bluuebunny/crossref_metadata_2025_split huggingface dataset:
LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice
Authors: Zhang Xin , Sun Jiang , Lu Yonggang , Zhang Jintao , Shimada Keisuke , Noda Taichi , Zhao Shuqin , Koyano Takayuki , Matsuyama Makoto , Zhou Shushu , Wu Jiayan , Ikawa Masahito , Liu Mingxi 
Published: June 2021

AbstractCilia and flagella are ancient structures that achieve controlled motor functions through the coordinated interaction of structures including dynein arms, radial spokes (RSs), microtubules, and the dynein regulatory complex (DRC). RSs facilitate the beating motion of these organelles by mediating signal transduction between dyneins and a central pair (CP) of singlet microtubules. RS complex isolation from Chlamydomonas axonemes enabled the detection of 23 different proteins (RSP1-23), with the roles of RSP13, RSP15, RSP18, RSP19, and RSP21 remained poorly understood. Herein, we show that Lrrc23 is an evolutionarily conserved testis-enriched gene encoding an RSP15 homolog in mice. Through immunoelectron microscopy, we demonstrate that LRRC23 localizes to the RS complex within murine sperm flagella. We further found that LRRC23 was able to interact with RSHP9 and RSPH3A/B. The knockout of Lrrc23 resulted in RS disorganization and impaired motility in murine spermatozoa, whereas the ciliary beating was unaffected by the loss of this protein. Spermatozoa lacking LRRC23 were unable to efficiently pass through the uterotubal junction and exhibited defective zona penetration. Together these data indicate that LRRC23 is a key regulator underpinning the integrity of RS complex within the flagella of mammalian spermatozoa, whereas it is dispensable in cilia.Author summary
Distinct phosphorylation signals drive acceptor versus self-ubiquitination selection by Parkin
Authors: Dunkerley Karen M. , Rintala-Dempsey Anne C. , Salzano Guilia , Tadayon Roya , Hadi Dania , Barber Kathryn R. , Walden Helen , Shaw Gary S. 
Published: June 2021

AbstractThe RBR E3 ligase parkin is recruited to the outer mitochondrial membrane (OMM) during oxidative stress where it becomes activated and ubiquitinates numerous proteins. Parkin activation involves binding of a phosphorylated ubiquitin (pUb), followed by phosphorylation of parkin itself, both mediated by the OMM kinase, PINK1. However, targeted mitochondrial proteins have little structural or sequence similarity, with the commonality between substrates being proximity to the OMM. Here, we demonstrate that parkin efficiently ubiquitinates a mitochondrial acceptor pre-ligated to pUb and phosphorylation of parkin triggers autoubiquitination activity. Mitochondrial target proteins, Miro1 or CISD1, tethered to pUb are ubiquitinated by parkin more efficiently than if alone or Ub-tethered and ubiquitin molecules are ligated to acceptor protein lysines and not pUb. Parkin phosphorylation is not required for acceptor-pUb ubiquitination. In fact, only phospho-parkin induced self-ubiquitination and deletion of Ubl or mutation at K211N inhibited self-ubiquitination. We propose divergent parkin mechanisms whereby parkin-mediated ubiquitination of acceptor proteins is driven by binding to pre-existing pUb and subsequent parkin phosphorylation triggers autoubiquitination. This finding is critical for understanding parkin’s role in mitochondrial homeostasis and has implications on targets for therapeutics.
Label-free imaging for quality control of cardiomyocyte differentiation
Authors: Qian Tongcheng , Heaster Tiffany M. , Houghtaling Angela R. , Sun Kexin , Samimi Kayvan , Skala Melissa C. 
Published: June 2021

AbstractHuman pluripotent stem cell (hPSC)-derived cardiomyocytes provide a promising regenerative cell therapy for cardiovascular patients and an important model system to accelerate drug discovery. However, cost-effective and time-efficient platforms must be developed to evaluate the quality of hPSC-derived cardiomyocytes during biomanufacturing. Here, we developed a non-invasive label-free live cell imaging platform to predict the efficiency of hPSC differentiation into cardiomyocytes. Autofluorescence imaging of metabolic co-enzymes was performed under varying differentiation conditions (cell density, concentration of Wnt signaling activator) across three hPSC lines. Live cell autofluorescence imaging and multivariate classification models provided high accuracy to separate low (< 50%) and high (≥ 50%) differentiation efficiency groups (quantified by cTnT expression on day 12) within 1 day after initiating differentiation (area under the receiver operating characteristic curve, 0.98). This non-invasive and label-free method could be used to avoid batch-to-batch and line-to-line variability in cell manufacturing from hPSCs.
Detection of Alzheimer’s associated microRNAs using a DNA-based smart
reagent
Authors: Chandrasekaran Arun Richard , Halvorsen Ken 
Published: June 2021

Abstract
Alzheimer’s disease (AD) is the most common neurodegenerative disorder, with
significant research efforts devoted to identifying new biomarkers for clinical
diagnosis and treatment. MicroRNAs have emerged as likely disease regulators and
biomarkers for AD, now implicated as having roles in several biological
processes related to progression of the disease. In this work, we use the
miRacles assay
(microRNA
activated conditional
looping of engineered
switches) for single-step detection of AD-related
microRNAs. The technology is based on conformationally responsive DNA
nanoswitches that loop upon recognition of a target microRNA and report their
on/off status through an electrophoretic readout. Unlike many other methods, our
approach directly detects native microRNAs without amplification or labeling,
eliminating the need for expensive enzymes, reagents, and equipment. We used
this assay to screen for AD-related microRNAs, demonstrate specificity within a
microRNA family, sensitivity of ∼ 8 fM, and multiplexing capability to
simultaneously detect four microRNA targets. Toward clinical use, we provide
proof-of-concept detection and quantifiable dysregulation of specific microRNAs
from total RNA extracts derived from healthy and AD brain samples. In the
context of AD, this “smart reagent” could facilitate biomarker discovery,
accelerate efforts to understand the role of microRNAs in AD, and have clinical
potential as a diagnostic or monitoring tool for validated biomarkers.
Structural mechanism of GTPase-powered ribosome-tRNA movement
Authors: Petrychenko Valentyn , Peng Bee-Zen , Schwarzer Ana C. de A. P. , Peske Frank , Rodnina Marina V. , Fischer Niels 
Published: June 2021

AbstractGTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP–Pi form stabilizes ribosomal subunit rotation and induces twisting of the sarcin-ricin loop of the 23S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a cascade of rearrangements and a large-scale rotation of EF-G that exerts force on the ribosome and ultimately drives tRNA movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.
Male age andWolbachiadynamics: Investigating how fast and why bacterial densities and cytoplasmic incompatibility strengths vary
Authors: Shropshire J. Dylan , Hamant Emily , Cooper Brandon S. 
Published: June 2021

AbstractEndosymbionts can influence host reproduction and fitness to favor their maternal transmission. For example, endosymbioticWolbachiabacteria often cause cytoplasmic incompatibility (CI) that kills uninfected embryos fertilized byWolbachia-modified sperm. Infected females can rescue CI, providing them a relative fitness advantage.Wolbachia-induced CI strength varies widely and tends to decrease as host males age. Since strong CI drivesWolbachiato high equilibrium frequencies, understanding how fast and why CI strength declines with male age is crucial to explaining age-dependent CI’s influence onWolbachiaprevalence. Here, we investigate ifWolbachiadensities and/or CI gene (cif) expression covary with CI-strength variation and explore covariates of age-dependentWolbachia-density variation in two classic CI systems.wRi CI strength decreases slowly withDrosophila simulansmale age (6%/ day), butwMel CI strength decreases very rapidly (19%/ day), yielding statistically insignificant CI after only three days ofD. melanogasteremergence.Wolbachiadensities andcifexpression in testes decrease aswRi-infected males age, but both surprisingly increase aswMel-infected males age, and CI strength declines. We then tested if phage lysis, Octomom copy number (which impactswMel density), or host immune expression covary with age-dependentwMel densities—only host immune expression correlated with density. Together, our results identify how fast CI strength declines with male age in two model systems and reveal unique relationships between male age,Wolbachiadensities,cifexpression, and host immunity. We discuss new hypotheses about the basis of age-dependent CI strength and its contributions toWolbachiaprevalence.ImportanceWolbachiaare the most common animal-associated endosymbionts due in large part to their manipulation of host reproduction. ManyWolbachiacause cytoplasmic incompatibility (CI) that kills uninfected host eggs. Infected eggs are protected from CI, favoringWolbachiaspread in natural systems and in transinfected mosquito populations where vector-control groups use strong CI to maintain pathogen-blockingWolbachiaat high frequencies for biocontrol of arboviruses. CI strength varies considerably in nature and declines as males age for unknown reasons. Here, we determine that CI strength weakens at different rates with age in two model symbioses.Wolbachiadensity and CI gene expression covary withwRi-induced CI strength inDrosophila simulans, but neither explain rapidly decliningwMel-induced CI in agingD. melanogastermales. Patterns of host immune gene expression suggest a candidate mechanism behind age-dependentwMel densities. These findings inform how age-dependent CI may contribute toWolbachiaprevalence in natural systems and potentially in transinfected systems.
Effects of the COVID-19 pandemic on publication landscape in chimeric antigen receptor-modified immune cell research
Authors: Yilmaz Ahmet , Yu Jianhua 
Published: June 2021

AbstractChimeric antigen receptors (CARs) are artificial receptors introduced mainly into T cells. CAR-induced immune cell (CARi) products have achieved impressive success rates in treating some difficult-to-treat hematological malignancies. Here, we describe effects of the global COVID-19 pandemic on CARi publication landscape. Due to the pandemic, the total number of publications decreased in 2020 compared to 2019 in all fields of cancer immunotherapy except CARi. Nearly exponential increases in the number of CARi publications slowed-down in 2020 for the first time in the past 11 years. There were more CARi than coronavirus publications until 2020 when coronavirus publications increased over 5,000% compared to 2019 (575 publications in 2019 vs. 30,390 in 2020). Unlike cancer immunotherapy where the majority of the publications consist of conference abstracts and review articles, majority of the coronavirus publications are original research articles. There are more coronavirus publications in Pubmed than Embase. The opposite is true for CARi publications. Our analysis of the data from the FDA Adverse Event Reporting System (FAERS) show significantly higher death rate in patients treated with Kymirah than Yescarta (28.14% vs. 16.02%). Kymirah and Yescarta are the two main CAR T cell products for treatment of DLBCL and/or B-ALL. However, despite being highly significant, this result is not easily interpretable due to multiple confounding variables in the FAERS data. Our analysis additionally suggest that the significant effects of co-stimulatory domains (4-1BB vs. CD28) consistently reported in preclinical studies do not translate into clinical results. Our manual curation of the CARi publications in PubMed shows that only 5.2% of the publications report results from CARi clinical trials, although we found 663 clinical trials listed on ClinicalTrials.gov database. In conclusion, publication landscape in CARi as well as other fields of cancer immunotherapy has changed due to the global COVID-19 pandemic. This trend will likely continue in the near future. CARi research is now in need of increased measures by publishers to reduce repetitive and/or duplicate publications and more stringent criteria for data entry into public databases including PubMed, Embase, ClinicalTrials.gov, and FAERS to advance this important field of medical research.
A three-sodium-to-glycine stoichiometry shapes the structural relationships of ATB0,+ with GlyT2 and GlyT1 in the SLC6 family
Authors: Le Guellec Bastien , Rousseau France , Bied Marion , Supplisson Stéphane 
Published: June 2021

AbstractATB0,+ (SLC6A14) absorbs all neutral and cationic amino acids in the distal colon and lung epithelia, and is part of the amino acid transporter branch I of the SLC6 family with GlyT1 (SLC6A9) and GlyT2 (SLC6A5), two glycine-specific transporters coupled to 2:1 and 3:1 Na+:Cl−, respectively. However, ATB0,+ stoichiometry that specifies its driving force and electrogenicity remains unsettled. Using the reversal potential slope method, here we demonstrate that ATB0,+-mediated glycine transport is coupled to 3 Na+ and 1 Cl− and has a charge coupling of 2.1 e/glycine. ATB0,+ behaves as a unidirectional transporter with limited efflux and exchange capabilities. Analysis and computational modeling of the pre-steady-state charge movement reveal higher sodium affinity of the apo-ATB0,+, and a locking trap preventing Na+ loss at depolarized potentials. A 3 Na+/ 1 Cl− stoichiometry substantiates ATB0,+ concentrative-uptake and trophic role in cancers and rationalizes its structural proximity with GlyT2 despite their divergent substrate specificity.
In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center at the Channel Entrance
Authors: Khara Pratick , Christie Peter J. , Hu Bo 
Published: June 2021

AbstractBacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as ‘minimized’ or ‘expanded’ based on whether assembly requires only a core set of signature subunits or additional system-specific components. The prototypical ‘minimized’ systems mediating Agrobacterium tumefaciens T-DNA transfer and conjugative transfer of plasmids pKM101 and R388 are built from 12 subunits generically named VirB1-VirB11 and VirD4. In this study, we visualized the pKM101-encoded T4SS in the native context of the bacterial cell envelope by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388, a large substructure of which was previously purified and analyzed by negative-stain electron microscopy (nsEM). The IMC of the in situ T4SSpKM101 machine is highly symmetrical and exhibits 6-fold symmetry, dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three ‘expanded’ T4SSs previously visualized by in situ CryoET, but strikingly differs from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Together, our findings support a unified architectural model for all T4SSs assembled in vivo regardless of their classification as ‘minimized’ or ‘expanded’: the signature VirB4-like ATPases invariably are arranged as central hexamers of dimers at the entrances to the T4SS channels.SignificanceBacterial type IV secretion systems (T4SSs) play central roles in antibiotic resistance spread and virulence. By cryoelectron tomography (CryoET), we solved the structure of the plasmid pKM101-encoded T4SS in the native context of the bacterial cell envelope. The inner membrane complex (IMC) of the in situ T4SS differs remarkably from that of a closely-related T4SS analyzed in vitro by single particle electron microscopy. Our findings underscore the importance of comparative in vitro and in vivo analyses of the T4SS nanomachines, and firmly support a unified model in which the signature VirB4 ATPases of the T4SS superfamily function as a central hexamer of dimers to regulate substrate entry into and passage through T4SS channels.
Hitting more birds with one stone: CD70 as an actionable immunotherapeutic target in recurrent glioblastoma
Authors: Seyfrid M , Maich W , Shaikh MV , Tatari N , Upreti D , Piyasena D , Subapanditha M , Savage N , McKenna D , Kuhlmann L , Khoo A , Salim SK , Bassey-Archibong B , Gwynne W , Chokshi C , Brown K , Murtaza N , Bakhshinyan D , Vora P , Venugopal C , Moffat J , Singh SK 
Published: June 2021

ABSTRACTPurposeGlioblastoma (GBM) patients suffer from a dismal prognosis, with standard of care therapy inevitably leading to therapy-resistant recurrent tumors. The presence of brain tumor initiating cells (BTICs) drives the extensive heterogeneity seen in GBM, prompting the need for novel therapies specifically targeting this subset of tumor-driving cells. Here we identify CD70 as a potential therapeutic target for recurrent GBM BTICs.Experimental DesignIn the current study, we identified the relevance and functional influence of CD70 on primary and recurrent GBM cells, and further define its function using established stem cell assays. We utilize CD70 knockdown studies, subsequent RNAseq pathway analysis, andin vivoxenotransplantation to validate CD70’s role in GBM. Next, we developed and tested an anti-CD70 CAR-T therapy, which we validatedin vitroandin vivousing our established preclinical model of human GBM. Lastly, we explored the importance of CD70 in the tumor immune microenvironment (TIME) by assessing the presence of its receptor, CD27, in immune infiltrates derived from freshly resected GBM tumor samples.ResultsCD70 expression is elevated in recurrent GBM and CD70 knockdown reduces tumorigenicity in vitro and in vivo. CD70 CAR-T therapy significantly improves prognosisin vivo. We also found CD27 to be present on the cell surface of multiple relevant GBM TIME cell populations.ConclusionCD70 plays a key role in recurrent GBM cell aggressiveness and maintenance. Immunotherapeutic targeting of CD70 significantly improves survival in animal models and the CD70/CD27 axis may be a viable poly-therapeutic avenue to co-target both GBM and its TIME.
Staring at the naked Goddess. Unraveling structure and reactivity of Artemis endonuclease interacting with a DNA double strand
Authors: Hognon Cécilia , Monari Antonio 
Published: June 2021

AbstractArtemis is an endonuclease responsible for breaking hairpin DNA strand during immune system adaptation and maturation as well as the processing of potentially toxic DNA lesions. Thus, Artemis may be an important target in the development of anticancer therapy, both for the sensitization of radiotherapy and for immunotherapy. Despite its importance its structure has been resolved only recently, and important questions concerning the arrangement of its active center, the interaction with the DNA substrate, or the catalytic mechanism remain unanswered. In this contribution, by performing extensive molecular dynamic simulation, both classically and at hybrid quantum mechanics/ molecular mechanics level, we evidence the stable interaction modes of Artemis with a model DNA strand. We also analyze the catalytic cycle providing the free energy profile and key transition states for the DNA cleavage reaction.
A Functional Menadione Biosynthesis Pathway is Required for Capsule Production by Staphylococcus aureus
Authors: Altwiley Dina , Brignoli Tarcisio , Edwards Andrew , Recker Mario , Lee Jean , Massey Ruth C. 
Published: June 2021

AbstractStaphylococcus aureus is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of S. aureus to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by S. aureus we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the menD gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of S. aureus often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV we functionally verified this association between menD and capsule production. Although the clinical isolates with polymorphisms in the menD gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type menD gene. Our work suggests that menadione plays a critical role in the production of the S. aureus capsule, and that amongst clinical isolates polymorphisms exist in the menD gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with SCV phenotype.
Relative Affinities of Protein-Cholesterol Interactions from Equilibrium Molecular Dynamics Simulations
Authors: Ansell T. Bertie , Curran Luke , Horrell Michael R. , Pipatpolkai Tanadet , Letham Suzanne C. , Song Wanling , Siebold Christian , Stansfeld Phillip J. , Sansom Mark. S. P. , Corey Robin. A. 
Published: June 2021

AbstractSpecific interactions of lipids with membrane proteins contribute to protein stability and function. Multiple lipid interactions surrounding a membrane protein are often identified in molecular dynamics (MD) simulations and are, increasingly, resolved in cryo-EM densities. Determining the relative importance of specific interaction sites is aided by determination of lipid binding affinities by experimental or simulation methods. Here, we develop a method for determining protein-lipid binding affinities from equilibrium coarse-grained MD simulations using binding saturation curves, designed to mimic experimental protocols. We apply this method to directly obtain affinities for cholesterol binding to multiple sites on a range of membrane proteins and compare our results with free energies obtained from density-based equilibrium methods and with potential of mean force calculations, getting good agreement with respect to the ranking of affinities for different sites. Thus, our binding saturation method provides a robust, high-throughput alternative for determining the relative consequence of individual sites seen in e.g. cryo-EM derived membrane protein structures surrounded by a plethora of ancillary lipid densities.