bluuebunny/crossref_metadata_2025_split huggingface dataset:
Parallel evolution and enhanced virulence upon in vivo passage of an RNA virus in Drosophila melanogaster
Authors: Lezcano Oscar M , Fuhrmann Lara , Ramakrishnan Gayatri , Beerenwinkel Niko , Huynen Martijn A , van Rij Ronald P 
Published: December 2023

Abstract
Virus evolution is strongly affected by antagonistic co-evolution of virus and host. Host immunity positively selects for viruses that evade the immune response, which in turn may drive counter-adaptations in host immune genes. We investigated how host immune pressure shapes virus populations, using the fruit fly Drosophila melanogaster and its natural pathogen Drosophila C virus (DCV), as a model. We performed an experimental evolution study in which DCV was serially passaged for ten generations in three fly genotypes differing in their antiviral RNAi response: wild-type flies and flies in which the endonuclease gene Dicer-2 was either overexpressed or inactivated. All evolved virus populations replicated more efficiently in vivo and were more virulent than the parental stock. The number of polymorphisms increased in all three host genotypes with passage number, which was most pronounced in Dicer-2 knockout flies. Mutational analysis showed strong parallel evolution, as mutations accumulated in a specific region of the VP3 capsid protein in every lineage in a host genotype-independent manner. The parental tyrosine at position ninety-five of VP3 was substituted with either one of five different amino acids in fourteen out of fifteen lineages. However, no consistent amino acid changes were observed in the viral RNAi suppressor gene 1A, nor elsewhere in the genome in any of the host backgrounds. Our study indicates that the RNAi response restricts the sequence space that can be explored by viral populations. Moreover, our study illustrates how evolution towards higher virulence can be a highly reproducible, yet unpredictable process.
Global value chains in EU law
Authors: Beckers Anna 
Published: December 2023

Abstract
There is a burgeoning legal literature about the legal underpinnings of global value chains (GVCs). This article contributes to this literature through the lens of European Union (EU) law and proposes a different conceptual way to read GVCs legally. To that end, the contribution proposes understanding the evolving EU law on GVCs as a process of institutionalization leading to at least three legal forms. In EU company law, GVCs manifest actor-centrically as corporate obligations to govern the value chain. This mostly happens in the policies related to sustainability, but also features in some digitalization policies. In EU consumer law, the value chain appears as a collective network of actors that also bears collective responsibility towards the consumer for the production process. Moreover, in EU market practices and trade law, the value chain is approached in a de-personified manner by, on the one hand, targeting products relating to the territory (import/internal market access) and, on the other hand, trading practices in relations characterised by power asymmetry. These findings suggests that, rather than identifying a uniform legal concept of GVCs in EU law, a fragmented picture emerges in which different sub-areas of EU law develop different, partly even opposing, legal understandings of GVCs.
An adeno‐associated viral labeling approach to visualize the meso‐ and microanatomy of mechanosensory afferents and autonomic innervation of the rat urinary bladder
Authors: Wiedmann Nicole M. , Fuller‐Jackson John‐Paul , Osborne Peregrine B. , Keast Janet R. 
Published: December 2023

AbstractThe urinary bladder is supplied by a rich network of sensory and autonomic axons, commonly visualized by immunolabeling for neural markers. This approach demonstrates overall network patterning but is less suited to understanding the structure of individual motor and sensory terminals within these complex plexuses. There is a further limitation visualizing the lightly myelinated (A‐delta) class of sensory axons that provides the primary mechanosensory drive for initiation of voiding. Whereas most unmyelinated sensory axons can be revealed by immunolabeling for specific neuropeptides, to date no unique neural marker has been identified to immunohistochemically label myelinated visceral afferents. We aimed to establish a non‐surgical method to visualize and map myelinated afferents in the bladder in rats. We found that in rats, the adeno‐associated virus (AAV), AAV‐PHP.S, which shows a high tropism for the peripheral nervous system, primarily transduced myelinated dorsal root ganglion neurons, enabling us to identify the structure and regional distribution of myelinated (mechanosensory) axon endings within the muscle and lamina propria of the bladder. We further identified the projection of myelinated afferents within the pelvic nerve and lumbosacral spinal cord. A minority of noradrenergic and cholinergic neurons in pelvic ganglia were transduced, enabling visualization and regional mapping of both autonomic and sensory axon endings within the bladder. Our study identified a sparse labeling approach for investigating myelinated sensory and autonomic axon endings within the bladder and provides new insights into the nerve‐bladder interface.
From biosynthesis and beyond—Loss or overexpression of the cytokinin synthesis gene, iptA, alters cytokinesis and mitochondrial and amino acid metabolism in Dictyostelium discoideum
Authors: Aoki Megan M. , Kisiala Anna B. , Mathavarajah Sabateeshan , Schincaglia Angela , Treverton Jared , Habib Elias , Dellaire Graham , Emery R. J. Neil , Brunetti Craig R. , Huber Robert J. 
Published: December 2023

AbstractCytokinins (CKs) are a class of growth‐promoting signaling molecules that affect multiple cellular and developmental processes. These phytohormones are well studied in plants, but their presence continues to be uncovered in organisms spanning all kingdoms, which poses new questions about their roles and functions outside of plant systems. Cytokinin production can be initiated by one of two different biosynthetic enzymes, adenylate isopentenyltransfases (IPTs) or tRNA isopentenyltransferases (tRNA‐IPTs). In this study, the social amoeba, Dictyostelium discoideum, was used to study the role of CKs by generating deletion and overexpression strains of its single adenylate‐IPT gene, iptA. The life cycle of D. discoideum is unique and possesses both single‐ and multicellular stages. Vegetative amoebae grow and divide while food resources are plentiful, and multicellular development is initiated upon starvation, which includes distinct life cycle stages. CKs are produced in D. discoideum throughout its life cycle and their functions have been well studied during the later stages of multicellular development of D. discoideum. To investigate potential expanded roles of CKs, this study focused on vegetative growth and early developmental stages. We found that iptA‐deficiency results in cytokinesis defects, and both iptA‐deficiency and overexpression results in dysregulated tricarboxylic acid (TCA) cycle and amino acid metabolism, as well as increased levels of adenosine monophosphate (AMP). Collectively, these findings extend our understanding of CK function in amoebae, indicating that iptA loss and overexpression alter biological processes during vegetative growth that are distinct from those reported during later development.
An efficient plasmid-based system for the recovery of recombinant vesicular stomatitis virus encoding foreign glycoproteins
Authors: Marqués María-Carmen , Andreu-Moreno Iván , Sanjuán Rafael , Elena Santiago F. , Geller Ron 
Published: December 2023

AbstractViral glycoproteins mediate entry into host cells, thereby dictating host range and pathogenesis. In addition, they constitute the principal target of neutralizing antibody responses, making them important antigens in vaccine development. Recombinant vesicular stomatitis virus (VSV) encoding foreign glycoproteins can provide a convenient and safe surrogate system to interrogate the function, evolution, and antigenicity of viral glycoproteins from viruses that are difficult to manipulate or those requiring high biosafety levels containment. However, the production of recombinant VSV can be technically challenging. In this work, we present an efficient and robust plasmid-based system for the production of recombinant VSV encoding foreign glycoproteins. We validate the system using glycoproteins from different viral families, including arenaviruses, coronaviruses, and hantaviruses, as well as highlight their utility for studying the effects of mutations on viral fitness. Overall, the methods described herein can facilitate the study of both native and recombinant VSV encoding foreign glycoproteins and can serve as the basis for the production of VSV-based vaccines.
Fertilisation of agricultural soils with municipal biosoilds: Part 2- Site properties, environmental factors, and crop identify influence soil bacterial communities more than municipal biosolid application
Authors: Blakney Andrew J.C. , Morvan Simon , Lucotte Marc , Moingt Matthieu , Charbonneau Ariane , Bipfubusa Marie , Gonzalez Emmanuel , Pitre Frédéric E. 
Published: December 2023

AbstractReducing the environmental impact of Canadian field crop agriculture, including the reliance on conventional synthesised fertilisers, are key societal targets for establishing long-term sustainable practices. Municipal biosolids (MSB) are an abundant, residual organic material, rich in phosphate, nitrogen and other oligo-nutrients, that could be used in conjunction with conventional fertilisers to decrease their use. Though MBS have previously been shown to be an effective fertiliser substitute for different crops, including corn and soybean, there remain key knowledge gaps concerning the impact of MBS on the resident soil bacterial communities in agro-ecosystems. We hypothesised that the MBS fertiliser amendment would not significantly impact the structure or function of the soil bacterial communities, nor contribute to the spread of human pathogenic bacteria, in corn or soybean agricultural systems. In field experiments, fertiliser regimes for both crops were amended with MBS, and compared to corn and soybean plots with standard fertiliser treatments. We repeated this across four different agricultural sites in Quebec, over 2021 and 2022. We sampled MBS-treated, and untreated soils, and identified the composition of the soil bacterial communities via 16S rRNA metabarcoding. We found no indication that the MBS fertiliser amendment altered the structure of the soil bacterial communities, but rather that the soil type and crop identities were the most significant factors in structuring the bacterial communities. Moreover, there was no evidence that the MBS-treated soils experienced a shift in functions, nor contributed potential human bacterial pathogens over the two years of our study. Our analysis indicates that not only can MBS function as substitutes for conventional, synthesised fertilisers, but that they also do not disrupt the structure, or function, of the resident soil bacterial communities in the short term. Finally, we suggest that the use of MBS in agro-ecosystems poses no greater concern to the public than existing soil bacterial communities.HighlightsMunicipal biosolids may represent a sustainable fertiliser substituteBut, the impact of biosolids on soil bacteria in agricultural fields is unknownUsing 16S rRNA metabarcoding we analysed community structure and functionsWe found no disruption of soil bacterial communities fertilised with biosolidsBiosolids are safe, sustainable fertilisers with little impact on soil bacteriaGraphical Abstract
miR-519a-3p, found to regulate cellular prion protein during Alzheimer’s disease pathogenesis, as a biomarker of asymptomatic stages
Authors: Jácome Dayaneth , Cotrufo Tiziana , Andrés-Benito Pol , Martí Eulàlia , Ferrer Isidre , del Río José Antonio , Gavín Rosalina 
Published: December 2023

AbstractMiRNAs induce post-transcriptional gene silencing by binding to the 3’-UTR of complementary messenger RNAs and causing either degradation or inhibition of translation.The clinical relevance of miRNAs as biomarkers is growing due to their stability and detection in biofluids. In this sense, diagnosis at asymptomatic stages of Alzheimer’s disease (AD) remains a challenge since it can only be made at autopsy according to Braak NFT staging. Achieving the objective of detecting AD at early stages would allow possible therapies to be addressed before the onset of cognitive impairment.Many studies have determined that the expression pattern of some miRNAs is deregulated in AD patients, but to date, none has been correlated with downregulated expression of cellular prion protein (PrPC) during disease progression. That is why, by means of cross studies of miRNAs up-regulated in AD within silicoidentification of potential miRNAs-binding to 3’UTR of humanPRNPgene, we selected miR-519a-3p for our study.Other family members of miR-519 have been shown to bind to the 3’UTR region ofPRNP in vitroand presumably degradePRNPmRNA. In addition, up-regulation of some of them has been reported in various tissues from AD patients, including cerebrospinal fluid, plasma, and blood serum. In fact, miR-519d-3p is marked as a bridge regulator between mild cognitive impairment and severe AD. However, none of the studies address the prodromal stages of the disease or the expression profile of miR-519 in other neurodegenerative diseases that also may present dementia. Therefore, in this study we analyzed miR-519a-3p expression in cerebral samples of AD at different stages of evolution as well as other neurodegenerative diseases such as other tauopathies and synucleinopathies. Our results show the specific and early upregulation of miR-519a-3p starting from Braak stage I of AD, suggesting its potential use as a biomarker of preclinical stages of the disease.
Benchmarking the identification of a single degraded protein to explore optimal search strategies for ancient proteins
Authors: Rodriguez Palomo Ismael , Nair Bharath , Chiang Yun , Dekker Joannes , Dartigues Benjamin , Mackie Meaghan , Evans Miranda , Macleod Ruairidh , Olsen Jesper V. , Collins Matthew J. 
Published: December 2023

AbstractPalaeoproteomics is a rapidly evolving discipline, and practitioners are constantly developing novel strategies for the analyses and interpretations of complex, degraded protein mixtures. The community has also established standards of good practice to interrogate our data. However, there is a lack of a systematic exploration of how these affect the identification of peptides, post-translational modifications (PTMs), proteins and their significance (through the False Discovery Rate) and correctness. We systematically investigated the performance of a wide range of sequencing tools and search engines in a controlled system: the experimental degradation of the single purified bovine β-lactoglobulin (BLG), heated at 95 °C and pH 7 for 0, 4 and 128 days. We target BLG since it is one of the most robust and ubiquitous proteins in the archaeological record. We tested different reference database choices, a targeted dairy protein one, and the whole bovine proteome and the three digestion options (tryptic-, semi-tryptic- and non-specific searches), in order to evaluate the effects of search space and the identification of peptides. We also explored alternative strategies, including open search that allows for the global identification of PTMs based upon wide precursor mass tolerance andde novosequencing to boost sequence coverage. We analysed the samples using Mascot, MaxQuant, Metamorpheus, pFind, Fragpipe and DeNovoGUI (pepNovo+, DirecTag, Novor), benchmarked these tools and discuss the optimal strategy for the characterisation of ancient proteins. We also studied physicochemical properties of the BLG that correlate with bias in the identification coverage.
Refinement of Cryo-EM 3D Maps with Self-Supervised Denoising Model: crefDenoiser
Authors: Agarwal Ishaant , Kaczmar-Michalska Joanna , Nørrelykke Simon F. , Rzepiela Andrzej J. 
Published: December 2023

AbstractCryogenic electron microscopy (cryo-EM) is a pivotal technique for imaging macromolecular structures. Despite extensive processing of large image sets collected in a cryo-EM experiment to amplify the signal-to-noise ratio, the reconstructed 3D protein density maps are often limited in quality due to residual noise, which in turn affects the accuracy of the macromolecular representation. In this paper, we introduce crefDenoiser, a denoising neural network model designed to enhance the signal in 3D cryo-EM maps produced with standard processing pipelines, beyond the current state of the art. crefDenoiser is trained without the need for ‘clean’, ground-truth target maps. Instead, we employ a custom dataset composed of real noisy protein half-maps sourced from the Electron Microscopy Data Bank repository. Strong model performance is achieved by optimizing for the theoretical noise-free map during self-supervised training. We demonstrate that our model successfully amplifies the signal across a wide variety of protein maps, outperforming a classical map denoiser and a network-based sharpening model. Without biasing the map, the proposed denoising method often leads to improved visibility of protein structural features, including protein domains, secondary structure elements, and amino-acid side chains.
Structural and mechanistic views of enzymatic, heme-dependent nitrogen-nitrogen bond formation
Authors: Higgins Melanie A. , Shi Xinjie , Soler Jordi , Harland Jill B. , Parkkila Taylor , Lehnert Nicolai , Garcia-Borràs Marc , Du Yi-Ling , Ryan Katherine S. 
Published: December 2023

SummaryMolecules with nitrogen-nitrogen (N-N) bonds constitute a large group of clinically important drugs, and various synthetic approaches have been developed to construct functional groups like hydrazines, diazos, pyrazoles, and N-nitrosos. While hundreds of N-N-containing specialized natural metabolites have also been discovered, little is known about the underlying enzymatic mechanisms that have evolved for N-N bond formation. In order to directly form a single N(sp3)-N(sp3) bond, enzymes must reverse the typical nucleophilicity of one nitrogen. Here, we report structural and mechanistic interrogations of the piperazate synthase PipS, a heme-dependent enzyme that catalyzes an N-N bond forming cyclization ofN5-OH-L-ornithine to give the non-proteinogenic amino acid L-piperazic acid. We show that PipS can process a variety ofN-substituted hydroxylamines, to give either an imine or an N-N bond, in a substrate-specific manner. Using a combination of structural and biochemical experiments, computational studies, and spectroscopic characterization, we propose that heme-dependent dehydration and N-N bond formation in PipS proceed through divergent pathways, which may stem from a shared nitrenoid intermediate that effectively reverses the nucleophilicity of the hydroxylamine nitrogen. Our work expands the current knowledge of enzymatic N-N bond formation, and delineates the catalytic versatility of a heme cofactor, paving the way for future development of genetically encoded biocatalysts for N-N bond formation.
Synergistic insulation of regulatory domains by developmental genes and clusters of CTCF sites
Authors: Ealo Thais , Sanchez-Gaya Victor , Respuela Patricia , Martín María Muñoz-San , Martin-Batista Elva , Haro Endika , Rada-Iglesias Alvaro 
Published: December 2023

AbstractThe specificity of gene expression during development requires the insulation of regulatory domains to avoid inappropriate enhancer-gene interactions. In vertebrates, this insulator function is mostly attributed to clusters of CTCF sites located at topologically associating domain (TAD) boundaries. However, TAD boundaries allow a certain level of physical crosstalk across regulatory domains, which is at odds with the highly specific and precise expression of developmental genes. Here we show that developmental genes and nearby clusters of CTCF sites synergistically foster the robust insulation of regulatory domains. Firstly, we found that the TADs containing developmental genes have distinctive features, including the sequential organization of developmental genes and CTCF clusters near TAD boundaries. Most importantly, by genetically dissecting representative loci in mouse embryonic stem cells, we showed that developmental genes and CTCF sites synergistically strengthened the insulation capacity of nearby boundaries through different mechanisms. Namely, while CTCF sites prevent undesirable enhancer-gene contacts (i.e.physical insulation), developmental genes preferentially contribute to regulatory insulation through non-structural mechanisms involving promoter competition rather than enhancer blocking. Overall, our work provides important insights into the specificity of gene regulation, which in turn might help interpreting the pathological consequences of certain structural variants.
Recent evolution of large offspring size and post-fertilization nutrient provisioning in swordtails
Authors: Payne Cheyenne Y. , Ly Derek , Rodriguez-Soto Rebecca A. , Powell Daniel L. , Robles Nim D. , Gunn Theresa , Bazcenas John J , Bergman Abby J. , Pollock Alexa , Moran Ben M. , Baker Julie C. , Reznick David , Schumer Molly 
Published: December 2023

AbstractOrganisms have evolved diverse reproductive strategies that impact the probability that their offspring survive to adulthood. Here, we describe divergence in reproductive strategy between two closely related species of swordtail fish (Xiphophorus). Swordtail fish and their relatives have evolved viviparity: they have internal fertilization and give birth to fully developed fry. We find that one species,X. malinche, which lives in high-elevation environments, has evolved larger offspring than its closest relativeX. birchmanniand dwarfs the offspring size of other species in the genus. The larger fry ofX. malincheare more resilient to starvation than theirX. birchmannirelatives, hinting that the evolution of large offspring size may be an adaptation to the particularly challenging environments in whichX. malincheare born. We find evidence thatX. malincheachieves larger offspring size in part by continuing to provision their offspring over the course of embryonic development after fertilization, the first time this process has been documented in theXiphophorusgenus. Moreover, we observe differential regulation in the ovary of genes associated with maternal nutrient provisioning in other species that use this reproductive strategy. Intriguingly, these reproductive differences may drive an asymmetric hybrid incompatibility, sinceX. birchmannimothers pregnant with F1embryos give birth to premature and stillborn fry at an exceptionally high rate.