bluuebunny/crossref_metadata_2025_split huggingface dataset:
CRISPR-Cas9 Screen Reveals PSMB3 Contributes to Gliomagenesis Through Proteasome-Dependent and Independent Mechanisms
Authors: Baisiwala Shivani , Budhiraja Shreya , Zolp Andrew J , Nandoliya Khizar , Chen Li , Park Cheol H. , Perrault Ella N , Saathoff Miranda R , Dmello Crismita , Shireman Jack M , Lin Peiyu , Dara Gabriel , McCortney Katy , Horbinski Craig , Sonabend Adam M , Ahmed Atique U 
Published: July 2021

AbstractGlioblastoma (GBM) is the most common adult malignant brain tumor, with a median survival of 21 months and a 100% recurrence rate. Even though many of the critical oncogenic drivers for GBM have been identified, the basis of gliomagenesis is still under investigation. To identify novel genes that contribute to GBM progression, we performed a genome-wide CRISPR-Cas9 knockout screen. We identified four previously unstudied genes – PSMB3, CHCHD4, SPDYE5, HSPA1 – which had elevated expression in cancer and demonstrated a significant positive correlation with respect to GBM growth and patient survival in vivo and patient datasets. Furthermore, overexpression of PSMB3 and HSPA5 in neural stem cells resulted in transformation to a cancer phenotype. Further investigation of PSMB3, a subunit of the proteasome, allowed us to identify both ubiquitin-mediated and non-ubiquitin-mediated mechanisms of oncogenesis. Ultimately, the data from our CRISPR screens suggests that these genes drive tumor progression, making them promising therapeutic targets for GBM.
Killer Knots: Molecular evolution of Inhibitor Cystine Knot toxins in wandering spiders (Araneae: Ctenidae)
Authors: Cole T. Jeffrey , Brewer Michael S. 
Published: July 2021

AbstractVenom expressed by the nearly 50,000 species of spiders on Earth largely remains an untapped reservoir of a diverse array of biomolecules with potential for pharmacological and agricultural applications. A large fraction of the noxious components of spider venoms are a functionally diverse family of structurally related polypeptides with an inhibitor cystine knot (ICK) motif. The cysteine-rich nature of these toxins makes structural elucidation difficult, and most studies have focused on venom components from the small handful of medically relevant spider species such as the highly aggressive Brazilian wandering spider Phoneutria nigriventer. To alleviate difficulties associated with the study of ICK toxins in spiders, we devised a comprehensive approach to explore the evolutionary patterns that have shaped ICK functional diversification using venom gland transcriptomes and proteomes from phylogenetically distinct lineages of wandering spiders and their close relatives. We identified 626 unique ICK toxins belonging to seven topological elaborations. Phylogenetic tests of episodic diversification revealed distinct regions between cysteine residues that demonstrated differential evidence of positive or negative selection, which may have structural implications towards the specificity and efficacy of these toxins. Increased taxon sampling and whole genome sequencing will provide invaluable insights to further understand the evolutionary processes that have given rise to this diverse class of toxins.
Functional Inference of Gene Regulation using Single-Cell Multi-Omics
Authors: Kartha Vinay K. , Duarte Fabiana M. , Hu Yan , Ma Sai , Chew Jennifer G. , Lareau Caleb A. , Earl Andrew , Burkett Zach D. , Kohlway Andrew S. , Lebofsky Ronald , Buenrostro Jason D. 
Published: July 2021

AbstractCells require coordinated control over gene expression when responding to environmental stimuli. Here, we apply scATAC-seq and scRNA-seq in resting and stimulated human blood cells. Collectively, we generate ∼91,000 single-cell profiles, allowing us to probe the cis-regulatory landscape of immunological response across cell types, stimuli and time. Advancing tools to integrate multi-omic data, we develop FigR - a framework to computationally pair scATAC-seq with scRNA-seq cells, connect distal cis-regulatory elements to genes, and infer gene regulatory networks (GRNs) to identify candidate TF regulators. Utilizing these paired multi-omic data, we define Domains of Regulatory Chromatin (DORCs) of immune stimulation and find that cells alter chromatin accessibility prior to production of gene expression at time scales of minutes. Further, the construction of the stimulation GRN elucidates TF activity at disease-associated DORCs. Overall, FigR enables the elucidation of regulatory interactions across single-cell data, providing new opportunities to understand the function of cells within tissues.
Single cell analysis of endometriosis reveals a coordinated transcriptional program driving immunotolerance and angiogenesis across eutopic and ectopic tissues
Authors: Tan Yuliana , Flynn William F. , Sivajothi Santhosh , Luo Diane , Bozal Suleyman B. , Luciano Anthony A. , Robson Paul , Luciano Danielle E. , Courtois Elise T. 
Published: July 2021

AbstractEndometriosis is characterized by growth of endometrial-like tissue outside of the uterus affecting many women in their reproductive age, causing years of pelvic pain and potential infertility. Its pathophysiology remains largely unknown, limiting diagnosis and treatment. We characterized peritoneal and ovarian lesions at single-cell transcriptome resolution and compared to matched eutopic endometrium, control endometrium, and organoids derived from these tissues, generating data on over 100,000 cells across 12 individuals. We spatially localized many of the cell types using imaging mass cytometry. We identify a perivascular mural cell unique to the peritoneal lesions with dual roles in angiogenesis promotion and immune cell trafficking. We define an immunotolerant peritoneal niche, fundamental differences in eutopic endometrium and between lesions microenvironments, and a novel progenitor-like epithelial cell subpopulation. Altogether, this study provides a holistic view of the endometriosis microenvironment representing the first comprehensive cell atlas of the disease, essential information for advancing therapeutics and diagnostics.
The epithelial-specific ER stress sensor IRE1β enables host-microbiota crosstalk to affect colon goblet cell development
Authors: Grey Michael J. , Luca Heidi De , Ward Doyle V. , Kreulen Irini A. M. , Foley Sage E. , Thiagarajah Jay R. , McCormick Beth A. , Turner Jerrold R. , Lencer Wayne I. 
Published: July 2021

ABSTRACTEpithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express IRE1β (Ern2), a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor IRE1α. How IRE1β functions at the host-environment interface and why a second IRE1 paralogue evolved remain incompletely understood. Using conventionally raised and germ-freeErn2-/-mice, we found that IRE1β was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced IRE1β expression. IRE1β acted by splicingXbp1mRNA to expand ER function and prevent ER stress in goblet cells. Although IRE1α can also spliceXbp1mRNA, it did not act redundantly to IRE1β in this context. By regulating assembly of the colon mucus layer, IRE1β further shaped the composition of the gut microbiota. Mice lacking IRE1β had a dysbiotic microbial community that failed to induce goblet cell development when transferred into germ-free wild type mice. These results show that IRE1β evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.
Patient-derived gene and protein expression signatures of NGLY1 deficiency
Authors: Rauscher Benedikt , Mueller William F. , Clauder-Münster Sandra , Jakob Petra , Saiful Islam M. , Sun Han , Ghidelli-Disse Sonja , Boesche Markus , Bantscheff Marcus , Pflaumer Hannah , Collier Paul , Haase Bettina , Chen Songjie , Wang Guangwen , Benes Vladimir , Snyder Michael , Drewes Gerard , Steinmetz Lars M. 
Published: July 2021

ABSTRACTN-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing 4 different cell types derived from 14 NGLY1 deficient patients and 17 controls. While gene and protein expression levels agreed well with each other, expression differences were more pronounced at the protein level. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared to parent controls, depending on the genotype, NGLY1 protein was still detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosomal mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cystein ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.
Transparency in peer review: Exploring the content and tone of reviewers’ confidential comments to editors
Authors: O’Brien Bridget C. , Artino Anthony R. , Costello Joseph A. , Driessen Erik , Maggio Lauren A. 
Published: July 2021

AbstractPurposeRecent calls to improve transparency in peer review have prompted examination of many aspects of the peer review process. Peer review systems often allow confidential comments to editors that could reduce transparency to authors, yet this option has escaped scrutiny. Our study explores 1) how reviewers use the confidential comments section and 2) alignment between comments to the editor and comments to authors with respect to content and tone.MethodsOur dataset included 358 reviews of 168 manuscripts submitted between January 1, 2019 and August 24, 2020 to a health professions education journal with a single blind review process. We first identified reviews containing comments to the editor. Then, for the reviews with comments, we used procedures consistent with conventional and directed qualitative content analysis to develop a coding scheme and code comments for content, tone, and section of the manuscript. For reviews in which the reviewer recommended “reject,” we coded for alignment between reviewers’ comments to the editor and to authors. We report descriptive statistics.Results49% of reviews contained comments to the editor (n=176). Most of these comments summarized the reviewers’ impression of the article (85%), which included explicit reference to their recommended decision (44%) and suitability for the journal (10%). The majority of comments addressed argument quality (56%) or research design/methods/data (51%). The tone of comments tended to be critical (40%) or constructive (34%). For the 48 reviews recommending “reject,” the majority of comments to the editor contained content that also appeared in comments to the authors (65%); additional content tended to be irrelevant to the manuscript. Tone frequently aligned (85%).ConclusionFindings indicate variability in how reviewers use the confidential comments to editor section in online peer review systems, though generally the way they use them suggests integrity and transparency to authors.
Developmentally-regulated impairment of parvalbumin interneuron synaptic transmission in an experimental model of Dravet syndrome
Authors: Kaneko Keisuke , Currin Christopher B. , Goff Kevin M. , Somarowthu Ala , Vogels Tim P. , Goldberg Ethan M. 
Published: July 2021

ABSTRACTDravet syndrome (DS) is a neurodevelopmental disorder defined by epilepsy, intellectual disability, and sudden death, due to heterozygous variants in SCN1A with loss of function of the sodium channel subunit Nav1.1. Nav1.1-expressing parvalbumin GABAergic interneurons (PV-INs) from pre-weanling Scn1a+/− mice show impaired action potential generation. A novel approach assessing PV-IN function in the same mice at two developmental time points showed that, at post-natal day (P) 16-21, spike generation was impaired all mice, deceased prior or surviving to P35. However, synaptic transmission was selectively dysfunctional in pre-weanling mice that did not survive. Spike generation in surviving mice normalized by P35, yet we again identified abnormalities in synaptic transmission. We conclude that combined dysfunction of PV-IN spike generation and synaptic transmission drives disease severity, while ongoing dysfunction of synaptic transmission contributes to chronic pathology. Modeling revealed that PV-IN axonal propagation is more sensitive to decreases in sodium conductance than spike generation.
Predictive modeling reveals that higher-order cooperativity drives transcriptional repression in a synthetic developmental enhancer
Authors: Kim Yang Joon , Rhee Kaitlin , Liu Jonathan , Jeammet Paul , Turner Meghan , Small Stephen , Garcia Hernan G. 
Published: July 2021

AbstractA challenge in quantitative biology is to predict output patterns of gene expression from knowledge of input transcription factor patterns and from the arrangement of binding sites for these transcription factors on regulatory DNA. We tested whether widespread thermodynamic models could be used to infer parameters describing simple regulatory architectures that inform parameter-free predictions of more complex enhancers in the context of transcriptional repression by Runt in the early fruit 2y embryo. By modulating the number and placement of Runt binding sites within an enhancer, and quantifying the resulting transcriptional activity using live imaging, we discovered that thermodynamic models call for higher-order cooperativity between multiple molecular players. This higher-order cooperativity capture the combinatorial complexity underlying eukaryotic transcriptional regulation and cannot be determined from simpler regulatory architectures, highlighting the challenges in reaching a predictive understanding of transcriptional regulation in eukaryotes and calling for approaches that quantitatively dissect their molecular nature.
Ethanol pre-exposure enhances alcohol-seeking behavior at cellular level by chemoattraction and exhibits bleb-driven cellular stress response in uniform ethanol concentration
Authors: Kar Neelakshi , Bellare Jayesh 
Published: July 2021

AbstractThis study investigates the effect of ethanol and its pre-exposure on cell migration. Here, fibroblast cells were first pre-treated with ethanol, and their migratory behavior was tested in both chemotaxis and chemokinesis setup. 1% ethanol was taken as a potential chemotactic agent. The study reveals that in presence of ethanol gradient cells display migration towards ethanol, and pre-exposure further augments this migratory behavior by altering their chemotactic responsiveness. In uniform ethanol concentration, cells first undergo three staged adaptations to the new environment: shrinking, blebbing, and recovery, where cells use bleb-driven cell protection machinery to adapt. Thus, migration is initially stalled. But once the cells resume locomotion, no significant difference in migratory parameters is observed. Overall, this study establishes ethanol as a chemoattractant for fibroblasts, with cells showing enhanced alcohol-seeking behavior upon pre-exposure. Such behavior is reminiscent of seeking and tolerance exhibited by alcohol-dependent addictive behavior in higher organisms including humans.
4D live-cell imaging of microgametogenesis in the human malaria parasite Plasmodium falciparum
Authors: Yahiya Sabrina , Jordan Sarah , Smith Holly X. , Gaboriau David C. A. , Famodimu Mufuliat T. , Dahalan Farah A. , Churchyard Alisje , Ashdown George W. , Baum Jake 
Published: July 2021

ABSTRACTFormation of gametes in the malaria parasite occurs in the midgut of the mosquito and is critical to onward parasite transmission. Transformation of the male gametocyte into microgametes, called microgametogenesis, is an explosive cellular event and one of the fastest eukaryotic DNA replication events known. The transformation of one microgametocyte into eight flagellated microgametes requires reorganisation of the parasite cytoskeleton, replication of the 22.9 Mb genome, axoneme formation and host erythrocyte egress, all of which occur simultaneously in <20 minutes. Whilst high-resolution imaging has been a powerful tool for defining stages of microgametogenesis, it has largely been limited to fixed parasite samples, given the speed of the process and parasite photosensitivity. Here, we have developed a live-cell fluorescence imaging workflow that captures the explosive dynamics of microgametogenesis in full. Using the most virulent human malaria parasite, Plasmodium falciparum, our live-cell approach combines three-dimensional imaging through time (4D imaging) and covers early microgametocyte development through to microgamete release. Combining live-cell stains for DNA, tubulin and the host erythrocyte membrane, 4D imaging enables definition of the positioning of newly replicated and segregated DNA. It also shows the microtubular cytoskeleton, location of newly formed basal bodies and elongation of axonemes, as well as behaviour of the erythrocyte membrane, including its specific perforation prior to microgamete egress. 4D imaging was additionally undertaken in the presence of known transmission-blocking inhibitors and the untested proteasomal inhibitor bortezomib. Here, for the first time we find that bortezomib inhibition results in a clear block of DNA replication, full axoneme nucleation and elongation. These data not only define a framework for understanding microgametogenesis in general but also suggest that the process is critically dependent on proteasomal activity, helping to identify potentially novel targets for transmission-blocking antimalarial drug development.
An open state of a voltage-gated sodium channel involving a π-helix and conserved pore-facing Asparagine
Authors: Choudhury Koushik , Kasimova Marina A. , McComas Sarah , Howard Rebecca J , Delemotte Lucie 
Published: July 2021

AbstractVoltage-gated sodium (Nav) channels play critical roles in propagating action potentials and otherwise manipulating ionic gradients in excitable cells. These channels open in response to membrane depolarization, selectively permeating sodium ions until rapidly inactivating. Structural characterization of the gating cycle in this channel family has proved challenging, particularly due to the transient nature of the open state. A structure from the bacterium Magnetococcus marinus Nav (NavMs) was initially proposed to be open, based on its pore diameter and voltage-sensor conformation. However, the functional annotation of this model, and the structural details of the open state, remain disputed. In this work, we used molecular modeling and simulations to test possible open-state models of NavMs. The full-length experimental structure, termed here the α-model, was consistently dehydrated at the activation gate, indicating an inability to conduct ions. Based on a spontaneous transition observed in extended simulations, and sequence/structure comparison to other Nav channels, we built an alternative π-model featuring a helix transition and the rotation of a conserved asparagine residue into the activation gate. Pore hydration, ion permeation and state-dependent drug binding in this model were consistent with an open functional state. This work thus offers both a functional annotation of the full-length NavMS structure, and a detailed model for a stable Nav open state, with potential conservation in diverse ion-channel families.
Retinal vibrations in bacteriorhodopsin are mechanically harmonic but electronically anharmonic: evidence from overtone and combination bands
Authors: Lorenz-Fonfria Victor A. , Yagi Kiyoshi , Ito Shota , Kandori Hideki 
Published: July 2021

AbstractVibrations of the chromophore in the membrane protein bacteriorhodopsin (BR), a protonated Schiff base retinal, have been studied for decades, both by resonance Raman and by infrared (IR) difference spectroscopy. In spite the light-induced IR difference spectrum between the K intermediate (13-cis retinal) and the initial BR state (all-trans retinal) being first published almost 40 years ago, we present here unreported bands in the 2500 to 1800 cm−1 region. We show that the bands between 2500 and 2300 cm−1 originate from overtone and combination transitions of retinal C-C stretches. We assigned some of the newly reported bands below 2300 cm−1 to the combination of retinal C-C stretches with methyl rocks and with hydrogen-out-of-plane vibrations. Remarkably, experimental C-C overtone bands appeared at roughly twice the wavenumber of their fundamentals, with anharmonic mechanical constants ≤ 3.5 cm−1, and in some cases of ∼1 cm−1. Comparison of combination and fundamental bands indicates that most of the mechanical coupling constants are also very small. Despite the mechanical quasi-harmonicity of the C-C stretches, the area of their overtone bands was only ∼50 to ∼100 times smaller than of their fundamental bands. We concluded that electronic anharmonicity, the second mechanism giving intensity to overtone bands, must be particularly high for the retinal C-C stretches. We corroborated the assignments of negative bands in the K-BR difference spectrum by ab initio anharmonic spectral calculations of all-trans retinal in BR, which also reproduced reasonably well the small experimental anharmonic and coupling mechanical constants. Yet, and in spite accounting for both mechanical and electronic anharmonicities, the intensity of overtone C-C transitions was underestimated by a factor of 4 to 20, indicating room for improvement in state-of-the-art anharmonic vibrational calculations.
Insights from spatial measures of intolerance to identify pathogenic variants in developmental and epileptic encephalopathies
Authors: Silk M , De Sá A , Olshansky M , Ascher D B 
Published: July 2021

AbstractDevelopmental and epileptic encephalopathies (DEEs) are a group of epilepsies with early onset and severe symptoms that sometimes lead to death. While a number of genes have been successfully implicated, it remains challenging to identify causative mutations within these genes from the background variation present in all individuals due to disease heterogeneity. Our ability to detect likely pathogenic variants has continued to improve as in silico predictors of deleteriousness have advanced. We investigate their use in prioritising likely pathogenic variants in epileptic encephalopathy patient whole exome sequences and show that the inclusion of structure-based predictors of intolerance improve upon previous attempts to demonstrate enrichment within epilepsy genes.