bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemical-Genetic Interrogation of Nuclear Size Control Reveals Cancer-Specific Effects on Cell Migration and Invasion
Authors: Rizzotto Andrea , Tollis Sylvain , Pham Nhan T. , Wildenhain Jan , Zuleger Nikolaj , Keys Jeremy T. , Batrakou Dzmitry , Culley Jayne , Zheng Sarah , Lammerding Jan , Carragher Neil O. , Brunton Valerie G. , Auer Manfred , Tyers Mike , Schirmer Eric C.
Published: January 2020

Lower survival rates for many cancer types correlate with increases or decreases in nuclear size/scaling in a tumor-type/tissue-specific manner. Postulating that nuclear size changes confer a fitness advantage on tumor cells, we screened for FDA/EMA-approved compounds that reverse tumor nuclear size changes in cell lines from three such tumor types: prostate adenocarcinoma, colonic adenocarcinoma, and small-cell squamous lung cancer. We found distinct, largely non-overlapping sets of compounds that either rectify or exacerbate nuclear size changes for each tumor type. Nuclear size phenotypes across cell lines clustered particular classes of compounds including serotonin uptake inhibitors, cyclo-oxygenase inhibitors, beta-adrenergic receptor agonists, monoamine oxidase inhibitors, and Na+/K+ATPase inhibitors. Nearly all compounds selected for further investigation inhibited cell migration and/or invasion, suggesting that targeting nuclear size control pathways in chemotherapy regimens could improve patient survival.
Antigen responsive CD4+T cell clones contribute to the HIV-1 latent reservoir
Authors: Mendoza Pilar , Jackson Julia R. , Oliveira Thiago , Gaebler Christian , Ramos Victor , Caskey Marina , Jankovic Mila , Nussenzweig Michel C. , Cohn Lillian B.
Published: January 2020

Antiretroviral therapy suppresses but does not cure HIV-1 infection due to the existence of a long-lived reservoir of latently infected cells. The reservoir has an estimated half-life of 44 months and is largely composed of clones of infected CD4+T cells. The long half-life appears to result in part from expansion and contraction of infected CD4+T cell clones. However, the mechanisms that govern this process are poorly understood. To determine whether the clones might result from, and be maintained by exposure to antigen, we measured responses of reservoir cells to a small subset of antigens from viruses that produce chronic or recurrent infections. Despite the limited panel of test antigens, clones of antigen responsive CD4+T cells containing defective or intact latent proviruses were found in 7 out of 8 individuals studied. Thus, chronic or repeated exposure to antigen may contribute to the longevity of the HIV-1 reservoir by stimulating the clonal expansion of latently infected CD4+T cells.
Efficient and accurate inference of microbial trajectories from longitudinal count data
Authors: Joseph Tyler A. , Pasarkar Amey P. , Pe’er Itsik
Published: January 2020

The recently completed second phase of the Human Microbiome Project has highlighted the relationship between dynamic changes in the microbiome and disease, motivating new microbiome study designs based on longitudinal sampling. Yet, analysis of such data is hindered by presence of technical noise, high dimensionality, and data sparsity. To address these challenges, we propose LUMINATE (LongitUdinal Microbiome INference And zero deTEction), a fast and accurate method for inferring relative abundances from noisy read count data. We demonstrate on synthetic data that LUMINATE is orders of magnitude faster than current approaches, with better or similar accuracy. This translates to feasibility of analyzing data at the requisite dimensionality for current studies. We further show that LUMINATE can accurately distinguish biological zeros, when a taxon is absent from the community, from technical zeros, when a taxon is below the detection threshold. We conclude by demonstrating the utility of LUMINATE for downstream analysis by using estimates of latent relative abundances to fit the parameters of a dynamical system, leading to more accurate predictions of community dynamics.

Code availability

https://github.com/tyjo/luminate
Music-selective neural populations arise without musical training
Authors: Boebinger Dana , Norman-Haignere Sam , McDermott Josh , Kanwisher Nancy
Published: January 2020

Recent work has shown that human auditory cortex contains neural populations anterior and posterior to primary auditory cortex that respond selectively to music. However, it is unknown how this selectivity for music arises. To test whether musical training is necessary, we measured fMRI responses to 192 natural sounds in 10 people with almost no musical training. When voxel responses were decomposed into underlying components, this group exhibited a music-selective component that was very similar in response profile and anatomical distribution to that previously seen in individuals with moderate musical training. We also found that musical genres that were less familiar to our participants (e.g., Balinesegamelan) produced strong responses within the music component, as did drum clips with rhythm but little melody, suggesting that these neural populations are broadly responsive to music as a whole. Our findings demonstrate that the signature properties of neural music selectivity do not require musical training to develop, showing that the music-selective neural populations are a fundamental and widespread property of the human brain.

NEW & NOTEWORTHY

We show that music-selective neural populations are clearly present in people without musical training, demonstrating that they are a fundamental and widespread property of the human brain. Additionally, we show music-selective neural populations respond strongly to music from unfamiliar genres as well as music with rhythm but little pitch information, suggesting that they are broadly responsive to music as a whole.
An amygdalo-parabrachial pathway regulates pain perception and chronic pain
Authors: Raver Charles , Uddin Olivia , Ji Yadong , Li Ying , Cramer Nathan , Jenne Carleigh , Morales Marisela , Masri Radi , Keller Asaf
Published: January 2020

The parabrachial (PB) complex mediates both ascending nociceptive signaling and descending pain modulatory information in the affective/emotional pain pathway. We have recently reported that chronic pain is associated with amplified activity of PB neurons in a rat model of neuropathic pain. Here we demonstrate that similar activity amplification occurs in mice, and that this is related to suppressed inhibition to PB neurons from the central nucleus of the amygdala (CeA). Animals with pain after chronic constriction injury of the infraorbital nerve (CCI-Pain) displayed higher spontaneous and evoked activity in PB neurons, and a dramatic increase in after-discharges—responses that far outlast the stimulus—compared to controls. PB neurons in CCI-Pain animals showed a reduction in inhibitory, GABAergic inputs. We show that—in both rats and mice—PB contains few GABAergic neurons, and that most of its GABAergic inputs arise from CeA. These CeA GABA neurons express dynorphin, somatostatin and/or corticotropin releasing hormone. We find that the efficacy of this CeA-LPB pathway is suppressed in chronic pain. Further, optogenetically stimulating this pathway suppresses acute pain, and inhibiting it, in naïve animals, evokes pain behaviors. These findings demonstrate that the CeA-LPB pathway is critically involved in pain regulation, and in the pathogenesis of chronic pain.

Significance Statement

We describe a novel pathway, consisting of inhibition by dynorphin, somatostatin and corticotropin-releasing hormone expressing neurons in the central nucleus of the amygdala that project to the parabrachial nucleus (PB). We show that this pathway regulates the activity of pain-related neurons in PB, and that, in chronic pain, this inhibitory pathway is suppressed, and that this suppression is causally related to pain perception. We propose that this amygdalo-parabrachial pathway is a key regulator of both chronic and acute pain, and a novel target for pain relief.
CRL5-dependent regulation of Arl4c and Arf6 controls hippocampal morphogenesis
Authors: Han Jisoo S. , Hino Keiko , Reyes Raenier V. , Canales Cesar P. , Miltner Adam M. , Haddadi Yasmin , Sun Junqing , Chen Chao-Yin , La Torre Anna , Simó Sergi
Published: January 2020

The small GTPase Arl4c participates in the regulation of cell migration, cytoskeletal rearrangements, and vesicular trafficking in epithelial cells. The Arl4c signaling cascade starts by the recruitment of the Arf-GEF cytohesins to the plasma membrane, which in turn engage the small GTPase Arf6. In the nervous system, Arf6 regulates dendrite outgrowth in vitro and neuronal migration in the developing cortex. However, the role of Arl4c-cytohesin-Arf6 signaling during brain development and particularly during hippocampal development remain elusive. Here, we report that the E3 ubiquitin ligase Cullin 5/Rbx2 (CRL5) controls the stability of Arl4c and its signaling effectors to regulate hippocampal morphogenesis. Rbx2 knock out causes hippocampal pyramidal neuron mislocalization and formation of multiple apical dendrites. The same phenotypes were observed when Cullin 5 was knocked down in pyramidal neurons by in utero electroporation. We used quantitative mass spectrometry to show that Arl4c, Cytohesin-1/3, and Arf6 accumulate in the telencephalon when Rbx2 is absent. Arl4c expression is post-transcriptionally regulated, with a peak in expression at early postnatal stages, and is localized at the plasma membrane and on intracellular vesicles in hippocampal pyramidal neurons. Furthermore, we show that depletion of Arl4c rescues the phenotypes caused by Cullin 5 knock down in the hippocampus, whereas depletion of Arf6 exacerbates over-migration. Finally, we show that Arl4c and Arf6 are necessary for the dendritic outgrowth of pyramidal neurons to the most superficial strata of the hippocampus. Overall, we identified CRL5 as a key regulator of hippocampal development and uncovered Arl4c and Arf6 as novel CRL5-regulated signaling effectors that control pyramidal neuron migration and dendritogenesis.
Advantages and limits of metagenomic assembly and binning of a giant virus
Authors: Schulz Frederik , Andreani Julien , Francis Rania , Khalil Jacques Yaacoub Bou , Lee Janey , La Scola Bernard , Woyke Tanja
Published: January 2020

Giant viruses have large genomes, often within the size range of cellular organisms. This distinguishes them from most other viruses and demands additional effort for the successful recovery of their genomes from environmental sequence data. Here we tested the performance of genome-resolved metagenomics on a recently isolated giant virus, Fadolivirus, by spiking it into an environmental sample from which two other giant viruses were isolated. At high spike-in levels, metagenome assembly and binning led to the successful genomic recovery of Fadolivirus from the sample. A complementary survey of viral hallmark genes indicated the presence of other giant viruses in the sample matrix, but did not detect the two isolated from this sample. Our results indicate that genome-resolved metagenomics is a valid approach for the recovery of near-complete giant virus genomes given that sufficient clonal particles are present. Our data also underline that a vast majority of giant viruses remain currently undetected, even in an era of terabase-scale metagenomics.
Modulation of the canonical Wnt activity by androgen signaling in prostate epithelial basal stem cells
Authors: Liu Yueli , Wang Jiawen , Horton Corrigan , Katzman Sol , Cai Tao , Wang Zhu A.
Published: January 2020

Both the canonical Wnt signaling and androgen signaling are important factors regulating prostate organogenesis. How these two pathways crosstalk to regulate prostate stem cell functions remain unclear. Here, we show that while canonical Wnt activity is required for prostate basal stem cell multipotency in vivo, ectopic Wnt activity does not promote basal-to-luminal cell differentiation. We provide evidence that androgen signaling may keep Wnt activity in check. In prostate organoid culture from basal cells, dihydrotestosterone (DHT) antagonizes R-spondin-stimulated organoid growth in a concentration-dependent manner. Molecular analyses of organoids under different treatment conditions showed that androgen signaling down-regulated the expressions of a Wnt reporter as well as many Wnt target genes. Pathway analysis and gene set enrichment analysis of organoid RNA-seq data also revealed the canonical Wnt signaling as a key pathway distinguishing organoids treated with or without DHT. Notably, DHT treatment enhanced AR and β–catenin binding in the nuclei of prostate organoids, providing possible mechanistic clues. Our results reveal a critical role of AR signaling in modulating canonical Wnt activity in prostate basal cells to regulate their multipotency.
BAF facilitates interphase nuclear membrane repair through recruitment of nuclear transmembrane proteins
Authors: Young Alexandra M. , Gunn Amanda L. , Hatch Emily M.
Published: January 2020

Nuclear membrane rupture during interphase occurs in a variety of cell contexts, both healthy and pathological. Membrane ruptures can be rapidly repaired, but these mechanisms are still unclear. Here we show BAF, a nuclear envelope protein that shapes chromatin and recruits membrane proteins in mitosis, also facilitates nuclear membrane repair in interphase, in part through recruitment of the nuclear membrane proteins emerin and LEMD2 to rupture sites. Characterization of GFP-BAF accumulation at nuclear membrane rupture sites confirmed BAF is a fast, accurate, and persistent mark of nucleus rupture whose kinetics are partially dictated by membrane resealing. BAF depletion significantly delayed nuclear membrane repair, with a larger effect on longer ruptures. This phenotype could be rescued by GFP-BAF, but not by a BAF mutant lacking the LEM-protein binding domain. Depletion of the BAF interactors LEMD2 or emerin, and to a lesser extent lamin A/C, increased the duration of nucleus ruptures, consistent with LEM-protein binding being a key function of BAF during membrane repair. Overall our results suggest a model where BAF is critical for timely repair of large ruptures in the nuclear membrane, potentially by facilitating membrane attachment to the rupture site.
Rare protein coding variants implicate genes involved in risk of suicide death
Authors: DiBlasi Emily , Shabalin Andrey A. , Monson Eric T. , Keeshin Brooks R. , Bakian Amanda V. , Kirby Anne V. , Ferris Elliott , Chen Danli , William Nancy , Gaj Eoin , Klein Michael , Jerominski Leslie , Callor W. Brandon , Christensen Erik , Gray Douglas , Smith Ken R. , Fraser Alison , Yu Zhe ,   PsychChip Investigators of the Psychiatric Genomics Consortium, Camp Nicola J. , Stahl Eli A. , Li Qingqin S. , Docherty Anna R. , Coon Hilary
Published: January 2020

Suicide death is a worldwide health crisis, claiming close to 800,000 lives per year. Recent evidence suggests that prediction and prevention challenges may be aided by discoveries of genetic risk factors. Here we focus on the role of rare (MAF <1%), putatively functional single nucleotide polymorphisms (SNPs) in suicide death using the large genetic resources available in the Utah Suicide Genetic Risk Study (USGRS). We conducted a single-variant association analysis of 30,377 rare putatively functional SNPs present on the PsychArray genotyping array in 2,672 USGRS suicides of non-Finnish European (NFE) ancestry and 51,583 publicly available NFE controls from gnomAD, with additional follow-up analyses using an independent control sample of 21,324 NFE controls from the Psychiatric Genomics Consortium. SNPs underwent rigorous quality control, and among SNPs meeting significance thresholds, we considered only those that were validated in sequence data. We identified five novel, high-impact, rare SNPs with significant associations with suicide death (SNAPC1, rs75418419;TNKS1BP1, rs143883793;ADGRF5, rs149197213;PER1, rs145053802; andESS2, rs62223875). BothPER1andSNAPC1have other supporting gene-level evidence of suicide risk, and an association with bipolar disorder has been reported forPER1and with schizophrenia forPER1, TNKS1BP1, andESS2. Three genes (PER1, TNKS1BP1, andADGRF5), with additional genes implicated by GWAS studies on suicidal behavior, showed significant enrichment in immune system, homeostatic and signal transduction processes. Pain, depression, and accidental trauma were the most prevalent phenotypes in electronic medical record data for the categories assessed. These findings suggest an important role for rare variants in suicide risk and provide new insights into the genetic architecture of suicide death. Furthermore, we demonstrate the added utility of careful assessment of genotyping arrays in rare variant discovery.
Spatially compartmentalized phase regulation of a Ca2+-cAMP-PKA oscillatory circuit
Authors: Tenner Brian , Getz Michael , Ross Brian , Ohadi Donya , Bohrer Christopher H. , Greenwald Eric , Mehta Sohum , Xiao Jie , Rangamani Padmini , Zhang Jin
Published: January 2020

Signaling networks are spatiotemporally organized in order to sense diverse inputs, process information, and carry out specific cellular tasks. In pancreatic β cells, Ca2+, cyclic adenosine monophosphate (cAMP), and Protein Kinase A (PKA) exist in an oscillatory circuit characterized by a high degree of feedback, which allows for specific signaling controls based on the oscillation frequencies. Here, we describe a novel mode of regulation within this circuit involving a spatial dependence of the relative phase between cAMP, PKA, and Ca2+. We show that nanodomain clustering of Ca2+-sensitive adenylyl cyclases drives oscillations of local cAMP levels to be precisely in-phase with Ca2+oscillations, whereas Ca2+-sensitive phosphodiesterases maintain out-of-phase oscillations outside of the nanodomain, representing a striking example and novel mechanism of cAMP compartmentation. Disruption of this precise in-phase relationship perturbs Ca2+oscillations, suggesting that the relative phase within an oscillatory circuit can encode specific functional information. This example of a signaling nanodomain utilized for localized tuning of an oscillatory circuit has broad implications for the spatiotemporal regulation of signaling networks.
ChiS is a noncanonical DNA-binding hybrid sensor kinase that directly regulates the chitin utilization program inVibrio cholerae
Authors: Klancher Catherine A. , Yamamoto Shouji , Dalia Triana N. , Dalia Ankur B.
Published: January 2020

Two-component signal transductionsystems (TCSs) represent a major mechanism that bacteria use to sense and respond to their environment. Prototypical TCSs are composed of a membrane-embedded histidine kinase (HK), which senses an environmental stimulus and subsequently phosphorylates a cognate partner protein called a response regulator (RR) that regulates gene expression in a phosphorylation-dependent manner.Vibrio choleraeuses the hybrid HK ChiS to activate the expression of the chitin utilization program, which is critical for the survival of this facultative pathogen in its aquatic reservoir. A cognate RR for ChiS has not been identified and the mechanism of ChiS-dependent signal transduction remains unclear. Here, we show that ChiS is a noncanonical membrane-embedded one-component system that can both sense chitin and directly regulate gene expression via a cryptic DNA binding domain. Unlike prototypical TCSs, we find that ChiS DNA binding is diminished, rather than stimulated, by phosphorylation. Finally, we provide evidence that ChiS likely activates gene expression by directly recruiting RNA polymerase. Together, this work addresses the mechanism of action for a major transcription factor inV. choleraeand highlights the versatility of signal transduction systems in bacterial species.

Significance Statement

From bacteria to humans, the ability to properly respond to environmental cues is critical for survival. The cholera pathogenVibrio choleraeuses one protein, ChiS, to sense chitin in its environmental reservoir to regulate the expression of genes that are critical for the survival and evolution of this pathogen in this niche. Here, we study how the chitin sensor ChiS works, and discover that it regulates gene expression in an unexpected and unorthodox manner. Thus, this study uncovers how the major regulator ChiS works in this important human pathogen and highlights the versatile mechanisms that living systems use to respond to their environment.
Bariatric surgery reveals a gut-restricted TGR5 agonist that exhibits anti-diabetic effects
Authors: Chaudhari Snehal N. , Harris David A. , Aliakbarian Hassan , Henke Matthew T. , Subramaniam Renuka , Vernon Ashley H. , Tavakkoli Ali , Sheu Eric G. , Devlin A. Sloan
Published: January 2020

Bariatric surgery, the most effective treatment for obesity and type 2 diabetes, is consistently associated with increased levels of the incretin hormone GLP-1 and changes in overall levels of circulating bile acids. The levels of individual bile acids in the GI tract following surgery, however, have remained largely unstudied. Using UPLC-MS-based quantification, we observed an increase in an endogenous bile acid, cholic acid-7-sulfate (CA7S), in the GI tract of both mice and humans after sleeve gastrectomy. We show that CA7S is a TGR5 agonist that induces GLP-1 secretion in vitro and in vivo and that CA7S administration increases glucose tolerance in insulin-resistant mice in a GLP-1 receptor-dependent manner. CA7S remains gut-restricted, minimizing off-target effects previously observed for TGR5 agonists absorbed into circulation. By studying changes in individual metabolites following surgery, this study has revealed a naturally occurring TGR5 agonist that exerts systemic glucoregulatory effects while remaining confined to the gut.
Highly Multiplexed Single-Cell Full-Length cDNA Sequencing of human immune cells with 10X Genomics and R2C2
Authors: Volden Roger , Vollmers Christopher
Published: January 2020

Single cell transcriptome analysis elucidates facets of cell biology that have been previously out of reach. However, the high-throughput analysis of thousands of single cell transcriptomes has been limited by sample preparation and sequencing technology. High-throughput single cell analysis today is facilitated by protocols like the 10X Genomics platform or Drop-Seq which generate cDNA pools in which the origin of a transcript is encoded at its 5’ or 3’ end. These cDNA pools are most often analyzed by short read Illumina sequencing which can identify the cellular origin of a transcript and what gene it was transcribed from. However, these methods fail to retrieve isoform information. In principle, cDNA pools prepared using these approaches can be analyzed with Pacific Biosciences and Oxford Nanopore long-read sequencers to retrieve isoform information but current implementations rely heavily on Illumina short-reads for analysis in addition to long reads. Here, we used R2C2 to sequence and demultiplex 12 million full-length cDNA molecules generated by the 10X Chromium platform from ∼3000 peripheral blood mononuclear cells (PBMCs). We used these reads to – independent from Illumina data – cluster cells into B cells, T cells, and Monocytes and generate isoform-level transcriptomes for these cell types. We also generated isoform-level transcriptomes for all single cells and used this information to identify a wide range of isoform diversity between genes. Finally, we also designed a computational workflow to extract paired adaptive immune receptors – T cell receptor and B cell receptor (TCR and BCR) – sequences unique to each T and B cell. This work represents a new, simple, and powerful approach that – using a single sequencing method – can extract an unprecedented amount of information from thousands of single cells.
Detection of urease and carbonic anhydrase activity using a rapid and economical field test to assess microbially-induced carbonate precipitation
Authors: Ferrer Fernando Medina , Hobart Kathryn , Bailey Jake V.
Published: January 2020

Microbial precipitation of calcium carbonate has diverse engineering applications, from building and soil restoration, to carbon sequestration. Urease-mediated ureolysis and CO2(de)hydration by carbonic anhydrase (CA) are known for their potential to precipitate carbonate minerals, yet many microbial community studies rely on marker gene or metagenomic approaches that are unable to determinein situactivity. Here, we developed fast and cost-effective tests for the field detection of urease and CA activity using pH-sensitive strips inside microcentrifuge tubes that change color in response to the reaction products of urease (NH3) and CA (CO2). Samples from a saline lake, a series of calcareous fens, and ferrous springs were assayed in the field, finding relatively high urease activity in lake samples, whereas CA activity was only detected in a ferrous spring. Incubations of lake microbes with urea resulted in significantly higher CaCO3precipitation compared to incubations with a urease inhibitor. Therefore, the rapid assay indicated an on-site active metabolism potentially mediating carbonate mineralization. Field urease and CA activity assays complement molecular approaches and facilitate the search for carbonate-precipitating microbes and theirin situactivity, which could be applied toward agriculture, engineering and carbon sequestration technologies.
Chaperone mediated coupling of subunit availability to activation of flagellar Type III Secretion
Authors: Bryant Owain J. , Chung Betty Y-W. , Fraser Gillian M.
Published: January 2020

Bacterial flagellar subunits are exported across the cell membrane by the flagellar Type III Secretion System (fT3SS), powered by the proton motive force (pmf) and a specialized ATPase that enables the flagellar export gate to utilise the pmf electric potential (ΔΨ). Export gate activation is mediated by the ATPase stalk, FliJ, but how this process is regulated to prevent wasteful dissipation of pmf in the absence of subunit cargo is not known. Here, we show that FliJ activation of the export gate is regulated by flagellar export chaperones. FliJ binds unladen chaperones and, using novel chaperone variants specifically defective for FliJ binding, we show that disruption of this interaction attenuates motility and cognate subunit export. We demonstrate in vitro that chaperones and the FlhA export gate component compete for binding to FliJ, and show in vivo that unladen chaperones, which would be present in the cell when subunit levels are low, sequester FliJ to prevent activation of the export gate and attenuate subunit export. Our data indicate a mechanism whereby chaperones couple availability of subunit cargo to pmf-driven export by the fT3SS.
Slow drift of neural activity as a signature of impulsivity in macaque visual and prefrontal cortex
Authors: Cowley Benjamin R. , Snyder Adam C. , Acar Katerina , Williamson Ryan C. , Yu Byron M. , Smith Matthew A.
Published: January 2020

An animal’s decision depends not only on incoming sensory evidence but also on its fluctuating internal state. This internal state is a product of cognitive factors, such as fatigue, motivation, and arousal, but it is unclear how these factors influence the neural processes that encode the sensory stimulus and form a decision. We discovered that, over the timescale of tens of minutes during a perceptual decision-making task, animals slowly shifted their likelihood of reporting stimulus changes. They did this unprompted by task conditions. We recorded neural population activity from visual area V4 as well as prefrontal cortex, and found that the activity of both areas slowly drifted together with the behavioral fluctuations. We reasoned that such slow fluctuations in behavior could either be due to slow changes in how the sensory stimulus is processed or due to a process that acts independently of sensory processing. By analyzing the recorded activity in conjunction with models of perceptual decision-making, we found evidence for the slow drift in neural activity acting as an impulsivity signal, overriding sensory evidence to dictate the final decision. Overall, this work uncovers an internal state embedded in the population activity across multiple brain areas, hidden from typical trial-averaged analyses and revealed only when considering the passage of time within each experimental session. Knowledge of this cognitive factor was critical in elucidating how sensory signals and the internal state together contribute to the decision-making process.
Machine learning classification can reduce false positives in structure-based virtual screening
Authors: Adeshina Yusuf , Deeds Eric , Karanicolas John
Published: January 2020

With the recent explosion in the size of libraries available for screening, virtual screening is positioned to assume a more prominent role in early drug discovery’s search for active chemical matter. Modern virtual screening methods are still, however, plagued with high false positive rates: typically, only about 12% of the top-scoring compounds actually show activity when tested in biochemical assays. We argue that most scoring functions used for this task have been developed with insufficient thoughtfulness into the datasets on which they are trained and tested, leading to overly simplistic models and/or overtraining. These problems are compounded in the literature because none of the studies reporting new scoring methods have validated their model prospectively within the same study. Here, we report a new strategy for building a training dataset (D-COID) that aims to generate highly-compelling decoy complexes that are individually matched to available active complexes. Using this dataset, we train a general-purpose classifier for virtual screening (vScreenML) that is built on the XGBoost framework of gradient-boosted decision trees. In retrospective benchmarks, our new classifier shows outstanding performance relative to other scoring functions. We additionally evaluate the classifier in a prospective context, by screening for new acetylcholinesterase inhibitors. Remarkably, we find that nearly all compounds selected by vScreenML show detectable activity at 50 µM, with 10 of 23 providing greater than 50% inhibition at this concentration. Without any medicinal chemistry optimization, the most potent hit from this initial screen has an IC50 of 280 nM, corresponding to a Ki value of 173 nM. These results support using the D-COID strategy for training classifiers in other computational biology tasks, and for vScreenML in virtual screening campaigns against other protein targets. Both D-COID and vScreenML are freely distributed to facilitate such efforts.
Reconstruction of motor control circuits in adultDrosophilausing automated transmission electron microscopy
Authors: Maniates-Selvin Jasper T. , Hildebrand David Grant Colburn , Graham Brett J. , Kuan Aaron T. , Thomas Logan A. , Nguyen Tri , Buhmann Julia , Azevedo Anthony W. , Shanny Brendan L. , Funke Jan , Tuthill John C. , Lee Wei-Chung Allen
Published: January 2020

Many animals use coordinated limb movements to interact with and navigate through the environment. To investigate circuit mechanisms underlying locomotor behavior, we used serial-section electron microscopy (EM) to map synaptic connectivity within a neuronal network that controls limb movements. We present a synapse-resolution EM dataset containing the ventral nerve cord (VNC) of an adult femaleDrosophila melanogaster. To generate this dataset, we developed GridTape, a technology that combines automated serial-section collection with automated high-throughput transmission EM. Using this dataset, we reconstructed 507 motor neurons, including all those that control the legs and wings. We show that a specific class of leg sensory neurons directly synapse onto the largest-caliber motor neuron axons on both sides of the body, representing a unique feedback pathway for fast limb control. We provide open access to the dataset and reconstructions registered to a standard atlas to permit matching of cells between EM and light microscopy data. We also provide GridTape instrumentation designs and software to make large-scale EM data acquisition more accessible and affordable to the scientific community.
Perfect mimicry betweenHeliconiusbutterflies is constrained by genetics and development
Authors: Van Belleghem Steven M. , Alicea Roman Paola A. , Gutierrez Heriberto Carbia , Counterman Brian A. , Papa Riccardo
Published: January 2020

Müllerian mimicry strongly exemplifies the power of natural selection. However, the exact measure of such adaptive phenotypic convergence and the possible causes of its imperfection often remain unidentified. The butterfly speciesHeliconius eratoandHeliconius melpomenehave a large diversity of co-mimicking geographic races with remarkable resemblance in melanic patterning across the mid-forewing that has been linked to expression patterns of the geneWntA. Recent CRISPR/Cas9 experiments have informed us on the exact areas of the wings in whichWntAaffects color pattern formation in bothH. eratoandH. melpomene, thus providing a unique comparative dataset to explore the functioning of a gene and its potential effect on phenotypic evolution. We therefore quantified wing color pattern differences in the mid-forewing region of 14 co-mimetic races ofH. eratoandH. melpomeneand measured the extent to which mimicking races are not perfectly identical. While the relative size of the mid-forewing pattern is generally nearly identical, our results highlight the areas of the wing that prevent these species from achieving perfect mimicry and demonstrate that this mismatch can be largely explained by constraints imposed by divergence in the gene regulatory network that define wing color patterning. Divergence in the developmental architecture of a trait can thus constrain morphological evolution even between relatively closely related species.
The novel reversible LSD1 inhibitor SP-2577 promotes anti-tumor immunity in SWItch/Sucrose-NonFermentable (SWI/SNF) complex mutated ovarian cancer
Authors: Soldi Raffaella , Halder Tithi Ghosh , Weston Alexis , Thode Trason , Drenner Kevin , Lewis Rhonda , Kaadige Mohan R. , Srivastava Shreyesi , Ampanattu Sherin Daniel , del Villar Ryan Rodriguez , Lang Jessica , Vankayalapati Hariprasad , Weissman Bernard , Trent Jeffrey M. , Hendricks William P.D. , Sharma Sunil
Published: January 2020

Chromatin remodeling SWItch/Sucrose-NonFermentable (SWI/SNF) complexes, initially identified in yeast 20 years ago, are evolutionarily conserved multi-subunit protein complexes that use the energy from hydrolysis of adenosine triphosphate (ATP) to remodel nucleosome structure and modulate transcription. Mutations in proteins of SWI/SNF complexes occur in 20% of human cancers including ovarian cancer (OC). Approximately 50% of ovarian clear cell carcinoma (OCCC) carries mutations in the SWI/SNF subunit ARID1A while small cell carcinoma of the ovary hypercalcemic type (SCCOHT) is driven primarily by genetic inactivation of the SWI/SNF ATPase SMARCA4 (BRG1) alongside epigenetic silencing of the homolog ATPase SMARCA2 (BRM). Dual loss of these ATPases disrupts SWI/SNF chromatin remodeling activity and may also interfere with the function of other histone-modifying enzymes that associate with or are dependent on SWI/SNF activity. One such enzyme is lysine-specific histone demethylase 1 (LSD1/KDM1A) which regulates the chromatin landscape and gene expression by demethylating proteins, including histone H3. LSD1 associates with epigenetic complexes such as the nucleosome remodeling deacetylase complex (NuRD) and SWI/SNF to inhibit the transcription of genes involved in tumor suppression and cell differentiation. TGCA analysis of human cancers shows that LSD1 is highly expressed in SWI/SNF-mutated tumors. Further, SCCOHT and OCCC cell lines show low nM IC50s for the reversible LSD1 inhibitor SP-2577 (Seclidemstat, currently in clinical phase I trials), supporting that these SWI/SNF-deficient ovarian cancers are dependent on LSD1 activity. Recently, it has been also shown that inhibition of LSD1 stimulates interferon (IFN)-dependent anti-tumor immunity through induction of Endogenous Retroviruses Elements (ERVs) and may thereby overcome resistance to checkpoint blockade. Additionally, SCCOHTs have been shown to exhibit an immune-active tumor microenvironment with PD-L1 expression in both tumor and stromal cells that strongly correlated with T cell infiltration. Thus, in this study we investigated the ability of SP-2577 to promote anti-tumor immunity and T cell infiltration in SWI/SNF-mutant SCCOHT and OCCC models. Our data shows that the reversible LSD1 inhibitor SP-2577 stimulates IFN-dependent anti-tumor immunity in SCCOHT cellsin vitroin a 3D immune-organoid platform. Additionally, SP-2577 promoted the expression of PD-L1 in both SCCOHT and OCCC models. Together our findings suggest that SP-2577 and checkpoint inhibitors as a therapeutic combination may induce or augment immunogenic responses in these tumors.
Ferrosomes are iron storage organelles formed by broadly conserved gene clusters in bacteria and archaea
Authors: Grant Carly R. , Komeili Arash
Published: January 2020

Cellular iron homeostasis is vital and maintained through tight regulation of iron import, efflux, storage, and detoxification1–3. The most common modes of iron storage employ proteinaceous compartments that are composed of ferritin or related proteins4,5. While lipid-bounded iron compartments have also been described, the basis for their formation and function remains unknown. Here, we focus on one such compartment, the ferrosome, which had been previously observed in the anaerobic bacteriumDesulfovibrio magneticus6. We identify three ferrosome-associated (Fez) proteins, encoded by a putative operon, that are associated with and responsible for forming ferrosomes inD. magneticus. Fez proteins include FezB, a P1B-6-ATPase found in phylogenetically and metabolically diverse species of bacteria and archaea with anaerobic lifestyles. In the majority of these species, two to ten genes define a cluster that encodes FezB. We show that two other species,Rhodopseudomonas palustrisandShewanella putrefaciens, make ferrosomes in anaerobic conditions through the action of their six-genefezoperon. Additionally, we find that theS. putrefaciens fezoperon is sufficient for ferrosome formation inEscherichia coli. UsingS. putrefaciensas a model, we find that ferrosomes likely play a role in the anaerobic adaptation to iron starvation. Overall, this work establishes ferrosomes as a new class of lipid-bounded iron storage organelles and sets the stage for studying ferrosome formation and structure in diverse microorganisms.
A 4-base pair core-enclosing helix in telomerase RNA is essential and binds to the TERT catalytic protein subunit
Authors: Mefford Melissa A. , Hass Evan P. , Zappulla David C.
Published: January 2020

The telomerase RNP counters the chromosome end-replication problem, completing genome replication to prevent cellular senescence in yeast, humans, and most other eukaryotes. The telomerase RNP core enzyme is composed of a dedicated RNA subunit and a reverse transcriptase (TERT). Although the majority of the 1157-ntSaccharomyces cerevisiaetelomerase RNA, TLC1, is rapidly evolving, the central catalytic core is largely conserved, containing the template, template-boundary helix, pseudoknot, and core-enclosing helix (CEH). Here, we show that 4-base pairs of core-enclosing helix is required for telomerase to be activein vitroand to maintain yeast telomeresin vivo, whereas ΔCEH, 1-bp, and 2-bp alleles do not support telomerase function. Using the CRISPR/dCas9-based “CARRY two-hybrid” assay to assess binding of our CEH mutant RNAs to TERT, we find that the 4-bp CEH RNA binds to TERT, but the shorter-CEH constructs do not, consistent with the telomerase activity andin vivocomplementation results. Thus, the CEH is essential in yeast telomerase RNA because it is needed to bind TERT to form the core RNP enzyme. Although the 8 nucleotides that form this 4-bp stem at the base of the CEH are nearly invariant amongSaccharomycesspecies, our results with sequence-randomized and truncated-CEH helices strongly suggest that this binding interaction with TERT is dictated more by secondary than primary structure. In summary, we have mapped an essential binding site in telomerase RNA for TERT that is crucial to form the catalytic core of this biomedically important RNP enzyme.
Expansion of Functional Regulatory T Cells Using Soluble RAGE Prevents Type 1 Diabetes
Authors: Leung Sherman S. , Borg Danielle J. , McCarthy Domenica A. , Boursalian Tamar E. , Cracraft Justen , Zhuang Aowen , Fotheringham Amelia K. , Flemming Nicole , Watkins Thomas , Miles John J. , Groop Per-Henrik , Scheijen Jean L. , Schalkwijk Casper G. , Steptoe Raymond J. , Radford Kristen J. , Knip Mikael , Forbes Josephine M.
Published: January 2020

Type 1 diabetes (T1D) is an autoimmune disease with no cure. Therapeutic translation has been hampered by preclinical reproducibility. Here, short-term administration of an antagonist to the receptor for advanced glycation end products (sRAGE) protected against murine diabetes at two independent centers. Treatment with sRAGE increased regulatory T cells (Tregs) within islets, pancreatic lymph nodes and spleen, increasing islet insulin expression and function. Diabetes protection was abrogated by Tregdepletion and shown to be dependent on antagonizing RAGE using knockout mice. Human Tregstreated with a RAGE ligand downregulated genes for suppression, migration and Treghomeostasis (FOXP3, IL7R, TIGIT, JAK1, STAT3, STAT5b, CCR4). Loss of suppressive function was reversed by sRAGE, where Tregsincreased proliferation and suppressed conventional T cell division, confirming that sRAGE expands functional human Tregs. These results highlight sRAGE as an attractive treatment to prevent diabetes, showing efficacy at multiple research centers and in human T cells.
The rifampicin-inactivating mono-ADP-ribosyl transferase ofMycobacterium smegmatissignificantly influences reactive oxygen species levels in the actively growing cells
Authors: Swaminath Sharmada , Pradhan Atul , Nair Rashmi Ravindran , Ajitkumar Parthasarathi
Published: January 2020

A classic example of antibiotic inactivating function in bacteria is theMycobacterium smegmatis(Msm) encoded rifampicin-inactivating mono-ADP-ribosyl transferase (arr). Since its probable biological role has been proposed to be in DNA damage response, which is inflicted by reactive oxygen species (ROS), in the present study, we examined whetherMsmArr influences ROS levels. For this purpose, the levels of the ROS, hydroxyl radical and superoxide, were determined in the mid-log phase (MLP) cells ofMsm arrknockout (arr-KO) strain, in comparison to those in the equivalently grownMsm arr+wild-type (WT) strain. The MLParr-KO cells generated significantly elevated levels of superoxide and hydroxyl radical, unlike the equivalently grown WT MLP cells. Complementation ofarr-KO witharr, but not with empty vector, restored the ROS levels comparable to those in the WT strain. Elevated ROS levels in thearr-KO strain enabled selection of rifampicin-resistant mutants at 10-7cfu/ml from the rifampicin-unexposed MLP cells ofarr-KO, which is one-log10higher than that for WT cells (10-8). Upon prolonged exposure to rifampicin, the susceptibility, persister formation, generation of elevated levels of hydroxyl radical by the persisters, rifampicin-resister generation frequency of the persisters and regrowth of the rifampicin-resistant mutants from the respective persisters were all comparable between thearr-KO and WT strains. These observations revealed that Arr influences ROS levels in the actively growingM. smegmatiscells but not in the rifampicin-exposed cells. We proposed the probable pathway through which Arr might be influencing ROS levels in the actively growingM. smegmatiscells.

IMPORTANCE

Diverse genera of bacteria consisting of pathogens, opportunistic pathogens and non-pathogens, possess Arr-type activities that confer equally efficient rifampicin resistance, thereby posing serious health hazard. Acquisition of this function by other bacteria through horizontal gene transfer enhances the hazard posed by the bacteria possessing it.M. smegmatisis an opportunistic human pathogen that causes infections of skin and soft tissues. Moreover,M. smegmatisis a genetically tractable model organism forM. tuberculosiswith the potential to function even as tuberculosis vaccine. In view of these significant aspects of Arr andM. smegmatis, the study to find out the natural physiological role of Arr inM. smegmatis, gains importance for designing strategies to prevent antibiotic inactivation and to target the cellular function to contain the bacterium. Above all, the three-dimensional structure ofM. smegmatisArr reveals significant structural homology with eukaryotic ADP-ribosyltransferases and bacterial toxins, thereby giving the study broad significance.
Visible Light Plasmon Excitation of Silver Nanoparticles Against Antibiotic-ResistantPseudomonas aeruginosa
Authors: da Silva Rafael T. P. , Petri Marcos V. , Valencia Estela Y. , Camargo Pedro H. C. , de Torresi Susana I. C. , Spira Beny
Published: January 2020

The interaction of metallic nanoparticles with light excites a local surface plasmon resonance (LSPR). This phenomenon enables the transfer of hot electrons to substrates that release Reactive Oxygen Species (ROS). In this context, the present study was aimed at enhancing the antibacterial effect of citrate-covered silver nanoparticles (AgNPs), which already possess excellent antimicrobial properties, via LSPR excitation with visible LED againstPseudomonas aeruginosa, one of the most refractory organisms to antibiotic treatment. The Minimum Inhibitory Concentration (MIC) of AgNPs was 10 μg/ml under dark conditions and 5 μg/ml under light conditions. The combination of light and AgNPs led to 100% cell death after 60 minutes. Quantification of ROS via flow cytometry showed that LSPR stimulated AgNPs increased intracellular ROS concentration by 4.8-fold, suggesting that light-exposed AgNPs caused cell death via ROS production. Light exposition caused a small release of silver ions (0.4%) reaching a maximum after 6 hours. This indicates that silver ions play at most a secondary role inP. aeruginosadeath. Overall, the results presented here show that LSPR generation from AgNPs by visible light enhances the antimicrobial activity of silver nanoparticles and can be an alternative for the treatment of topic infections caused by antibiotic-resistant bacteria such asP. aeruginosa.