñas Isabel , Huber Wolfgang , Anders Simon , Martin-Villalba Ana
Published: August 2020

Autism spectrum disorder (ASD) is a neurodevelopmental disease affecting social behavior. Many of the high-confident ASD risk genes relate to mRNA translation. Specifically, many of these genes are involved in regulation of gene expression for subcellular compartmentalization of proteins1. Cis-regulatory motifs that often localize to 3’- and 5’-untranslated regions (UTRs) offer an additional path for posttranscriptional control of gene expression. Alternative cleavage and polyadenylation (APA) affect 3’UTR length thereby influencing the presence or absence of regulatory elements. However, APA has not yet been addressed in the context of neurodevelopmental disorders. Here we used single cell 3’end sequencing to examine changes in 3’UTRs along the differentiation from neural stem cells (NSCs) to neuroblasts within the adult brain. We identified many APA events in genes involved in neurodevelopment, many of them being high confidence ASD risk genes. Further, analysis of 3’UTR lengths in single cells from ASD and healthy individuals detected longer 3’UTRs in ASD patients. Motif analysis of modulated 3’UTRs in the mouse adult neurogenic lineage and ASD-patients revealed enrichment of the cytoplasmic and polyadenylation element (CPE). This motif is bound by CPE binding protein 4 (CPEB4). In human and mouse data sets we observed co-regulation of CPEB4 and the CPEB-binding synaptic adhesion molecule amyloid beta precursor-like protein 1 (APLP1). We show that mice deficient in APLP1 show aberrant regulation of APA, decreased number of neural stem cells, and autistic-like traits. Our findings indicate that APA is used for control of gene expression along neuronal differentiation and is altered in ASD patients.
Differential thermotolerance adaptation between species ofCoccidioides
Authors: Mead Heather L. , Hamm Paris S. , Shaffer Isaac N. , de Melo Teixeira Marcus , Wendel Christopher S. , Wiederhold Nathan P. , Thompson George R. , Muñiz-Salazar Raquel , Castañón-Olivares Laura Rosio , Keim Paul , Plude Carmel , Terriquez Joel , Galgiani John N. , Orbach Marc J. , Barker Bridget M.
Published: August 2020

Coccidioidomycosis, or Valley fever, is caused by two species of dimorphic fungi. Based on molecular phylogenetic evidence, the genusCoccidioidescontains two reciprocally monophyletic species:C. immitisandC. posadasii.However, phenotypic variation between species has not been deeply investigated. We therefore explored differences in growth rate under various conditions. A collection of 39C. posadasiiand 46C. immitisisolates, representing the full geographical range of the two species, were screened for mycelial growth rate at 37°C and 28°C on solid media. The radial growth rate was measured over 16 days on yeast extract agar. A linear mixed effect model was used to compare the growth rate ofC. posadasiiandC. immitisat 37°C and 28°C respectively.C. posadasiigrew significantly faster at 37°C, when compared toC. immitis;whereas both species had similar growth rates at 28°C. These results indicate thermotolerance differs between these two species. As the ecological niche has not been well-described forCoccidioidesspp., and disease variability between species has not been shown, the evolutionary pressure underlying the adaptation is unclear. However, this research reveals the first significant phenotypic difference between the two species that directly applies to ecological and clinical research.
Significantly improving the quality of genome assemblies through curation
Authors: Howe Kerstin , Chow William , Collins Joanna , Pelan Sarah , Pointon Damon-Lee , Sims Ying , Torrance James , Tracey Alan , Wood Jonathan
Published: August 2020

Background

Genome sequence assemblies provide the basis for our understanding of biology. Generating error-free assemblies is therefore the ultimate, but sadly still unachieved goal of a multitude of research projects. Despite the ever-advancing improvements in data generation, assembly algorithms and pipelines, no automated approach has so far reliably generated near error-free genome assemblies for eukaryotes.

Results

Whilst working towards improved data sets and fully automated pipelines, assembly evaluation and curation is actively employed to bridge this shortcoming and significantly reduce the number of assembly errors. In addition to this increase in product value, the insights gained from assembly curation are fed back into the automated assembly strategy and contribute to notable improvements in genome assembly quality.

Conclusions

We describe our tried and tested approach for assembly curation using gEVAL, the genome evaluation browser. We outline the procedures applied to genome curation using gEVAL and also our recommendations for assembly curation in an gEVAL-independent context to facilitate the uptake of genome curation in the wider community.
Regulated delivery controls Drosophila Hedgehog, Wingless and Decapentaplegic signaling
Authors: Hatori Ryo , Kornberg Thomas B.
Published: August 2020

Morphogen signaling proteins disperse across tissues to activate signal transduction in target cells. We investigated dispersion of Hedgehog (Hh), Wingless (Wg), and Bone morphogenic protein homolog Decapentaplegic (Dpp) in the Drosophila wing imaginal disc, and found that delivery to targets is regulated. Cells take up <5% Hh produced, and neither amounts taken up nor extent of signaling changes under conditions of Hh production from 50-200% normal amounts. Similarly, cells take up <25% Wg produced, and variation in Wg production from 50-700% normal has no effect on amounts taken up or signaling. Similar properties were observed for Dpp. Wing disc-produced Hh signals to disc-associated tracheal and myoblast as well as an approximately equal number of disc cells, but the extent of signaling in the disc is unaffected by the presence or absence of the tracheal cells and myoblasts. These findings show that target cells do not take up signaling proteins from a common pool and that both the amount and destination of delivered morphogens are regulated..

The extent of Hh, Wg, and Dpp signaling is independent of the amount of signal produced or the number of recipient cells.
TRIM28 regulates SARS-CoV-2 cell entry by targeting ACE2
Authors: Wang Yinfang , Fan Yingzhe , Huang Yitong , Du Tao , Liu Zongjun , Huang Dekui , Wang Ying , Wang Nanping , Zhang Peng
Published: August 2020

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19), it binds to angiotensin-converting enzyme 2 (ACE2) to enter into human cells. The expression level of ACE2 potentially determine the susceptibility and severity of COVID-19, it is thus of importance to understand the regulatory mechanism of ACE2 expression. Tripartite motif containing 28 (TRIM28) is known to be involved in multiple processes including antiviral restriction, endogenous retrovirus latency and immune response, it is recently reported to be co-expressed with SARS-CoV-2 receptor in type II pneumocytes; however, the roles of TRIM28 in ACE2 expression and SARS-CoV-2 cell entry remain unclear. This study showed that knockdown of TRIM28 induces ACE2 expression and increases pseudotyped SARS-CoV-2 cell entry of A549 cells and primary pulmonary alveolar epithelial cells (PAEpiCs). In a co-culture model of NK cells and lung epithelial cells, our results demonstrated that NK cells inhibit TRIM28 and promote ACE2 expression in lung epithelial cells, which was partially reversed by depletion of interleukin-2 and blocking of granzyme B in the co-culture medium. Furthermore, TRIM28 knockdown enhanced interferon-γ (IFN-γ)-induced ACE2 expression through a mechanism involving upregulating IFN-γ receptor 2 (IFNGR2) in both A549 and PAEpiCs. Importantly, the upregulated ACE2 induced by TRIM28 knockdown and co-culture of NK cells was partially reversed by dexamethasone in A549 cells but not PAEpiCs. Our study identified TRIM28 as a novel regulator of ACE2 expression and SARS-CoV-2 cell entry.
KSHV-encoded vCyclin can modulate HIF1α levels to promote DNA replication in hypoxia
Authors: Singh Rajnish Kumar , Pei Yonggang , Lamplugh Zachary L , Sun Kunfeng , Yuan Yan , Lieberman Paul , You Jianxin , Robertson Erle S
Published: August 2020

The cellular adaptive response to hypoxia, mediated by high HIF1α levels includes metabolic reprogramming, restricted DNA replication and cell division. In contrast to healthy cells, the genome of cancer cells, and Kaposi’s sarcoma associated herpesvirus (KSHV) infected cells maintains replication in hypoxia. We show that KSHV infection, despite promoting expression of HIF1α in normoxia, can also restricts transcriptional activity, and promoted its degradation in hypoxia. KSHV-encoded vCyclin, expressed in hypoxia, mediated HIF1a cytosolic translocation, and its degradation through a non-canonical lysosomal pathway. Attenuation of HIF1α levels by vCyclin allowed cells to bypass the block to DNA replication and cell proliferation in the hypoxia. These results demonstrated that KSHV utilizes a unique strategy to balance HIF1α levels to overcome replication arrest and induction of the oncogenic phenotype, which are dependent on the levels of oxygen in the microenvironment.
Midkine in chick and mouse retinas: neuroprotection, glial reactivity and the formation of Müller glia-derived progenitor cells
Authors: Campbell Warren A. , Fritsch-Kelleher Amanda , Palazzo Isabella , Hoang Thanh , Blackshaw Seth , Fischer Andy J.
Published: August 2020

Recent studies have shown that midkine (MDK), a basic heparin-binding growth factor, is involved in the development and regeneration of the zebrafish retina. However, very little is known about MDK in the retinas of warm-blooded vertebrates. We investigate the expression patterns of MDK and related factors, roles in neuronal survival, and influence upon the formation of Müller glia-derived progenitor cells (MGPCs) in chick and mouse model systems. By using single-cell RNA-sequencing, we find thatMDKis upregulated during Müller glia (MG) maturation in chick development and when stimulated to reprogram into MGPCs after NMDA damage or FGF2/Insulin treatment. Interestingly,MDKis significantly up-regulated by MG in damaged chick retinas, but down-regulated by MG in damaged mouse retinas. In both chick and mouse retinas, exogenous MDK selectively up-regulates cFOS and pS6 (a readout of mTOR-signaling) in MG. In the chick, intraocular injections of MDK before injury is neuroprotective with an observed decrease in dying neurons and microglial reactivity, inducing fewer proliferating MGPCs. Blocking MDK signaling with Na3VO4following blocks neuroprotective effects with an increase the number of dying cells and negates the pro-proliferative effects on MGPCs. Inhibitors of PP2A and Pak1 associated with MDK integrin β1 signaling had MG specific inhibitory effects on MGPC formation. In mice, MDK administration with NMDA damage drives a small but significant increase in MGPCs. We conclude thatMDKexpression is dynamically regulated in reactive Müller glia and during reprogramming into MGPCs. MDK acts to coordinate glial activity, neuronal survival, and may act in an autocrine manner to influence the re-programming of Müller glia into proliferating MGPCs.
A genome-wide screen in macrophages identifies new regulators of IFNγ-inducible MHCII that contribute to T cell activation
Authors: Kiritsy Michael C. , Ankley Laurisa M. , Trombley Justin D. , Huizinga Gabrielle P. , Lord Audrey E. , Orning Pontus , Elling Roland , Fitzgerald Katherine A. , Olive Andrew J.
Published: August 2020

Abstract

Cytokine-mediated activation of host immunity is central to the control of pathogens. A key cytokine in protective immunity is interferon-gamma (IFNγ), which is a potent activator of antimicrobial and immunomodulatory effectors within the host. A major role of IFNγ is to induce major histocompatibility complex class II molecules (MHCII) on the surface of cells, which is required for CD4+T cell activation. Despite its central role in host immunity, the complex and dynamic regulation of IFNγ-induced MHCII is not well understood. Here, we integrated functional genomics and transcriptomics to comprehensively define the genetic control of IFNγ-mediated MHCII surface expression in macrophages. Using a genome-wide CRISPR-Cas9 library we identified genes that control MHCII surface expression, many of which have yet to be associated with MHCII. Mechanistic studies uncovered two parallel pathways of IFNγ-mediated MHCII control that require the multifunctional glycogen synthase kinase 3 beta (GSK3β) or the mediator complex subunit MED16. Both pathways are necessary for IFNγ-mediated induction of the MHCII transactivator CIITA, MHCII expression, and CD4+T cell activation. Using transcriptomic analysis, we defined the regulons controlled by GSK3β and MED16 in the presence and absence of IFNγ and identified unique networks of the IFNγ-mediated transcriptional landscape that are controlled by each gene. Our analysis suggests GSK3β and MED16 control distinct aspects of the IFNγ-response and are critical for macrophages to respond appropriately to IFNγ. Our results define previously unappreciated regulation of MHCII expression that is required to control CD4+T cell responses by macrophages. These discoveries will aid in our basic understanding of macrophage-mediated immunity and will shed light on mechanisms of failed adaptive responses pervasive in infectious disease, autoimmunity, and cancer.
Gene expression associated with disease resistance and long-term growth in a reef-building coral
Authors: Kelley Emma R. , Sleith Robin S. , Matz Mikhail V. , Wright Rachel M.
Published: August 2020

Rampant coral disease, exacerbated by climate change and other anthropogenic stressors, threatens reefs worldwide, especially in the Caribbean. Physically isolated yet genetically connected reefs such as Flower Garden Banks National Marine Sanctuary (FGBNMS) may serve as potential refugia for degraded Caribbean reefs. However, little is known about the mechanisms and trade-offs of pathogen resistance in reef-building corals. Here we measure pathogen resistance inMontastraea cavernosafrom FGBNMS. We identified individual colonies that demonstrated resistance or susceptibility toVibrio spp. in a controlled laboratory environment. Long-term growth patterns suggest no trade-off between disease resistance and calcification. Predictive (pre-exposure) gene expression highlights subtle differences between resistant and susceptible genets, encouraging future coral disease studies to investigate associations between resistance and replicative age and immune cell populations. Predictive gene expression associated with long-term growth underscores the role of cation transporters and extracellular matrix remodelers, contributing to the growing body of knowledge surrounding genes that influence calcification in reef-building corals. Together these results demonstrate that coral genets from isolated sanctuaries such as FGBNMS can withstand pathogen challenges and potentially aid restoration efforts in degraded reefs. Furthermore, gene expression signatures associated with resistance and long-term growth help inform strategic assessment of coral health parameters.
Increased copy number couples the evolution of plasmid horizontal transmission and antibiotic resistance
Authors: Dimitriu Tatiana , Matthews Andrew , Buckling Angus
Published: August 2020

Antimicrobial resistance (AMR) in bacteria is commonly encoded on conjugative plasmids, mobile elements which can spread horizontally between hosts. Conjugative transfer disseminates AMR in communities but it remains unclear when and how high transfer rates evolve, and with which consequences. Here we studied experimentally the evolution of two antibiotic resistance encoding plasmids when confronted to different immigration rates of susceptible, plasmid-free hosts. While plasmid RP4 did not evolve detectably, plasmid R1 rapidly evolved up to 1000-fold increased transfer rates in the presence of susceptible hosts, at a cost to its host. Unexpectedly, most evolved plasmids also conferred to their hosts the ability to grow at high concentrations of antibiotics. The most common mutations in evolved plasmids were contained within thecopAgene which controls plasmid replication and copy number. EvolvedcopAvariants had elevated copy number, leading to both higher transfer rates and AMR. Due to these pleiotropic effects, host availability and antibiotics were each sufficient to select for highly transmissible plasmids conferring high levels of antibiotic resistance.
Aβ initiates brain hypometabolism and network dysfunction via NOX2 activation: a potential onset mechanism of Alzheimer’s disease
Authors: Malkov Anton , Popova Irina , Ivanov Anton , Jang Sung-Soo , Yoon Seo Yeon , Osypov Alexander , Huang Yadong , Zilberter Yuri , Zilberter Misha
Published: August 2020

A predominant trigger and driver of sporadic Alzheimer’s disease (AD) is the synergy of brain oxidative stress and glucose hypometabolism starting at early preclinical stages. Oxidative stress damages macromolecules, while glucose hypometabolism impairs cellular energy supply and antioxidant defense. However, the exact cause of AD-associated glucose hypometabolism and its network consequences has remained unknown. Here we report NADPH oxidase 2 (NOX2) activation as the main initiating mechanism behind Aβ1-42-related glucose hypometabolism and network dysfunction. We utilize a combination of electrophysiology with real-time recordings of metabolic transients bothex- andin-vivoto show that Aβ1-42induces oxidative stress and acutely reduces cellular glucose consumption followed by long-lasting network hyperactivity and abnormalities in the animal behavioral profile. Critically, all of these pathological changes were prevented by the novel bioavailable NOX2 antagonist GSK2795039. Our data provide the first direct experimental evidence for causes and consequences of AD-related brain glucose hypometabolism, and suggest that targeting NOX2-mediated oxidative stress is a promising approach to both the prevention and treatment of AD.

Single sentence summary

Beta-amyloid induces brain hypometabolism, network hyperactivity, and behavioral changes via NADPH oxidase-mediated oxidative stress, suggesting a novel therapeutic target for Alzheimer’s disease treatment.
Structural basis for transcription complex disruption by the Mfd translocase
Authors: Kang Jin Young , Llewellyn Eliza , Chen James , Olinares Paul Dominic B. , Brewer Joshua , Chait Brian T. , Campbell Elizabeth A. , Darst Seth A.
Published: August 2020

Transcription-coupled repair (TCR) is a sub-pathway of nucleotide excision repair (NER) that preferentially removes lesions from the template-strand (t-strand) that stall RNA polymerase (RNAP) elongation complexes (EC). Mfd mediates TCR in bacteria by removing the stalled RNAP concealing the lesion and recruiting Uvr(A)BC. We used cryo-electron microscopy to visualize Mfd engaging with a stalled EC and attempting to dislodge the RNAP. We visualized seven distinct Mfd-EC complexes in both ATP and ADP-bound states. The structures explain how Mfd is remodeled from its repressed conformation, how the UvrA-interacting surface of Mfd is hidden during most of the remodeling process to prevent premature engagement with the NER pathway, how Mfd alters the RNAP conformation to facilitate disassembly, and how Mfd forms a processive translocation complex after dislodging the RNAP. Our results reveal an elaborate mechanism for how Mfd kinetically discriminates paused from stalled ECs and disassembles stalled ECs to initiate TCR.
ASH2L drives proliferation and sensitivity to bleomycin and other genotoxins in Hodgkin’s lymphoma and testicular cancer cells
Authors: Constantin Daniel , Widmann Christian
Published: August 2020

It is of clinical importance to identify biomarkers predicting the efficacy of DNA damaging drugs (genotoxins) so that non-responders are not unduly exposed to the deleterious effects of otherwise inefficient drugs. Using a whole genome CRISPR/Cas9 gene knockout approach we have identified that low levels of ASH2L cause resistance to genotoxins. ASH2L is a core component of the H3K4 methyl transferase complex. We show that ASH2L absence decreases cell proliferation and favors DNA repair upon genotoxin exposure. The cell models we have used are derived from cancers currently treated either partially (Hodgkin’s lymphoma), or entirely (testicular cancer) with genotoxins. For such cancers, ASH2L levels could be used as a biomarker to predict the response to genotoxins. Our data also indicate that patients with low ASH2L expressing tumors do not develop resistance to ATR inhibitors. In these patients, such inhibitors may represent an alternative treatment to DNA damaging drugs.
Creb5 establishes the competence forPrg4expression in articular cartilage
Authors: Zhang Cheng-Hai , Gao Yao , Jadhav Unmesh , Hung Han-Hwa , Holton Kristina M , Grodzinsky Alan J. , Shivdasani Ramesh A. , Lassar Andrew B.
Published: August 2020

A hallmark of cells comprising the superficial zone of articular cartilage is their expression of lubricin, encoded by thePrg4gene, that lubricates the joint and protects against the development of arthritis. Here, we identify Creb5 as a transcription factor that is specifically expressed in superficial zone articular chondrocytes and is required for TGF-β and EGFR signaling to inducePrg4expression. Notably, forced expression of Creb5 in chondrocytes derived from the deep zone of the articular cartilage confers the competence for TGF-β and EGFR signals to inducePrg4expression. Chromatin-IP and ATAC-Seq analyses have revealed that Creb5 directly binds to twoPrg4promoter-proximal regulatory elements, that display an open chromatin conformation specifically in superficial zone articular chondrocytes; and which work in combination with a more distal regulatory element to drive induction ofPrg4by TGF-β. Our results indicate that Creb5 is a critical regulator ofPrg4/lubricin expression in the articular cartilage.
Multiscale simulations examining glycan shield effects on drug binding to influenza neuraminidase
Authors: Seitz Christian , Casalino Lorenzo , Konecny Robert , Huber Gary , Amaro Rommie E. , McCammon J. Andrew
Published: August 2020

Influenza neuraminidase is an important drug target. Glycans are present on neuraminidase, and are generally considered to inhibit antibody binding via their glycan shield. In this work we studied the effect of glycans on the binding kinetics of antiviral drugs to the influenza neuraminidase. We created all-atomin silicosystems of influenza neuraminidase with experimentally-derived glycoprofiles consisting of four systems with different glycan conformations and one system without glycans. Using Brownian dynamics simulations, we observe a two- to eight-fold decrease in the rate of ligand binding to the primary binding site of neuraminidase due to the presence of glycans. These glycans are capable of covering much of the surface area of neuraminidase, and the ligand binding inhibition is derived from glycans sterically occluding the primary binding site on a neighboring monomer. Our work also indicates that drugs preferentially bind to the primary binding site (i.e. the active site) over the secondary binding site, and we propose a binding mechanism illustrating this. These results help illuminate the complex interplay between glycans and ligand binding on the influenza membrane protein neuraminidase.

Statement of Significance

The influenza glycoprotein neuraminidase is the target for three FDA-approved influenza drugs in the US. However, drug resistance and low drug effectiveness merits further drug development towards neuraminidase, which is hindered by our limited understanding of glycan effects on ligand binding. Generally, drug developers do not include glycans in their development pipelines. Here, we show that even though glycans can reduce drug binding towards neuraminidase, we recommend future drug development work to focus on strong binders with a long lifetime. Furthermore, we examine the binding competition between the primary and secondary binding sites on neuraminidase, leading us to propose a new, to the best of our knowledge, multivalent binding mechanism.