bluuebunny/crossref_metadata_2025 huggingface dataset:
A macroscopic free-swimming medusa from the middle Cambrian Burgess Shale
Authors: Moon Justin , Caron Jean-Bernard , Moysiuk Joseph
Published: August 2023

Cnidarians are regarded as one of the earliest-diverging animal phyla. One of the hallmarks of the cnidarian body plan is the evolution of a free-swimming medusa in some medusozoan classes, but the origin of this innovation remains poorly constrained by the fossil record and molecular data. Previously described macrofossils, putatively representing medusa stages of crown-group medusozoans from the Cambrian of Utah and South China, are here reinterpreted as ctenophore-grade organisms. Other putative Ediacaran to Cambrian medusozoan fossils consist mainly of microfossils and tubular forms. Here we describe
Burgessomedusa phasmiformis
gen. et sp. nov., the oldest unequivocal macroscopic free-swimming medusa in the fossil record. Our study is based on 182 exceptionally preserved body fossils from the middle Cambrian Burgess Shale (Raymond Quarry, British Columbia, Canada).
Burgessomedusa
possesses a cuboidal umbrella up to 20 cm high and over 90 short, finger-like tentacles. Phylogenetic analysis supports a medusozoan affinity, most likely as a stem group to Cubozoa or Acraspeda (a group including Staurozoa, Cubozoa and Scyphozoa).
Burgessomedusa
demonstrates an ancient origin for the free-swimming medusa life stage and supports a growing number of studies showing an early evolutionary diversification of Medusozoa, including of the crown group, during the late Precambrian–Cambrian transition.
Spontaneous helping in pigs is mediated by helper's social attention and distress signals of individuals in need
Authors: Moscovice Liza R. , Eggert Anja , Manteuffel Christian , Rault Jean-Loup
Published: August 2023

Helping behaviour is of special interest for prosociality because it appears to be motivated by the needs of others. We developed a novel paradigm to investigate helping in pigs (
Sus scrofa domesticus
) and tested 75 individuals in eight groups in their home pens. Two identical compartments were attached to the pen, equipped with a window, and a door that could be opened from the outside by lifting a handle. Pigs in all groups spontaneously opened doors during a 5-day familiarization. During testing, each pig was isolated once from its group and placed in one of the two compartments, in a counter-balanced order. In 85% of cases, pigs released a trapped group member from the test compartment within 20 min (median latency = 2.2 min). Pigs were more likely and quicker to open a door to free the trapped pig than to open a door to an empty compartment. Pigs who spent more time looking at the window of the compartment containing the trapped pig were more likely to help. Distress signals by the trapped pig increased its probability of being helped. Responses are consistent with several criteria for identifying targeted helping, but results can also be explained by selfish motivations.
Genomic characterization of the antiviral arsenal of Actinobacteria
Authors: Georjon Héloïse , Tesson Florian , Shomar Helena , Bernheim Aude
Published: August 2023

Phages are ubiquitous in nature, and bacteria with very different genomics, metabolisms, and lifestyles are subjected to their predation. Yet, the defence systems that allow bacteria to resist their phages have rarely been explored experimentally outside a very limited number of model organisms. Actinobacteria (Actinomycetota) are a phylum of GC-rich Gram-positive bacteria, which often produce an important diversity of secondary metabolites. Despite being ubiquitous in a wide range of environments, from soil to fresh and sea water but also the gut microbiome, relatively little is known about the anti-phage arsenal of Actinobacteria. In this work, we used DefenseFinder to systematically detect 131 anti-phage defence systems in 22803 fully sequenced prokaryotic genomes, among which are 2253 Actinobacteria of more than 700 species. We show that, like other bacteria, Actinobacteria encode many diverse anti-phage systems that are often encoded on mobile genetic elements. We further demonstrate that most detected defence systems are absent or rarer in Actinobacteria than in other bacteria, while a few rare systems are enriched (notably gp29-gp30 and Wadjet). We characterize the spatial distribution of anti-phage systems on

Streptomyces

chromosomes and show that some defence systems (e.g. RM systems) tend to be encoded in the core region, while others (e.g. Lamassu and Wadjet) are enriched towards the extremities. Overall, our results suggest that Actinobacteria might be a source of novel anti-phage systems and provide clues to characterize mechanistic aspects of known anti-phage systems.
Sale of critically endangered sharks in the United States
Authors: Ryburn Savannah J. , Yu Tammy , Ong Kelly Jia-Wei , Alston Meggan A. , Howie Ella , LeRoy Peyton , Giang Sarah Elizabeth , Ball William , Benton Jewel , Calhoun Robert , Favreau Isabella , Gutierrez Ana , Hallac Kayla , Hanson Dakota , Hibbard Teagan , Loflin Bryson , Lopez Joshua , Mock Gracie , Myers Kailey , Pinos-Sánchez Andrés , Suarez Garcia Alejandra Maria , Romero Adriana Retamales , Thomas Audrey , Williams Rhiannon , Zaldivar Anabel , Bruno John Francis
Published: August 2023

Shark meat is widely available in the United States in grocery stores and seafood markets. The meat is often mislabeled or generically labeled as “shark”. The ambiguity of these generic labels makes it challenging to assess the conservation implications of this practice and for consumers to avoid species with high mercury concentrations. For this study we purchased and DNA barcoded 30 shark products purchased in the United States to determine their species identity and conservation status. These samples consisted of 19 filets sold in grocery stores, seafood markets, and Asian specialty markets (mostly in North Carolina) and 11 ordered online as “jerky”. 70% of samples were “soft mislabeled” (i.e., labeled generically as shark but not as a specific species). Of the nine samples labeled to species, eight were mislabeled (e.g., spinner shark labeled as mako shark). Only one sample was correctly labeled. All 30 samples were identified as shark and came from 11 different species, including three species listed by the IUCN as Critically Endangered: great hammerhead, scalloped hammerhead, and tope. The first two species have been found to contain very high levels of mercury, illustrating the implications of seafood mislabeling for human health. The widespread availability of shark meat in U.S. grocery stores is surprising given the dramatic decline of shark populations globally. Moreover, the fact that nearly all shark meat is either mislabeled or not labeled to species amplifies the problem. Accurate, verified product labels for shark meat would benefit consumers and shark conservation efforts, and should be a priority for the seafood industry.
Surface modification of multilayer graphene neural electrodes by local printing of platinum nanoparticles using spark ablation†
Authors: Bakhshaee Babaroud Nasim , Rice Samantha J. , Perez Maria Camarena , Serdijn Wouter A. , Vollebregt Sten , Giagka Vasiliki
Published: August 2023

In this paper, we present the surface modification of multilayer graphene neural electrodes with platinum (Pt) nanoparticles (NPs) using spark ablation. This method yields an individually selective local printing of NPs on an electrode surface at room temperature in a dry process. NP printing is performed as a post-process step to enhance the electrochemical characteristics of graphene electrodes. The NP-printed electrode shows significant improvements in impedance, charge storage capacity (CSC), and charge injection capacity (CIC), versus the equivalent electrodes without NPs. Specifically, electrodes with 40% NP surface density demonstrate 4.5 times lower impedance, 15 times higher CSC, and 4 times better CIC. Electrochemical stability, assessed via continuous cyclic voltammetry (CV) and voltage transient (VT) tests, indicated minimal deviations from the initial performance, while mechanical stability, assessed via ultrasonic vibration, is also improved after the NP printing. Importantly, NP surface densities up to 40% maintain the electrode optical transparency required for compatibility with optical imaging and optogenetics. These results demonstrate selective NP deposition and local modification of electrochemical properties in neural electrodes for the first time, enabling the cohabitation of graphene electrodes with different electrochemical and optical characteristics on the same substrate.
SARS-CoV-2 Viral Clearance and Evolution Varies by Extent of Immunodeficiency
Authors: Li Yijia , Choudhary Manish C. , Regan James , Boucau Julie , Nathan Anusha , Speidel Tessa , Liew May Yee , Edelstein Gregory E. , Kawano Yumeko , Uddin Rockib , Deo Rinki , Marino Caitlin , Getz Matthew A. , Reynold Zahra , Barry Mamadou , Gilbert Rebecca F. , Tien Dessie , Sagar Shruti , Vyas Tammy D. , Flynn James P. , Hammond Sarah P. , Novack Lewis A. , Choi Bina , Cernadas Manuela , Wallace Zachary S. , Sparks Jeffrey A. , Vyas Jatin M. , Seaman Michael S. , Gaiha Gaurav D. , Siedner Mark J. , Barczak Amy K. , Lemieux Jacob E. , Li Jonathan Z.
Published: August 2023

Despite vaccination and antiviral therapies, immunocompromised individuals are at risk for prolonged SARS-CoV-2 infection, but the immune defects that predispose to persistent COVID-19 remain incompletely understood. In this study, we performed detailed viro-immunologic analyses of a prospective cohort of participants with COVID-19. The median time to nasal viral RNA and culture clearance in the severe hematologic malignancy/transplant group (S-HT) were 72 and 40 days, respectively, which were significantly longer than clearance rates in the severe autoimmune/B-cell deficient (S-A), non-severe, and non-immunocompromised groups (P<0.001). Participants who were severely immunocompromised had greater SARS-CoV-2 evolution and a higher risk of developing antiviral treatment resistance. Both S-HT and S-A participants had diminished SARS-CoV-2-specific humoral, while only the S-HT group had reduced T cell-mediated responses. This highlights the varied risk of persistent COVID-19 across immunosuppressive conditions and suggests that suppression of both B and T cell responses results in the highest contributing risk of persistent infection.
Human iPSC-derived Microglial Cells Integrated into Mouse Retina and Recapitulated Features of Endogenous Microglia
Authors: Ma Wenxin , Zhao Lian , Xu Biying , Fariss Robert N. , Redmond T. Michael , Zou Jizhong , Wong Wai T. , Li Wei
Published: August 2023

Microglia exhibit both maladaptive and adaptive roles in the pathogenesis of neurodegenerative diseases and have emerged as a cellular target for central nervous system (CNS) disorders, including those affecting the retina. Replacing maladaptive microglia, such as those impacted by aging or over-activation, with exogenous microglia that can enable adaptive functions has been proposed as a potential therapeutic strategy for neurodegenerative diseases. To investigate microglia replacement as an approach for retinal diseases, we first employed a protocol to efficiently generate human-induced pluripotent stem cells (hiPSC)-derived microglia in quantities sufficient for in vivo transplantation. These cells demonstrated expression of microglia-enriched genes and showed typical microglial functions such as LPS-induced responses and phagocytosis. We then performed xenotransplantation of these hiPSC-derived microglia into the subretinal space of adult mice whose endogenous retinal microglia have been pharmacologically depleted. Long-term analysis post-transplantation demonstrated that transplanted hiPSC-derived microglia successfully integrated into the neuroretina as ramified cells, occupying positions previously filled by the endogenous microglia and expressed microglia homeostatic markers such as P2ry12 and Tmem119. Further, these cells were found juxtaposed alongside residual endogenous murine microglia for up to eight months in the retina, indicating their ability to establish a stable homeostatic statein vivo. Following retinal pigment epithelial (RPE) cell injury, transplanted microglia demonstrated responses typical of endogenous microglia, including migration, proliferation, and phagocytosis. Our findings indicate the feasibility of microglial transplantation and integration in the retina and suggest that modulating microglia through replacement may be a therapeutic strategy for treating neurodegenerative retinal diseases.
Identifying repurposed drugs with moderate anti-influenza virus activity through computational prioritization of drug-target pairs
Authors: Taye Biruhalem , Thünauer Roland , Sugrue Richard J , Maurer-Stroh Sebastian , Kosinski Jan
Published: August 2023

Influenza A virus (IAV) causes up to five million cases of severe illness and half a million deaths worldwide each year. While there are a few clinically approved drugs for treating IAV, they are challenged by the rapid evolution of the virus leading to emergence of drug resistance and the adverse effects of the drugs. Targeting host cellular factors that support virus replication could limit resistance, increase the broad-spectrum antiviral properties of drugs, and benefit from repurposing drugs already existing against those factors. However, selecting the right drug-target pairs with low toxicity and minimal adverse effects has been challenging, even though hundreds of cellular host factors have been identified. In this study, we applied a computational and knowledge-based drug-target prioritization approach to identify promising drug-target pairs. We selected five pairs for experimental validation: telmisartan-Angiotensin II receptor, type 1 (AGTR1), metoclopramide hydrochloride-Cholinergic receptor muscarinic 1 (CHRM1), cefepime hydrochloride-phosphogluconate dehydrogenase (PGD), ranolazine dihydrochloride-sodium channel voltage-gated type v alpha subunit (SCN5A), and ofloxacin-topoisomerase II alpha 170kDa (TOP2A). Except for cefepime hydrochloride, all four drugs showed significant plaque reduction in Madin Darby canine kidney (MDCK) cells. In the immunofluorescence assay, metoclopramide hydrochloride, ranolazine dihydrochloride, and telmisartan showed antiviral activity in MDCK and/or adenocarcinoma human alveolar basal epithelial (A549) cell lines. In conclusion, our approach can prioritize and identify drugs with antiviral activity against influenza virus. Refining and strengthening such approaches could be valuable for rapid antiviral discovery and pandemic preparedness.

Highlights

Computational drug-target prioritization indicated drugs for validation

Telmisartan showed anti-influenza virus activity in MDCK cells

Metoclopramide and ranolazine showed anti-influenza activity in A549 and MDCK cells
Humoral correlates of protection againstMycobacterium tuberculosisfollowing intravenous Bacille Calmette-Guérin vaccination in rhesus macaques
Authors: Irvine Edward B. , Darrah Patricia A. , Wang Shu , Wang Chuangqi , McNamara Ryan P. , Roederer Mario , Seder Robert A. , Lauffenburger Douglas A. , Flynn JoAnne L. , Fortune Sarah M. , Alter Galit
Published: August 2023

Altering the route of Bacille Calmette-Guérin (BCG) immunization from low-dose intradermal vaccination to high-dose intravenous (IV) vaccination resulted in a high level of protection againstMycobacterium tuberculosis(Mtb) infection, providing an opportunity to uncover immune correlates and mechanisms of protection. In addition to strong T cell immunity, IV BCG vaccination was associated with a robust expansion of humoral immune responses that tracked with bacterial control. However, given the near complete protection afforded by high-dose IV BCG immunization, a precise correlate of immune protection was difficult to define. Here we leveraged plasma and bronchoalveolar lavage fluid (BAL) from a cohort of rhesus macaques that received decreasing doses of IV BCG and aimed to define the correlates of immunity across macaques that experienced immune protection or breakthrough infection followingMtbchallenge. We show an IV BCG dose-dependent induction of mycobacterial-specific humoral immune responses, both in the plasma and in the airways. Moreover, antibody responses at peak immunogenicity significantly predicted bacterial control following challenge. Multivariate analyses revealed antibody-mediated complement and NK cell activating humoral networks as key functional signatures associated with protective immunity. Collectively, this work extends our understanding of humoral biomarkers and potential mechanisms of IV BCG mediated protection againstMtb.
Epi-microRNA mediated metabolic reprogramming ensures affinity maturation
Authors: Nakagawa Rinako , Llorian Miriam , Varsani-Brown Sunita , Chakravarty Probir , Camarillo Jeannie M. , Barry David , George Roger , Blackledge Neil P. , Duddy Graham , Kelleher Neil L. , Klose Robert J. , Turner Martin , Calado Dinis P.
Published: August 2023

To increase antibody affinity against pathogens, positively selected GC-B cells initiate cell division in the light zone (LZ) of germinal centres (GCs). Among those, higher-affinity clones migrate to the dark zone (DZ) and vigorously proliferate by relying on oxidative phosphorylation (OXPHOS). However, it remains unknown how positively selected GC-B cells adapt their metabolism for cell division in the glycolysis-dominant, cell cycle arrest-inducing, hypoxic LZ microenvironment. Here, we show that microRNA (miR)-155 mediates metabolic reprogramming during positive selection to protect high-affinity clones. Transcriptome examination and mass spectrometry analysis revealed that miR-155 regulates H3K36me2 levels by directly repressing hypoxia-induced histone lysine demethylase,Kdm2a. This is indispensable for enhancing OXPHOS through optimizing the expression of vital nuclear mitochondrial genes under hypoxia. The miR-155-Kdm2ainteraction is crucial to prevent excessive production of reactive oxygen species and apoptosis. Thus, miR-155-mediated epigenetic regulation promotes mitochondrial fitness in high-affinity clones, ensuring their expansion and consequently affinity maturation.
Biophysical basis for the induction of glioblastoma-like phenotype in astrocytes
Authors: Starck Laurent , Sarem Melika , Heimrich Bernd , Sawarkar Ritwick , Ritz Marie-Françoise , Hutter Gregor , Shastri V. Prasad
Published: August 2023

While the direct biological factors underlying the progression of GBM, an aggressive form of brain cancer, have been extensively studied, emerging evidence suggests that indirect biological triggers, such as traumatic brain injury (TBI), may also have a role. Since reactive astrocytes are associated with TBI, and astroglial cells are the source of proteoglycans which contribute to changes in biophysical characteristics (stochastic topography, stiffness) of the brain, we postulated a role for stochastic nanoroughness in the induction of glioma. Using a model system to emulate such physical cues, we demonstrate that human cortical astrocytes undergo spontaneous organization into spheroids in response to nanoroughness and retain the spheroid phenotype even upon withdrawal of the physical cues. Furthermore, spheroids serve as aggregation foci for naïve astrocytes; express activated MMP2, and disseminate upon implantation in mouse brain. RNA-seq revealed a tumoral phenotype with a gene expression pattern involving p53, ADAMTS proteases and fibronectin. Moreover, nanoroughness mediates a cross-talk between cancer cells and astrocytes through induced senescence. These findings implicate a role for stochastic biophysical cues in driving a potential malignant transformation of astrocytes.
Psychopathic traits predict neural responses to emotional movies in the general population
Authors: Aksiuto Anna , Lahnakoski Juha M. , Kauttonen Janne , Saarimäki Heini , Volynets Sofia , Nummenmaa Lauri , Sams Mikko , Jääskeläinen Iiro P.
Published: August 2023

Psychopathic personality traits such as callousness, manipulativeness, and lack of empathy are continuously distributed in non-institutionalized populations. Here, we show that brain responses to emotional movies in healthy females vary as a function of primary (PP) and secondary psychopathy (SP) traits. Healthy female volunteers (n=50) with variable scorings in Levenson Self-Report Psychopathy Scale watched a set of emotional movie clips (n=39) during functional magnetic resonance imaging of brain activity. We found that participants low on primary and secondary psychopathy traits showed higher cortical and subcortical activity when watching emotional movies, what possibly indicates the somatosensory simulation and empathizing with the emotions of movie characters. On the other hand, the individuals with high primary and secondary psychopathy traits presented a lack of vicarious engagement during watching movies and they manifested brainstem-based heightened response towards thread-related stimuli, which could indicate potent autonomic reactivity. Overall, our results indicate that variability in psychopathic traits in the general population is manifested as differential neural responses to emotional signals, and confirm previous findings of there being separate PP and SP personality characteristics due to the dissociable brain activity patterns while viewing emotional movies.
Elucidating the role of water in collagen self assembly by isotopically modulating collagen hydration
Authors: Giubertoni Giulia , Feng Liru , Klein Kevin , Giannetti Guido , Choi Yeji , van der Net Anouk , Castro-Linares Gerard , Caporaletti Federico , Micha Dimitra , Hunger Johannes , Deblais Antoine , Bonn Daniel , Šarić Andela , Ilie Ioana M. , Koenderink Gijsje H. , Woutersen Sander
Published: August 2023

Water is known to play an important role in collagen self assembly, but it is still largely unclear how water-collagen interactions influence the assembly process and determine the fibril network properties. Here, we use the H2O/D2O isotope effect on the hydrogen-bond strength in water to investigate the role of hydration in collagen self assembly. We dissolve collagen in H2O and D2O, and compare the growth kinetics and the structure of the collagen assemblies formed in these water isotopomers. Surprisingly, collagen assembly occurs ten times faster in D2O than in H2O, and collagen in D2O self assembles into much thinner fibrils, that form a more inhomogeneous and softer network, with a fourfold reduction in elastic modulus compared to H2O. Combining spectroscopic measurements with atomistic simulations, we show that collagen in D2O is less hydrated than in H2O. This partial dehydration lowers the enthalpic penalty for water removal and reorganization at the collagen-water interface, increasing the self assembly rate and the number of nucleation centers, leading to thinner fibrils and a softer network. Coarse-grained simulations show that the acceleration in the initial nucleation rate can be reproduced by the enhancement of electrostatic interactions, which appear to be crucial in determining the acceleration of the initial nucleation rate. These results show that water acts as a mediator between collagen monomers, by moderating their interactions so as to optimize the assembly process and, thus, the final network properties. We believe that isotopically modulating the hydration of proteins can be a valuable method to investigate the role of water in protein structural dynamics and protein self assembly.
Perceptual axioms are irreconcilable with Euclidean geometry
Authors: Zeki Semir , Hale Zachary F , Beyh Ahmad , Rasche Samuel E
Published: August 2023

There are different definitions of axioms, but the one that seems to have general approval is that axioms are statements whose truths are universally accepted but cannot be proven; they are the foundation from which further propositional truths are derived. Previous attempts, led by David Hilbert, to show that all of mathematics can be built into an axiomatic system that is complete and consistent failed when Kurt Gödel proved that there will always be statements which are known to be true but can never be proven within the same axiomatic system. But Gödel and his followers took no account of brain mechanisms that generate and mediate logic. In this largely theoretical paper, but backed by previous experiments and our new ones reported below, we show that in the case of so-called “optical illusions” there exists a significant and irreconcilable difference between their visual perception and their description according to Euclidean geometry; when participants are asked to adjust, from an initial randomised state, the perceptual geometric axioms to conform to the Euclidean description, the two never match, although the degree of mismatch varies between individuals. These results provide evidence that perceptual axioms, or statements known to be perceptually true, cannot be described mathematically. Thus the logic of the visual perceptual system is irreconcilable with the cognitive (mathematical) system and cannot be updated even when knowledge of the difference between the two is available. Hence no one brain reality is more “objective” than any other.
Optimising CNT-FET Biosensor Design: Predictive Modelling of Biomolecular Electrostatic Gating and its Application to Beta-Lactamase Detection
Authors: Gwyther Rebecca E.A. , Côté Sébastien , Lee Chang-Seuk , Ramakrishnan Krithika , Palma Matteo , Jones D. Dafydd
Published: August 2023

Carbon nanotube field effect transistor (CNT-FET) setups hold great promise for constructing next generation miniaturised biosensors whereby a biomolecular event gates conductance. The main issue is predicting how proteins, with their innate mosaic and distinctive electrostatic surfaces, interact with and thus modulate conductance of the CNT-FET. To overcome this barrier, we used advanced sampling molecular dynamics combined with non-canonical amino acid chemistry, to model the protein electrostatic potential imparted on SWCNTs. Here, we focused our efforts using β-lactamase binding protein (BLIP2) as the receptor due to its potential as a biosensor for the most common antibiotic degrading enzymes, the β-lactamases (BLs). Modelling was confirmed experimentally by attaching BLIP2 at single designed residues positions directly to SWCNTs using genetically encoded phenyl azide photochemistry. Our devices were able to successfully detect the two different BLs, TEM-1 and KPC-2, with each BL generating distinct conductance profiles due to differences in their unique surface electrostatic profiles presented close to the SWCNT surface. The changes in conductance closely matched the predicted electrostatic profile sampled by the SWCNTs on BL binding. Thus, our modelling approach combined with new and straight-forward residue-specific receptor attachment techniques, provides a general approach for more effective and optimal CNT-FET biosensor construction.
A dynamical limit to evolutionary adaptation
Authors: Melissa Matthew J. , Desai Michael M.
Published: August 2023

Natural selection makes evolutionary adaptation possible even if the overwhelming majority of new mutations are deleterious. However, in rapidly evolving populations where numerous linked mutations occur and segregate simultaneously, clonal interference and genetic hitchhiking can limit the efficiency of selection, allowing deleterious mutations to accumulate over time. This can in principle overwhelm the fitness increases provided by beneficial mutations, leading to an overall fitness decline. Here, we analyze the conditions under which evolution will tend to drive populations to higher versus lower fitness. Our analysis focuses on quantifying the boundary between these two regimes, as a function of parameters such as population size, mutation rates, and selection pressures. This boundary represents a state in which adaptation is precisely balanced by Muller’s ratchet, and we show that it can be characterized by rapid molecular evolution without any net fitness change. Finally, we consider the implications of global fitness-mediated epistasis, and find that under some circumstances this can drive populations towards the boundary state, which can thus represent a long-term evolutionary attractor.
RNA binding protein IMP1/ZBP1 drivesActbmRNA localization and local translation in microglial peripheral processes and mediates morphological changes, motility and phagocytosis in response to inflammation
Authors: Imaz-Iruretagoyena Josune , Blanco-Urrejola Maite , Núñez-García Irene , García-Toledo Irene , Fernández-Beltrán Luis C , Márquez Mar , Corrochano Silvia , Sierra Amanda , Baleriola Jimena
Published: August 2023

Polarized cells in the brain, such as neurons and glia, rely on the asymmetric distribution of their proteins compartmentalizing the function of dendrites, axons, glial projections and endfeet. Subcellular proteomes can be assembled either by the transport of proteins synthesized in the cell soma or by the delivery of mRNAs to target compartments where they are locally translated into protein. This latter mechanism is known as local protein synthesis or local translation, and it has been best studied in neurons. Increasing evidence suggest it is also required to maintain local protein homeostasis in glial cells, however, in microglia, local translation remains largely unexplored. Given the scant evidence, we aimed at exploring the existence of local translation in peripheral microglial processes (PeMPs) and unravel its functional significance in response to inflammation, a major hallmark of neurodegenerative diseases. We report that local translation in PeMPs is enhanced by triggering a microglial inflammatory response with bacterial lipopolysaccharides (LPS). We found thatActbmRNA polarizes to PeMPs and is locally translated upon LPS exposure. mRNA localization in eukaryotic cells is driven by RNA binding proteins. Interestingly, downregulation of theActbbinding protein IMP1/ZBP1 impairedActbmRNA polarization and its localized translation, and led to defects in filopodia distribution, PeMP motility, lamellar directed migration and phagocytosis in microglia. Thus, our work contributes to recent findings that mRNA localization and localized translation occur in microglia and gives a mechanistic insight into the relevance of this molecular mechanism in fundamental microglial functions in response to inflammation.
Selective inhibition of hsp90 paralogs: Uncovering the role of helix 1 in Grp94-selective ligand binding
Authors: Que Nanette L. S. , Seidler Paul M. , Aw Wen J. , Chiosis Gabriela , Gewirth Daniel T.
Published: August 2023

Grp94 is the endoplasmic reticulum paralog of the hsp90 family of chaperones, which have been targeted for therapeutic intervention via their highly conserved ATP binding sites. The design of paralog-selective inhibitors relies on understanding the protein structural elements that drive higher affinity in selective inhibitors. Here, we determined the structures of Grp94 and Hsp90 in complex with the Grp94-selective inhibitor PU-H36, and of Grp94 with the non-selective inhibitor PU-H71. In Grp94, PU-H36 derives its higher affinity by utilizing Site 2, a Grp94-specific side pocket adjoining the ATP binding cavity, but in Hsp90 PU-H36 occupies Site 1, a side pocket that is accessible in all paralogs with which it makes lower affinity interactions. The structure of Grp94 in complex with PU-H71 shows only Site 1 binding. While changes in the conformation of helices 4 and 5 in the N-terminal domain occur when ligands bind to Site 1 of both Hsp90 and Grp94, large conformational shifts that also involve helix 1 are associated with the engagement of the Site 2 pocket in Grp94 only. Site 2 in Hsp90 is blocked and its helix 1 conformation is insensitive to ligand binding. To understand the role of helix 1 in ligand selectivity, we tested the binding of PU-H36 and other Grp94-selective ligands to chimeric Grp94/Hsp90 constructs. These studies show that helix 1 is the major determinant of selectivity for Site 2 targeted ligands, and also influences the rate of ATPase activity in Hsp90 paralogs.
Intra-host variation in the spike S1/S2 region of a feline coronavirus type-1 in a cat with persistent infection
Authors: Olarte-Castillo Ximena A. , Licitra Beth. N. , André Nicole M. , Sierra Maria A. , Mason Christopher E. , Goodman Laura B. , Whittaker Gary R.
Published: August 2023

Feline coronavirus type 1 (FCoV-1) is widely known for causing feline infectious peritonitis (FIP), a systemic infection that is often fatal, with the virus known as the FIPV biotype. However, subclinical disease also occurs, in which cats may not show signs and intermittently shed the virus, including in feces, possibly for long periods of time. This virus is known as the FECV biotype. Progression of FECV to FIPV has been linked to several genomic changes, however a specific region of the viral spike protein at the interface of the spike S1 and S2 domains has been especially implicated. In this study, we followed a cat (#576) for six years from 2017, at which time FCoV-1 was detected in feces and conjunctival swabs, until 2022, when the animal was euthanized based on a diagnosis of alimentary small cell lymphoma. Over this time period, the cat was clinically diagnosed with inflammatory bowel disease and chronic rhinitis, and cardiac problems were also suspected. Using hybridization capture targeting the spike (S) gene of FCoV followed by next-generation sequencing, we screened 27 clinical samples. We detected FCoV-1 in 4 samples taken in 2017 (intestine and nasal tissue, feces, and conjunctiva), and 3 samples taken in 2022 (feces, and intestinal and heart tissue), but not in fecal samples taken in 2019 and 2020. Next, we focused on the S1/S2 region within S, which contains the furin cleavage site (FCS), a key regulator of viral transmission and pathogenesis. We show that the FCoV-1 variants obtained from feces in 2017 and 2022 were identical, while the ones from conjunctiva (2017), heart (2022), and intestine (2017 and 2022) were distinct. Sequence comparison of all the variants obtained showed that most of the non-synonymous changes in the S1/S2 region occur within the FCS. In the heart, we found two variants that differed by a single nucleotide, resulting in distinct FCS motifs that differ in one amino acid. It is predicted that one of these FCS motifs will down-regulate spike cleavability. The variant from the conjunctiva (2017) had a 6-nucleotide in-frame insertion that resulted in a longer and more exposed S1/S2 loop, which is predicted to be more accessible to the furin protease. Our studies indicate that FCoV-1 can independently persist in the gastrointestinal tract and heart of a cat over a long period of time without evidence of typical FIP signs, with intermittent viral shedding from the gastrointestinal and respiratory tracts.
IST1 regulates select recycling pathways
Authors: Clippinger Amy K. , Naismith Teresa V. , Yoo Wonjin , Jansen Silvia , Kast David J. , Hanson Phyllis I.
Published: August 2023

ESCRTs (Endosomal Sorting Complex Required for Transport) are a modular set of protein complexes with membrane remodeling activities that include the formation and release of intralumenal vesicles (ILVs) to generate multivesicular endosomes. While most of the 12 ESCRT-III proteins are known to play roles in ILV formation, IST1 has been associated with a wider range of endosomal remodeling events. Here, we extend previous studies of IST1 function in endosomal trafficking and confirm that IST1, along with its binding partner CHMP1B, contributes to scission of early endosomal carriers. Depleting IST1 impaired delivery of transferrin receptor from early/sorting endosomes to the endocytic recycling compartment and instead increased its rapid recycling to the plasma membrane via peripheral endosomes enriched in the clathrin adaptor AP-1. IST1 is also important for export of mannose 6-phosphate receptor from early/sorting endosomes. Examination of IST1 binding partners on endosomes revealed that IST1 interacts with the MIT domain-containing sorting nexin SNX15, a protein previously reported to regulate endosomal recycling. Our kinetic and spatial analyses establish that SNX15 and IST1 occupy a clathrin-containing subdomain on the endosomal perimeter distinct from those previously implicated in cargo retrieval or degradation. Using live-cell microscopy we see that SNX15 and CHMP1B alternately recruit IST1 to this subdomain or the base of endosomal tubules. These findings indicate that IST1 contributes to a subset of recycling pathways from the early/sorting endosome.

Abstract Figure
The ciliary MBO2 complex targets assembly of inner arm dynein b and reveals additional doublet microtubule asymmetries
Authors: Fu Gang , Augspurger Katherine , Sakizadeh Jason , Reck Jaimee , Bower Raqual , Tritschler Douglas , Gui Long , Nicastro Daniela , Porter Mary E
Published: August 2023

Ciliary motility requires the spatiotemporal coordination of multiple dynein motors by regulatory complexes located within the 96 nm axoneme repeat. Many organisms can alter ciliary waveforms in response to internal or external stimuli, but little is known about the specific polypeptides and structural organization of complexes that regulate waveforms. In Chlamydomonas, several mutations convert the ciliary waveform from an asymmetric, ciliary-type stroke to a symmetric, flagellar-type stroke. Some of these mutations alter subunits located at the inner junction of the doublet microtubule and others alter interactions between the dynein arms and the radial spokes. These and other axonemal substructures are interconnected by a network of poorly characterized proteins. Here we re-analyze several motility mutants (mbo, fap57, pf12/pacrg) to identify new components in this network. The mbo (move backwards only) mutants are unable to swim forwards with an asymmetric waveform. Proteomics identified more than 19 polypeptides that are missing or reduced in mbo mutants, including one inner dynein arm, IDA b. Several MBO2-associated proteins are also altered in fap57 and pf12/parcg mutants, suggesting overlapping networks. Two subunits are highly conserved, coiled coil proteins found in other species with motile cilia and others contain potential signaling domains. Cryo-electron tomography and epitope tagging revealed that the MBO2 complex is found on specific doublet microtubules and forms a large, L-shaped structure that contacts the base of IDA b that interconnects multiple dynein regulatory complexes and varies in a doublet microtubule specific fashion.
MicroED as a powerful tool for structure determination of macrocyclic drug compounds directly from their powder formulations
Authors: Danelius E , Bu G , Wieske H , Gonen T
Published: August 2023

Macrocycles are important drug leads with many advantages including the ability to target flat and featureless binding sites as well as act as molecular chameleons and thereby reach intracellular targets. However, due to their complex structures and inherent flexibility, macrocycles are difficult to study structurally and there are limited structural data available. Herein, we use the cryo-EM method MicroED to determine the novel atomic structures of several macrocycles which have previously resisted structural determination. We show that structures of similar complexity can now be obtained rapidly from nanograms of material, and that different conformations of flexible compounds can be derived from the same experiment. These results will have impact on contemporary drug discovery as well as natural product exploration.
PvDBPII-Matrix M elicits polyfunctional antibodies that limit parasite growth in a challenge trial
Authors: Martinez Francisco J. , White Michael , Guillotte-Blisnick Micheline , Huon Christèle , Boucharlat Alix , Agou Fabrice , England Patrick , Popovici Jean , Hou Mimi M. , Silk Sarah E. , Barrett Jordan R. , Nielsen Carolyn M. , Reimer Jenny M. , Mukherjee Paushali , Chauhan Virander S. , Minassian Angela M. , Draper Simon J. , Chitnis Chetan E.
Published: August 2023

The receptor-binding domain, region II, ofPlasmodium vivaxDuffy binding protein (PvDBPII) binds the Duffy antigen on reticulocytes to mediate invasion. A heterologous vaccine challenge trial recently showed that a delayed dosing regimen with recombinant PvDBPII SalI formulated with adjuvant Matrix-MTMreduced thein vivoparasite multiplication rate (PMR) challenged with theP. vivaxThai isolate PvW1. We describe extensive analysis of the polyfunctional antibody responses elicited by PvDBPII immunization and identify immune correlates for PMR reduction. A classification algorithm identified antibody features that contribute significantly to PMR reduction. These included antibody titre, receptor-binding inhibitory titre, dissociation constant for PvDBPII-antibody interaction, complement C1q and Fc gamma receptor binding and specific IgG subclasses. These data suggest that multiple immune mechanisms elicited by PvDBPII immunization are associated with protection. The identified immune correlates could guide the development of an effective vaccine forP. vivaxmalaria. Importantly, all the polyfunctional antibody features that correlated with protection cross-reacted with both PvDBPII SalI and PvW1 variants, suggesting that immunization with PvDBPII should protect against diverseP. vivaxisolates.
Strong immunogenicity & protection in mice with PlaCCine: A COVID-19 DNA vaccine formulated with a functional polymer
Authors: Sood Subeena , Matar Majed M. , Kim Jessica , Kinsella Meredyth , Rayavara Kempaiah , Signer Olivia , Henderson John , Rogers Joseph , Chawla Bhavna , Narvaez Brandon , Van Ry Alex , Kar Swagata , Arnold Austin , Rice Jennifer S. , Smith Alanna M. , Su Daishui , Sparks Jeff , Goff Corinne Le , Boyer Jean D. , Anwer Khursheed
Published: August 2023

DNA- based vaccines have demonstrated the potential as a safe and effective modality. PlaCCine, a DNA-based vaccine approach described subsequently relies on a synthetic DNA delivery system and is independent of virus or device. The synthetic functionalized polymer combined with DNA demonstrated stability over 12 months at 4C and for one month at 25C. Transfection efficiency compared to naked DNA increased by 5-15-fold in murine skeletal muscle. Studies of DNA vaccines expressing spike proteins from variants D614G (pVAC15), Delta (pVAC16), or a D614G + Delta combination (pVAC17) were conducted. Mice immunized intramuscular injection (IM) with pVAC15, pVAC16 or pVAC17 formulated with functionalized polymer and adjuvant resulted in induction of spike-specific humoral and cellular responses. Antibody responses were observed after one immunization. And endpoint IgG titers increased to greater than 1x 105two weeks after the second injection. Neutralizing antibodies as determined by a pseudovirus competition assay were observed following vaccination with pVAC15, pVAC16 or pVAC17. Spike specific T cell immune responses were also observed following vaccination and flow cytometry analysis demonstrated the cellular immune responses included both CD4 and CD8 spike specific T cells. The immune responses in vaccinated mice were maintained for up to 14 months after vaccination. In an immunization and challenge study of K18 hACE2 transgenic mice pVAC15, pVAC16 and pVAC17 induced immune responses lead to decreased lung viral loads by greater than 90% along with improved clinical score. These findings suggest that PlaCCine DNA vaccines are effective and stable and further development against emerging SARS-CoV-2 variants is warranted.
Structure of an open KATPchannel reveals tandem PIP2binding sites mediating the Kir6.2 and SUR1 regulatory interface
Authors: Driggers Camden M. , Kuo Yi-Ying , Zhu Phillip , ElSheikh Assmaa , Shyng Show-Ling
Published: August 2023

ATP-sensitive potassium (KATP) channels, composed of four pore-lining Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) subunits, control insulin secretion in pancreatic β-cells. KATPchannel opening is stimulated by PIP2and inhibited by ATP. Mutations that increase channel opening by PIP2reduce ATP inhibition and cause neonatal diabetes. Although considerable evidence has implicated a role for PIP2in KATPchannel function, previously solved open-channel structures have lacked bound PIP2, and mechanisms by which PIP2regulates KATPchannels remain unresolved. Here, we report cryoEM structure of a KATPchannel harboring the neonatal diabetes mutation Kir6.2-Q52R, bound to natural C18:0/C20:4 long-chain PI(4,5)P2in open conformation. The structure reveals two adjacent PIP2molecules between SUR1 and Kir6.2. The first PIP2binding site is conserved among PIP2-gated Kir channels. The second site forms uniquely in KATPat the interface of Kir6.2 and SUR1. Functional studies demonstrate both binding sites determine channel activity. Kir6.2 pore opening is associated with a twist of the Kir6.2 cytoplasmic domain and a rotation of the N-terminal transmembrane domain of SUR1, which widens the inhibitory ATP binding pocket to disfavor ATP binding. The open conformation is particularly stabilized by the Kir6.2-Q52R residue through cation-π bonding with SUR1 - W51. Together, these results uncover the cooperation between SUR1 and Kir6.2 in PIP2binding and gating, explain the antagonistic regulation of KATPchannels by PIP2and ATP, and provide the mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.
Wheat bZIPC1 interacts with FT2 and contributes to the regulation of spikelet number per spike
Authors: Glenn Priscilla , Woods Daniel P. , Zhang Junli , Gabay Gilad , Odle Natalie , Dubcovsky Jorge
Published: August 2023

Loss-of-function mutations and natural variation in the geneFLOWERING LOCUS T2(FT2) in wheat have previously been shown to affect spikelet number per spike (SNS). However, while other FT-like wheat proteins interact with bZIP-containing transcription factors from the A-group, FT2 does not interact with any of them. In this study, we used a yeast-two-hybrid screen with FT2 as bait and identified a grass-specific bZIP-containing transcription factor from the C-group, designated here as bZIPC1. Within the C-group, we identified four clades including wheat proteins that show Y2H interactions with different sets of FT-like and CEN-like encoded proteins.bZIPC1andFT2expression partially overlap in the developing spike, including the inflorescence meristem. Combined loss-of-function mutations inbZIPC-A1andbZIPC-B1(bzipc1) in tetraploid wheat resulted in a drastic reduction in SNS with a limited effect on heading date. Analysis of natural variation in thebZIPC-B1(TraesCS5B02G444100) region revealed three major haplotypes (H1-H3), with the H1 haplotype showing significantly higher SNS, grain number per spike and grain weight per spike than both the H2 and H3 haplotypes. The favorable effect of the H1 haplotype was also supported by its increased frequency from the ancestral cultivated tetraploids to the modern durum and common wheat varieties. We developed markers for the two non-synonymous SNPs that differentiate thebZIPC-B1ballele in the H1 haplotype from the ancestralbZIPC-B1aallele present in all other haplotypes. These diagnostic markers are useful tools to accelerate the deployment of the favorablebZIPC-B1ballele in pasta and bread wheat breeding programs.

Key Message

The wheat transcription factor bZIPC1 interacts with FT2 and affects spikelet and grain number per spike. We identified a natural allele with positive effects on these two economically important traits.