bluuebunny/crossref_metadata_2025_split huggingface dataset:
Adenovirus-α-defensin complexes induce NLRP3-associated maturation of human phagocytes via TLR4 engagement
Authors: Eichholz Karsten , Tran Tuan Hiep , Chéneau Coraline , Tran Thi Thu Phuong , Paris Océane , Pugniere Martine , Kremer Eric J 
Published: October 2021

AbstractIntramuscular delivery of human adenovirus (HAdV)-based vaccines leads to rapid recruitment of neutrophils, which then release antimicrobial peptides/proteins (AMPs). How these AMPs influence vaccine efficacy over the subsequent 24 hr is poorly understood. In this study, we asked if human neutrophil protein 1 (HNP-1), an α-defensin that influences the direct and indirect innate immune responses to a range of pathogens, impacts the innate response of human phagocytes to three HAdV species/types (HAdV-C5, -D26, -B35). We show that HNP-1 binds to the capsids, redirects HAdV-C5, -D26, -B35 to Toll-like receptor 4 (TLR4), which leads to internalization, an NLRP3-mediated inflammasome response, and IL-1β release. Surprisingly, IL-1β release was not associated with notable disruption of plasma membrane integrity. These data further our understanding of HAdV innate immunogenicity and may provide pathways to extend HAdV-based vaccines efficacy.
Hepatitis C Virus Infects and Perturbs Liver Stem Cells
Authors: Meyers Nathan L , Ashuach Tal , Lyons Danielle E , Simoneau Camille R , Erickson Ann L , Bouhaddou Mehdi , Nguyen Thong T. , Khalid Mir M , Taha Taha Y , Natarajan Vaishaali , Baron Jody L , Neff Norma , Zanini Fabio , Mahmoud Tokameh , Quake Stephen R , Krogan Nevan J , Cooper Stewart , McDevitt Todd C , Yosef Nir , Ott Melanie 
Published: October 2021

SummaryHepatitis C virus (HCV) is the leading cause of death from liver disease. How HCV infection causes lasting liver damage and increases cancer risk beyond viral clearance remains unclear. We identify bipotent liver stem cells as novel targets for HCV infection, and their erroneous differentiation as the potential cause of impaired liver regeneration and cancer development. We show 3D organoids generated from liver stem cells from actively HCV-infected individuals carry replicating virus and maintain low-grade infection over months. Organoids can be infected with a primary HCV isolate. Virus-inclusive single-cell RNA-sequencing uncovered extensive transcriptional reprogramming in HCV+ cells supporting hepatocytic differentiation, cancer stem cell development and viral replication while stem cell proliferation and interferon signaling are disrupted. Our data adds a pathogenesis factor – infection of liver stem cells – to the biology of HCV infection that explains persistent liver damage and enhanced cancer risk through an altered stem cell state.
Centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly
Authors: Wong Siu-Shing , Wilmott Zachary M. , Saurya Saroj , Alvarez-Rodrigo Ines , Zhou Felix Y. , Chau Kwai-Yin , Goriely Alain , Raff Jordan W. 
Published: October 2021

AbstractMitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome “maturation” requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd-2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. We show here that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process—peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. We propose that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process.
The glutathione import system satisfies the Staphylococcus aureus nutrient sulfur requirement and promotes interspecies competition
Authors: Lensmire Joshua M. , Wischer Michael R. , Sosinski Lo M. , Ensink Elliot , Dodson Jack P. , Shook John C. , Delekta Phillip C. , Cooper Christopher C. , Havlichek Daniel , Mulks Martha H. , Lunt Sophia Y. , Ravi Janani , Hammer Neal D. 
Published: October 2021

ABSTRACTSulfur is an indispensable element for proliferation of bacterial pathogens. Prior studies indicated that the human pathogen, Staphylococcus aureus utilizes glutathione (GSH) as a source of nutrient sulfur; however, mechanisms of GSH acquisition are not defined. Here, we identify a previously uncharacterized five-gene locus comprising a putative ABC-transporter and γ–glutamyl transpeptidase (ggt) that promotes S. aureus proliferation in medium supplemented with either reduced or oxidized GSH (GSSG) as the sole source of nutrient sulfur. Based on these phenotypes, we name this transporter the Glutathione import system (GisABCD). We confirm that Ggt is capable of cleaving GSH and GSSG γ–bonds and that this process is required for their use as nutrient sulfur sources. Additionally, we find that the enzyme is cell associated. Bioinformatic analyses reveal that only Staphylococcus species closely related to S. aureus encode GisABCD-Ggt homologues. Homologues are not detected in Staphylococcus epidermidis. Consequently, we establish that GisABCD-Ggt provides a competitive advantage for S. aureus over S. epidermidis in a GSH-dependent manner. Overall, this study describes the discovery of a nutrient sulfur acquisition system in S. aureus that targets GSH and promotes competition against other staphylococci commonly associated with the human microbiota.
Architecture of the linker-scaffold in the nuclear pore
Authors: Petrovic Stefan , Samanta Dipanjan , Perriches Thibaud , Bley Christopher J. , Thierbach Karsten , Brown Bonnie , Nie Si , Mobbs George W. , Stevens Taylor A. , Liu Xiaoyu , Hoelz André 
Published: October 2021

AbstractThe nuclear pore complex (NPC) is the sole bidirectional gateway for nucleocytoplasmic transport. Despite recent progress in elucidating the arrangement of the structured scaffold building blocks in the NPC symmetric core, their cohesion by multivalent unstructured linker proteins remained elusive. Combining biochemical reconstitution, high resolution structure determination, docking into cryo-electron tomographic reconstructions, and physiological validation, we elucidated the architecture of the entire linker-scaffold, yielding a near-atomic composite structure of the symmetric core accounting for ∼77 MDa of the human NPC. Whereas linkers generally play a rigidifying role, the linker-scaffold of the NPC provides the plasticity and robustness necessary for the reversible constriction and dilation of its central transport channel. Our results complete the structural characterization of the NPC symmetric core, providing a rich foundation for future functional studies.One sentence summaryAn interdisciplinary analysis established the near-atomic molecular architecture and evolutionary conservation of the linker-scaffold of the human nuclear pore complex.
Cyclic uniaxial mechanical load enhances chondrogenesis through entraining the molecular circadian clock
Authors: Vágó Judit , Katona Éva , Takács Roland , Dócs Klaudia , Hajdú Tibor , Kovács Patrik , Zákány Róza , van der Veen Daan R. , Matta Csaba 
Published: October 2021

AbstractThe biomechanical environment plays a key role in regulating cartilage formation, but current understanding of mechanotransduction pathways in chondrogenic cells is incomplete. Amongst the combination of external factors that control chondrogenesis are temporal cues that are governed by the cell-autonomous circadian clock. However, mechanical stimulation has not yet directly been proven to modulate chondrogenesis via entraining the circadian clock in chondroprogenitor cells. The purpose of this study was to establish whether mechanical stimuli entrain the core clock in chondrogenic cells, and whether augmented chondrogenesis caused by mechanical loading was at least partially mediated by the synchronised, rhythmic expression of the core circadian clock genes, chondrogenic transcription factors, and cartilage matrix constituents at both transcript and protein levels. We report here, for the first time, that cyclic uniaxial mechanical load applied for 1 hour for a period of 6 days entrains the molecular clockwork in chondroprogenitor cells during chondrogenesis in limb bud-derived micromass cultures. In addition to the several core clock genes and proteins, the chondrogenic markers SOX9 and ACAN also followed a robust sinusoidal rhythmic expression pattern. These rhythmic conditions significantly enhanced cartilage matrix production and upregulated marker gene expression. The observed chondrogenesis-promoting effect of the mechanical environment was at least partially attributable to its entraining effect on the molecular clockwork, as co-application of the small molecule clock modulator longdaysin attenuated the stimulatory effects of mechanical load. This study suggest that an optimal biomechanical environment enhances tissue homeostasis and histogenesis during chondrogenesis at least partially through entraining the molecular clockwork.
Development of ultrafast camera-based imaging of single fluorescent molecules and live-cell PALM
Authors: Fujiwara Takahiro K. , Takeuchi Shinji , Kalay Ziya , Nagai Yosuke , Tsunoyama Taka A. , Kalkbrenner Thomas , Iwasawa Kokoro , Ritchie Ken P. , Suzuki Kenichi G.N. , Kusumi Akihiro 
Published: October 2021

AbstractThe spatial resolution of fluorescence microscopy has recently been greatly improved. However, its temporal resolution has not been improved much, despite its importance for examining living cells. Here, by developing an ultrafast camera system, we achieved the world’s highest time resolutions for single fluorescent-molecule imaging of 33 (100) µs (multiple single molecules simultaneously) with a single-molecule localization precision of 34 (20) nm for Cy3 (best dye found), and for PALM data acquisition of a view-field of 640×640 pixels at 1 kHz with a single-molecule localization precision of 29 nm for mEos3.2. Both are considered the ultimate rates with available probes. This camera system (1) successfully detected fast hop diffusion of membrane molecules in the plasma membrane, detectable previously only by using less preferable 40-nm gold probes and bright-field microscopy, and (2) enabled PALM imaging of the entire live cell, while revealing meso-scale dynamics and structures, caveolae and paxillin islands in the focal adhesion, proving its usefulness for cell biology research.SummaryAn ultrafast camera developed by Fujiwara et al. allows single fluorescent-molecule imaging every 33 μs with a localization precision of 34 nm (every 100 μs; 20 nm), and enables ultrafast PALM imaging of whole live cells.
miRNA mediated downregulation of non-muscle Cyclase associated protein 1 is required for myogenic differentiation
Authors: Singh Anurag Kumar , Rai Amrita , Weber Anja , Posern Guido 
Published: October 2021

AbstractMyoblast fusion is crucial for the formation, growth and regeneration of healthy skeletal muscle, but the molecular mechanisms that govern fusion and myofiber formation remain poorly understood. Here we report that Cyclase-associated protein 1 (Cap1), a regulator of actin dynamics, plays a critical role in cytoskeletal remodeling during myoblast fusion and formation of myotubes. Cap1 mRNA and protein are expressed in murine C2C12 and human LHCN-M2 myoblasts, but its abundance decreases during myogenic differentiation. Perturbing the temporally controlled expression of Cap1 by overexpression or Crispr-Cas9 mediated knockout impaired actin rearrangement, myoblast alignment, expression of profusion molecules, differentiation into multinucleated myotubes and myosin heavy chain expression. Endogenous Cap1 expression is posttranscriptionally downregulated during differentiation by canonical myomiRs miR-1, miR-133 and miR-206, which have conserved binding sites in the 3’ UTR of the Cap1 mRNA. Deletion of the endogenous 3’ UTR in C2C12 cells phenocopies overexpression of Cap1 by inhibiting myotube formation. Our findings implicate Cap1 and its myomiR-mediated downregulation in the myoblast fusion process and the generation of skeletal muscle.
Chromosome-scale assemblies of Acanthamoeba castellanii genomes provide insights into Legionella pneumophila infection-related chromatin re-organization
Authors: Matthey-Doret Cyril , Colp Morgan J. , Escoll Pedro , Thierry Agnès , Curtis Bruce , Sarrasin Matt , Gray Michael W. , Lang B. Franz , Archibald John M. , Buchrieser Carmen , Koszul Romain 
Published: October 2021

AbstractThe unicellular amoeba Acanthamoeba castellanii is ubiquitous in aquatic environments, where it preys on bacteria. The organism also hosts bacterial endosymbionts, some of which are parasitic, including human pathogens such as Chlamydia and Legionella spp. Here we report complete, high quality genome sequences for two extensively studied A. castellanii strains, Neff and C3. Combining long- and short-read data with Hi-C, we generated near chromosome-level assemblies for both strains with 90% of the genome contained in 29 scaffolds for the Neff strain and 31 for the C3 strain. Comparative genomics revealed strain-specific functional enrichment, most notably genes related to signal transduction in the C3 strain, and to viral replication in Neff. Furthermore, we characterized the spatial organization of the A. castellanii genome and showed that it is reorganized during infection by Legionella pneumophila. Infection-dependent chromatin loops were found to be enriched in genes for signal transduction and phosphorylation processes. In genomic regions where chromatin organization changed during Legionella infection, we found functional enrichment for genes associated with metabolism, organelle assembly, and cytoskeleton organization, suggesting that changes in chromosomal folding are associated with host cell biology during infection.
The Mycobacterium tuberculosis DNA-repair helicase UvrD1 is activated by redox-dependent dimerization via a 2B domain cysteine conserved in other Actinobacteria
Authors: Chadda Ankita , Jensen Drake , Tomko Eric J. , Manzano Ana Ruiz , Nguyen Binh , Lohman Timothy M. , Galburt Eric A. 
Published: October 2021

AbstractMycobacterium tuberculosis (Mtb) causes Tuberculosis and, during infection, is exposed to reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) from the host immune response that can cause DNA damage. UvrD-like proteins are involved in DNA repair and replication and belong to the SF1 family of DNA helicases that use ATP hydrolysis to catalyze DNA unwinding. In Mtb, there are two UvrD-like enzymes where UvrD1 is most closely related to other family members. Previous studies have suggested that UvrD1 is exclusively monomeric, however it is well-known that E. coli UvrD and other UvrD-family members exhibit monomer-dimer equilibria and unwind as dimers in the absence of accessory factors. Here, we reconcile these incongruent studies by showing that Mtb UvrD1 exists in monomer, dimer, and tetramer oligomeric forms where dimerization is regulated by redox potential. We identify a 2B domain cysteine, conserved in many Actinobacteria, that underlies this effect. We also show that UvrD1 DNA unwinding activity correlates specifically with the dimer population and is thus titrated directly via increasing positive (i.e. oxidative) redox potential. Consistent with the regulatory role of the 2B domain and the dimerization-based activation of DNA unwinding in UvrD-family helicases, these results suggest that UvrD1 is activated under oxidizing conditions when it may be needed to respond to DNA damage during infection.
Suitability of GRK antibodies for individual detection and quantification of GRK isoforms in western blots
Authors: Reichel Mona , Weitzel Verena , Klement Laura , Hoffmann Carsten , Drube Julia 
Published: October 2021

AbstractG protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This leads to the recruitment of arrestins resulting in desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffold. This multifunctionality is also reflected by their diverse roles in pathological conditions like cancer, influenza infection, malaria, and metabolic disease.Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRK2, GRK3, GRK5, and GRK6 in western blot analysis. We thereby identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross reactivity. Thus, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained western blot results.Utilizing the most suitable antibodies we established the western blot-based, cost-effective, simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in five commonly used cell lines revealing differential isoform expression.
RNA compensation: A positive feedback insulation strategy for RNA-based networks
Authors: Liu Baiyang , Samaniego Christian Cuba , Bennett Matthew , Chappell James , Franco Elisa 
Published: October 2021

AbstractThe lack of signalling modularity of biomolecular systems poses major challenges toward engineering complex networks. An important problem is posed by the consumption of signaling molecules upon circuit interconnection, which makes it possible to control a downstream circuit but compromises the performance of the upstream circuit. This issue has been previously addressed with insulation strategies including high-gain negative feedback and phosphorylation-dephosphorylation reaction cycle. In this paper, we focus on RNA-based circuits and propose a new positive-feedback insulation strategy to mitigate signal consumption. An RNA input is added in tandem with transcription output to compensate the RNA consumption, leading to concentration robustness of the input RNA molecule regardless of the amount of downstream modules. We term this strategy RNA compensation, and it can be applied to systems that have a stringent input-output gain, such as Small Transcription Activating RNAs (STARs). Our analysis shows that RNA compensation not only eliminates the signaling consumption in individual STAR-based regulators, but also improves the composability of STAR cascades and the modularity of RNA bistable systems.
PDGFR dimer-specific activation, trafficking and downstream signaling dynamics
Authors: Rogers Madison A. , Fantauzzo Katherine A. 
Published: October 2021

AbstractSignaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in multiple cellular processes during development. The two PDGFRs, PDGFRα and PDGFRβ, dimerize to form homodimers and/or heterodimers. Here, we overcome previous limitations in studying PDGFR dimer-specific dynamics by generating cell lines stably expressing C-terminal fusions of each PDGFR with bimolecular fluorescence complementation (BiFC) fragments corresponding to the N-terminal or C-terminal regions of the Venus fluorescent protein. We find that PDGFRβ receptors homodimerize more quickly than PDGFRα receptors in response to PDGF ligand, with increased levels of autophosphorylation. Further, we demonstrate that PDGFRα homodimers are trafficked and degraded more quickly, while PDGFRβ homodimers are more likely to be recycled back to the cell membrane. We show that PDGFRβ homodimer activation results in a greater amplitude of phospho-ERK1/2 and phospho-AKT signaling, as well as increased proliferation and migration. Collectively, our findings provide significant insight into how biological specificity is introduced to generate unique responses downstream of PDGFR engagement.SummaryThe authors utilize a novel bimolecular fluorescence complementation approach to investigate PDGFR homodimer-specific dynamics. They uncover differences in the timing and extent of receptor dimerization, activation and trafficking, which lead to changes in downstream signaling and cellular activity.
Cell wall damage reveals spatial flexibility in peptidoglycan synthesis and a non-redundant role for RodA in mycobacteria
Authors: Melzer Emily S. , Kado Takehiro , García-Heredia Alam , Gupta Kuldeepkumar Ramnaresh , Meniche Xavier , Morita Yasu S. , Sassetti Christopher M. , Rego E. Hesper , Siegrist M. Sloan 
Published: October 2021

AbstractCell wall peptidoglycan is a heteropolymeric mesh that protects the bacteria from internal turgor and external insults. In many rod-shaped bacteria, peptidoglycan synthesis for normal growth is achieved by two distinct pathways: the Rod complex, comprised of MreB, RodA and a cognate class B PBP, and the class A PBPs. In contrast to laterally-growing bacteria, pole-growing mycobacteria do not encode an MreB homolog and do not require SEDS protein RodA forin vitrogrowth. However, RodA contributes to survival ofMycobacterium tuberculosisin some infection models, suggesting that the protein could have a stress-dependent role in maintaining cell wall integrity. Under basal conditions, we find here that the subcellular distribution of RodA largely overlaps with that of the aPBP PonA1, and that both RodA and the aPBPs promote polar peptidoglycan assembly. Upon cell wall damage, RodA fortifiesM. smegmatisagainst lysis and, unlike aPBPs, contributes to a shift in peptidoglycan assembly from the poles to the sidewall. Neither RodA nor PonA1 relocalize; instead, the redistribution of nascent cell wall parallels that of peptidoglycan precursor synthase MurG. Our results support a model in which mycobacteria balance polar growth and cell-wide repair via spatial flexibility in precursor synthesis and extracellular insertion.ImportancePeptidoglycan synthesis is a highly successful target for antibiotics. The pathway has been extensively studied in model organisms under laboratory-optimized conditions. In natural environments, bacteria are frequently under attack. Moreover the vast majority of bacterial species are unlikely to fit a single paradigm because of differences in growth mode and/or envelope structure. Studying cell wall synthesis under non-optimal conditions and in non-standard species may improve our understanding of pathway function and suggest new inhibition strategies.Mycobacterium smegmatis,a relative of several notorious human and animal pathogens, has an unusual polar growth mode and multi-layered envelope. In this work we challengedM. smegmatiswith cell wall-damaging enzymes to characterize the roles of cell wall-building enzymes when the bacterium is under attack.