bluuebunny/crossref_metadata_2025_split huggingface dataset:
Myosin VI regulates ciliogenesis by promoting the turnover of the centrosomal/satellite protein OFD1
Authors: Magistrati Elisa , Maestrini Giorgia , Lince-Faria Mariana , Beznoussenko Galina , Mironov Alexandre , Maspero Elena , Bettencourt-Dias Mónica , Polo Simona 
Published: June 2021

AbstractThe actin motor protein myosin VI is a multivalent protein with diverse functions. Here, we identified and characterised a myosin VI ubiquitous interactor, the oral-facial-digital syndrome 1 (OFD1) protein, whose mutations cause malformations of the face, oral cavity, digits, and polycystic kidney disease. We found that myosin VI regulates the localisation of OFD1 at the centrioles and, as a consequence, the recruitment of the distal appendage protein cep164. Myosin VI depletion in non-tumoural cell lines causes an aberrant localisation of OFD1 along the centriolar walls, which is due to a reduction in the OFD1 mobile fraction. Finally, loss of myosin VI triggers a severe defect in ciliogenesis that could be causally linked to an impairment in the autophagic removal of OFD1 from satellites. Altogether, our results highlight an unprecedent layer of regulation of OFD1 and a pivotal role of myosin VI in coordinating the formation of the distal appendages and primary cilium with important implications for the genetic disorders known as ciliopathies.
The Caenorhabditis elegans TDRD5/7-like protein, LOTR-1, interacts with the helicase ZNFX-1 to balance epigenetic signals in the germline
Authors: Marnik Elisabeth A. , Almeida Miguel V. , Cipriani P. Giselle , Chung George , Caspani Edoardo , Karaulanov Emil , Butter Falk , Sharp Catherine S. , Zinno John , Gan Hin Hark , Piano Fabio , Ketting René F , Gunsalus Kristin C. , Updike Dustin L. 
Published: June 2021

AbstractLOTUS and Tudor domain containing proteins have critical roles in the germline. Proteins that contain these domains, such as Tejas/Tapas in Drosophila, help localize Vasa to the germ granules and facilitate piRNA-mediated transposon silencing. The homologous proteins in mammals, TDRD5 and TDRD7, are required during spermiogenesis. Until now, proteins containing both LOTUS and Tudor domains in Caenorhabditis elegans have remained elusive. Here we describe LOTR-1 (D1081.7), which derives its name from its LOTUS and Tudor domains. Interestingly, LOTR-1 docks next to P granules to colocalize with the broadly conserved Z-granule helicase, ZNFX-1. LOTR-1’s Z-granule association requires its Tudor domain, but both LOTUS and Tudor deletions affect brood size when coupled with a knockdown of the Vasa homolog glh-1. In addition to interacting with the germ-granule components WAGO-1, PRG-1 and DEPS-1, we identified a Tudor-dependent association with ZNFX-1. Like znfx-1 mutants, lotr-1 mutants lose small RNAs from the 3’ ends of WAGO and Mutator targets, reminiscent of the loss of piRNAs from the 3’ ends of piRNA precursor transcripts in mouse Tdrd5 mutants. Our work suggests that LOTR-1 acts in a conserved mechanism that brings small RNA generating mechanisms towards the 3’ ends of small RNA templates or precursors.
Influence of viral transport media and freeze-thaw cycling on the sensitivity of qRT-PCR detection of SARS-CoV-2 nucleic acids
Authors: Holohan Cian , Hanarahan Sophia , Feely Nathan , Li Peng , O’Connell John , Moss Catherine , Carr Michael , Tagit Oya , Lee Gil U 
Published: June 2021

AbstractThe events of the last year have highlighted the complexity of implementing large-scale molecular diagnostic testing for novel pathogens. The purpose of this study was to determine the chemical influences of sample collection media and storage on the stability and detection of viral nucleic acids by qRT-PCR. We studied the mechanism(s) through which viral transport media (VTM) and number of freeze-thaw cycles influenced the analytical sensitivity of qRT-PCR detection of SARS-CoV-2. Our goal is to reinforce testing capabilities and identify weaknesses that could arise in resource-limited environments that do not have well-controlled cold chains. The sensitivity of qRT-PCR analysis was studied in four VTM for synthetic single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) simulants of the SARS-CoV-2 genome. The sensitivity and reproducibility of qRT-PCR for the synthetic ssRNA and dsDNA were found to be highly sensitive to VTM with the best results observed for ssRNA in HBSS and PBS-G. Surprisingly, the presence of epithelial cellular material with the ssRNA increased the sensitivity of the qRT-PCR assay. Repeated freeze-thaw cycling decreased the sensitivity of the qRT-PCR with two noted exceptions. The choice of VTM is critically important to defining the sensitivity of COVID-19 molecular diagnostics assays and this study suggests they can impact upon the stability of the SARS-CoV-2 viral genome. This becomes increasingly important if the virus structure is destabilised before analysis, which can occur due to poor storage conditions. This study suggests that COVID-19 testing performed with glycerol-containing PBS will produce a high level of stability and sensitivity. These results are in agreement with clinical studies reported for patient-derived samples.
GRAND: A database of gene regulatory network models across human conditions
Authors: Guebila Marouen Ben , Lopes-Ramos Camila M , Weighill Deborah , Sonawane Abhijeet Rajendra , Burkholz Rebekka , Shamsaei Behrouz , Platig John , Glass Kimberly , Kuijjer Marieke L , Quackenbush John 
Published: June 2021

AbstractGene regulation plays a fundamental role in shaping tissue identity, function, and response to perturbation. Regulatory processes are controlled by complex networks of interacting elements, including transcription factors, miRNAs and their target genes. The structure of these networks helps to determine phenotypes and can ultimately influence the development of disease or response to therapy. We developed GRAND (https://grand.networkmedicine.org) as a database for gene regulatory network models that can be compared between biological states, or used to predict which drugs produce changes in regulatory network structure. The database includes 12,468 genome-scale networks covering 36 human tissues, 28 cancers, 1,378 unperturbed cell lines, as well as 173,013 TF and gene targeting scores for 2,858 small molecule-induced cell line perturbation paired with phenotypic information. GRAND allows the networks to be queried using phenotypic information and visualized using a variety of interactive tools. In addition, it includes a web application that matches disease states to potentially therapeutic small molecule drugs using regulatory network properties.Graphical abstractModeling gene regulation across human conditions integrates cancer tissues and cell lines, small molecules, and normal tissue networks.
Extensive protein dosage compensation in aneuploid human cancers
Authors: Schukken Klaske M. , Sheltzer Jason M. 
Published: June 2021

AbstractAneuploidy is a hallmark of human cancers, but the effects of aneuploidy on protein expression remain poorly understood. To uncover how chromosome copy number changes influence the cancer proteome, we have conducted an analysis of hundreds of human cancer cell lines with matched copy number, RNA expression, and protein expression data. We found that a majority of proteins exhibit dosage compensation and fail to change by the degree expected based on chromosome copy number alone. We uncovered a variety of gene groups that were recurrently buffered upon both chromosome gain and loss, including protein complex subunits and cell cycle genes. Several genetic and biophysical factors were predictive of protein buffering, highlighting complex post-translational regulatory mechanisms that maintain appropriate gene product dosage. Finally, we established that chromosomal aneuploidy has an unexpectedly moderate effect on the expression of oncogenes and tumor suppressors, demonstrating that these key cancer drivers can be subject to dosage compensation as well. In total, our comprehensive analysis of aneuploidy and dosage compensation across cancers will help identify the key driver genes encoded on altered chromosomes and will shed light on the overall consequences of aneuploidy during tumor development.
Functional characterization of odor responses and gene expression changes in olfactory co-receptor mutants inDrosophila
Authors: Vulpe Alina , Mohapatra Pratyajit , Menuz Karen 
Published: June 2021

AbstractTwo large families of olfactory receptors, the Odorant Receptors (ORs) and the Ionotropic Receptors (IRs), mediate responses to most odors in the insect olfactory system. Individual odor binding “tuning” OR receptors are expressed by olfactory neurons in basiconic and trichoid sensilla and require the co-receptor Orco to function. The situation for IRs is more complex. Different tuning IR receptors are expressed by olfactory neurons in coeloconic sensilla and rely on either the Ir25a or Ir8a co-receptors; some evidence suggests that Ir76b may also act as a coreceptor, but its function has not been systematically examined. This is particularly important as recent data indicate that nearly all coeloconic olfactory neurons co-express Ir25a, Ir8a, and Ir76b. Here, we report the effects ofDrosophilaolfactory co-receptor mutants on odor detection by coeloconic olfactory neurons and determine their broader impact on gene expression through RNASeq analysis. We demonstrate that Ir76b and Ir25a function together in all amine-sensing olfactory receptor neurons. In most neurons, loss of either co-receptor abolishes amine responses, whereas in ac1 sensilla, amine responses persist in the absence of Ir76b or Ir25a, but are lost in a double-mutant. Such responses do not require Ir8a. Conversely, acid-sensing ORNs require Ir8a, but not Ir76b or Ir25a. Using antennal transcriptional profiling, we find that the expression of acid-sensing IR receptors is significantly reduced inIr8amutants, but is unaffected by the loss ofIr25aorIr76b.Similarly, select OR tuning receptors are also downregulated inOrco2mutants. In contrast, expression of amine-sensing IR receptors is mostly unchanged inIr25aandIr76bmutants. Together, our data reveal new aspects of co-receptor function in the olfactory system.SummaryInsect vectors of human disease rely on their sense of smell to locate humans for blood meals. A critical first step in olfaction is the odorant-induced activation of receptors on olfactory neurons. There are two major olfactory receptor families in insects, with each species having dozens of different odorant-binding “tuning” receptors. The receptor complexes also contain non-tuning co-receptors, which are highly conserved across insect species and are required for function. Here we characterize co-receptor mutants with electrophysiological recordings and transcriptome analysis inDrosophila. Our findings resolve the differential co-receptor dependence of olfactory neuron responses to volatile amines and acids. We also report changes in antennal gene expression that result from the absence of these co-receptors. Most notably, the absence of some co-receptors leads to a selective loss of transcript expression for the tuning olfactory receptors whose function depends on the missing co-receptors. Together our data provide new insight into the roles of co-receptors in insect olfaction.
Single nucleus pituitary transcriptomic and epigenetic landscape reveals human stem cell heterogeneity with diverse regulatory mechanisms
Authors: Zhang Zidong , Zamojski Michel , Smith Gregory R. , Willis Thea L. , Yianni Val , Mendelev Natalia , Pincas Hanna , Seenarine Nitish , Amper Mary Anne S. , Vasoya Mital , Nair Venugopalan D. , Turgeon Judith L. , Bernard Daniel J. , Troyanskaya Olga G. , Andoniadou Cynthia L. , Sealfon Stuart C. , Ruf-Zamojski Frederique 
Published: June 2021

AbstractDespite their importance in tissue homeostasis and renewal, human pituitary stem cells (PSCs) are incompletely characterized. We describe a human single nucleus (sn) RNAseq and ATACseq resource from pediatric, adult, and aged pituitaries (snpituitaryatlas.princeton.edu) and characterize cell type-specific gene expression and chromatin accessibility programs for all major pituitary cell lineages. We identify uncommitted PSCs, committing progenitor cells, and sex differences. Pseudotime trajectory analysis indicates that early life PSCs are distinct from the other age groups. Linear modeling of same-cell multiome data identifies regulatory domain accessibility sites and transcription factors (TFs) that are significantly associated with gene expression in PSCs compared to other cell types and within PSCs. Modeling the heterogeneous expression of two markers for committing cell lineages among PSCs shows significant correlation with regulatory domain accessibility forGATA3, but with TF expression forPOMC. These findings characterize human stem cell lineages and reveal diverse mechanisms regulating key PSC genes.
2.5D Tractions in monocytes reveal mesoscale mechanics of podosomes during substrate indenting cell protrusion
Authors: Schürmann H , Russo A , Hofemeier AD , Brandt M , Roth J , Vogl T , Betz T 
Published: June 2021

AbstractDegradation and protrusion are key to cellular barrier breaching in cancer metastasis and leukocyte extravasation. Cancerous invadopodia and myelomonocytic podosomes are widely considered as structural tools facilitating these processes and are thus summarized under the term invadosomes. Despite similar behaviour on the individual scale, substantial differences have been reported to arise on the collective scale. They are considered to be a result of podosome mesoscale-connectivity. In this study, we investigated global in-plane and out-of-plane mechanical forces of podosome clusters in ER-Hoxb8 cell derived monocytes. We are able to correlate these forces with the interpodosomal connectivity. The observed traction and protrusion patterns fail to be explained by summation of single podosome mechanics. Instead, they appear to originate from superimposed mesoscale effects. Based on mechanistic and morphological similarities with epithelial monolayer mechanics, we propose a spatiotemporal model of podosome cluster mechanics capable of relating single to collective podosome mechanical behaviour. Our results suggest that network contraction-driven (in-plane) tractions lead to a buckling instability that contributes to the out-of-plane indentation into the substrate. First assigning an active mechanical role to the dorsal podosome actomyosin network, we aim at translating actomyosin hierarchy into scale dependency of podosome mechanics.
Endothelial Resolution of Inflammation is Delayed Following JAK-driven but not NFκB-dependent Activation
Authors: Valenzuela Nicole M 
Published: June 2021

AbstractBlood endothelial cells actively regulate egress of leukocytes into peripheral tissues in response to inflammatory insult. The resolution of inflammation is critical for healing and return to homeostasis, but the timing and mechanisms involved in return to a non-inflamed state are not well-understood. We examined vascular endothelial activation comparing NFκB-driven TNFα and JAK/STAT-mediated IFNγ. Pro-adhesive gene expression, phenotype and secretome of human endothelial cells from 6 vascular beds were measured under chronic cytokine stimulation, and after short-term cytokine priming followed by withdrawal. The majority of inducible TNFα effectors require continuous exposure for reinforcement of the altered phenotype. NFκB and target genes are quickly down-regulated in the absence of cytokine. In contrast, the consequences of even short exposure to IFNγ are long-lasting and broad, with sustained elevation of adhesion molecules and chemokines up to 48hr later. JAK/STAT and interferon response factor expression are likewise durable, dependent on new transcription and autonomous of continuous IFNγ. Finally, intact persistent STAT expression and JAK signaling in the endothelium is required to maintain a pro-adhesive phenotype after IFNγ withdrawal, which could be prevented by the JAK1/2 inhibitor ruxolitinib. Our results reveal a sustained JAK-dependent perturbation of endothelial function after exposure to IFNγ, but not after NFκB-driven inflammation.
Preferential lipolysis of DGAT1 over DGAT2 generated triacylglycerol in Huh7 hepatocytes
Authors: Selvaraj Rajakumar , Zehnder Sarah V. , Watts Russell , Lian Jihong , Nelson Randal , Lehner Richard 
Published: June 2021

AbstractHepatic steatosis is defined by accumulation of neutral lipids in lipid droplets (LDs) including triacylglycerol (TG) and steryl esters. Two distinct diacylglycerol acyltransferases (DGAT1 and DGAT2) catalyze synthesis of TG in hepatocytes. TG formed through either DGAT1 or DGAT2 appears to be preferentially directed to distinct intercellular fates, such as fatty acid production for oxidation or very-low density lipoprotein assembly, respectively. Because of the preferential use of TG generated by DGAT1 and DGAT2, we hypothesized that targeting/association of lipolytic machinery to LDs would differ depending on whether the TG stores were generated through DGAT1 or DGAT2 activities. Inhibition of DGAT1 or DGAT2 in human hepatoma cells (Huh7) incubated with oleic acid resulted in only a small change in TG accretion suggesting that the two DGATs can compensate for each other in fatty acid esterification. This compensation was not accompanied by changes in DGAT1 or DGAT2 mRNA expression. DGAT1 inhibition (TG synthesized by DGAT2) resulted in large LDs, whereas DGAT2 inhibition (TG synthesized by DGAT1) caused the accumulation of numerous small LDs. Oleic acid treatment increased mRNA and protein expression of the LD-associated protein PLIN2 but not PLIN5 or the lipase ATGL and its activator ABHD5/CGI-58. Inactivation of DGAT1 or DGAT2 did not alter expression (mRNA or protein) of ATGL, ABHD5/CGI-58, PLIN2 or PLIN5, but inactivation of both DGATs increased PLIN2 abundance despite a dramatic reduction in the number of LDs. ATGL localized preferentially to DGAT1-made LDs rather than to DGAT2-made LDs, and TG in these LDs was preferentially used for fatty acid (FA) oxidation. A combination of DGAT2 inhibitor and the pan lipase inhibitor E600 resulted in large LDs, suggesting that the small size of DGAT1-made LDs is due to a lipolytic process.
The non-canonical interaction between calmodulin and calcineurin contributes to the differential regulation of plant-derived calmodulins on calcineurin
Authors: Sun Bin , Fang Xuan , Johnson Christopher N. , Hauck Garrett , Davis Jonathan P. , Kekenes-Huskey Peter M. 
Published: June 2021

AbstractCalmodulin (CaM) is an important Ca2+ signaling hub that regulates many protein signaling pathways. In recent years, several CaM homologs expressed in plants have been shown to regulate mammalian targets and they are attractive for gene therapy. However, the molecular basis of how the CaM homologs mutations impact target activation is unclear, which limits efforts to engineer their functional properties. To understand these mechanisms, we examined two CaM isoforms found in soybean plants that differentially regulate a mammalian target, calcineurin (CaN). These CaM isofroms, sCaM-1 and sCaM-4 share >90% and ~ 78% identity with the mammalian CaM (mCaM), respectively, activate CaN with comparable or reduced activity relative to mCaM. We used molecular simulations and experimental assays to probe whether calcium and protein-protein binding interactions are altered in plant CaMs relative to mCaM as a basis for differential CaN regulations. We found that the two sCaMs’ Ca2+-binding properties such as coordination and affinity are comparable to mCaM. Further, the binding of CaM to the CaM binding region (CaMBR) in CaN is also comparable among the three CaMs, as evidenced by calculated binding free energies and experimental measured EC50 [CaM]. However, mCaM and sCaM-1 exhibited stronger binding with a secondary region of CaN’s regulatory domain that is weakened for sCaM-4. This secondary interaction is likely to affect the turnover rate (kcat) of CaN based on our modeling of enzyme activity and is consistent with our experimental data. Together, our data show how plant-derived CaM variants can alter target activation through interactions beyond Ca2+-binding and canonical CaMBR binding, which may extend beyond the mammalian CaN target.
LD-CNV: rapid and simple discovery of chromosomal translocations using linkage disequilibrium between copy number variable loci
Authors: Comai Luca , Amundson Kirk , Ordonez Benny , Zhao Xin , Braz Guilherme Tomaz , Jiang Jiming , Henry Isabelle 
Published: June 2021

AbstractLarge scale structural variations, such as chromosomal translocations, can have profound effects on fitness and phenotype, but are difficult to identify and characterize. Here, we describe a simple and effective method aimed at identifying translocations using only the dosage of sequence reads mapped on the reference genome. We binned reads on genomic segments sized according to sequencing coverage and identified instances when copy number segregated in populations. For each dosage-polymorphic 1Mb bin, we tested linkage disequilibrium with other variable bins. In nine potato (Solanum tuberosum) dihaploid families translocations affecting pericentromeric regions were common and in two cases were due to genomic misassembly. In two populations, we found evidence for translocation affecting euchromatic arms. In cv. PI 310467, a non-reciprocal translocation between chromosome 7 and 8 resulted in a 5-3 copy number change affecting several Mb at the respective chromosome tips. In cv. “Alca Tarma”, the terminal arm of chromosome 4 translocated to the tip of chromosome 1. Using oligonucleotide-based fluorescent in situ hybridization painting probes (oligo-FISH), we tested and confirmed the predicted arrangement in PI 310467. In 192 natural accessions of Arabidopsis thaliana, dosage haplotypes tended to vary continuously and resulted in higher noise, but we identified pericentromeric LD suggesting the effect of repeats. This method should be useful in species where translocations are suspected because it tests linkage without the need for genotyping.
Mechanism of CFTR correction by type I folding correctors
Authors: Fiedorczuk Karol , Chen Jue 
Published: June 2021

SUMMARYSmall molecule chaperones have been exploited as therapeutics for the hundreds of diseases caused by protein misfolding. The most successful examples are the CFTR correctors, which transformed cystic fibrosis therapy. These molecules revert folding defects of the ΔF508 mutant and are widely used to treat patients. However, their mechanism of action is unknown. Here we present cryo-electron microscopy structures of CFTR in complex with two FDA-approved correctors: lumacaftor and tezacaftor. Both drugs insert into a hydrophobic pocket in the first transmembrane domain (TMD1), linking together four helices that are thermodynamically unstable. Mutating residues at the binding site rendered ΔF508-CFTR insensitive to lumacaftor and tezacaftor, underscoring the functional significance of the structural discovery. These results support a mechanism in which the correctors stabilize TMD1 at an early stage of biogenesis, prevent its pre-mature degradation, and thereby allosterically rescue a large number of disease-causing mutations.