bluuebunny/crossref_metadata_2025_split huggingface dataset:
Metabolic analysis in intact human-derived cerebral organoids by high-resolution magic-angle spinning NMR spectroscopy
Authors: Castilla Bolanos Maria Alejandra , Chinchalongporn Vorapin , Biswas Rajshree Ghosh , Bailey Colleen , Wu Maggie , Soong Ronald , Saleh Fermisk , Simpson Andre , Schuurmans Carol , Near Jamie 
Published: June 2024

AbstractHuman-derived cerebral organoids (COs) are an emerging model system for the study of human neural development and physiology. Here, we describe the assessment of metabolism in human-derived CO’s using high-resolution magic-angle spinning (HR-MAS) NMR spectroscopy. Metabolic changes during development are assessed by studying COs at various stages of maturity. Our results suggest that COs exhibit a metabolic profile similar toin vivohuman brain metabolism, albeit with a few notable metabolic differences.
Comparison of the CDC2-like kinase family across eukaryotes highlights the functional conservation of these unique biological thermometers
Authors: Ogle Rachel A. , Netherton Jacob K. , Robinson Benjamin R. , Heyd Florian , Zhang Xu Dong , Baker Mark A. 
Published: June 2024

AbstractThe family of CDC2-like kinases (CLKs) play a crucial role in regulating alternative splicing (AS), a process fundamental to eukaryotic gene expression and adaptation. Of particular interest, these enzymes exhibit unique responsiveness to minor temperature shifts, enabling them to modulate AS accordingly. Dysregulated CLK expression is linked to a wide variety of human diseases, establishing them as promising therapeutic targets. Despite the importance of CLKs, limited research has explored the genetic and functional diversification of this gene family. This report investigates the evolutionary origins, diversification, and functional implications of CLKs across major eukaryotic lineages through phylogenetic and structural comparisons. Our data demonstrate these kinases are prevalent throughout eukaryotes, with the original gene (which shares orthology to human CLK2), dating back to the Last Eukaryotic Common Ancestor. We identified three key duplication events in vertebrates, highlighting how this gene family has expanded and diversified in complex metazoans. Despite two instances of CLK paralog loss in vertebrate lineages, CLKs remain prevalent throughout metazoans, suggesting they are essential for complex eukaryotic life. Structural comparisons across diverse eukaryotes demonstrate kinase domain conservation, which is in line with their maintained function in AS regulation. While their N-terminal regions vary significantly in amino acid sequence, the function of this domain to regulate phosphorylation of AS factors is conserved, albeit in a species-specific manner. CLKs exhibit unique thermo-sensitive properties across diverse species, challenging conventional enzymatic behaviour. This temperature regulation, mediated by their kinase activation segment, is characterised by increased activity at lower physiological temperatures. The conservation of this structure, and a thermo-sensitive amino acid motif within it, suggests this was an ancient adaptation for responding to environmental cues. Species-specific temperature profiles highlight the adaptive evolution of CLKs, enabling organisms to thrive in diverse environmental conditions including extreme temperatures. Our analysis expands the understanding of CLK biology across diverse eukaryotes and connects insights from model organisms to human biology.
Essentiality of LD-Transpeptidation inAgrobacterium tumefaciens
Authors: Aliashkevich Alena , Guest Thomas , Alvarez Laura , Gilmore Michael C. , Amstutz Jennifer , Mateus André , Schiffthaler Bastian , Ruiz Iñigo , Typas Athanasios , Savitski Mikhail M. , Brown Pamela J. B. , Cava Felipe 
Published: June 2024

AbstractPeptidoglycan (PG), a mesh-like structure which is the primary component of the bacterial cell wall, is crucial to maintain cell integrity and shape. While most bacteria rely on penicillin binding proteins (PBPs) for crosslinking, some species employ LD-transpeptidases (LDTs). Unlike PBPs, the essentiality and biological functions of LDTs remain largely unclear. The Hyphomicrobiales order of the Alphaproteobacteria, known for their polar growth, have PG which is unusually rich in LD-crosslinks, suggesting that LDTs may play a more significant role in PG synthesis in these bacteria. Here, we investigated LDTs in the plant pathogenAgrobacterium tumefaciensand found that LD-transpeptidation, resulting from at least one of 14 putative LDTs present in this bacterium, is essential for its survival. Notably, a mutant lacking a distinctive group of 7 LDTs which are broadly conserved among the Hyphomicrobiales exhibited reduced LD-crosslinking and tethering of PG to outer membrane β-barrel proteins. Consequently, this mutant suffered severe fitness loss and cell shape rounding, underscoring the critical role played by these Hyphomicrobiales-specific LDTs in maintaining cell wall integrity and promoting elongation. Tn-sequencing screens further revealed non-redundant functions forA. tumefaciensLDTs. Specifically, Hyphomicrobiales-specific LDTs exhibited synthetic genetic interactions with division and cell cycle proteins, and a single LDT from another group. Additionally, our findings demonstrate that strains lacking all LDTs except one displayed distinctive phenotypic profiles and genetic interactions. Collectively, our work emphasizes the critical role of LD-crosslinking inA. tumefacienscell wall integrity and growth and provides insights into the functional specialization of these crosslinking activities.
Simultaneous polyclonal antibody sequencing and epitope mapping by cryo electron microscopy and mass spectrometry – a perspective
Authors: Schulte Douwe , Šiborová Marta , Käll Lukas , Snijder Joost 
Published: June 2024

AbstractAntibodies are a major component of adaptive immunity against invading pathogens. Here we explore possibilities for an analytical approach to characterize the antigen-specific antibody repertoire directly from the secreted proteins in convalescent serum. This approach aims to perform simultaneous antibody sequencing and epitope mapping using a combination of single particle cryo-electron microscopy (cryoEM) and bottom-up proteomics techniques based on mass spectrometry (LC-MS/MS). We evaluate the performance of the deep-learning tool ModelAngelo in determiningde novoantibody sequences directly from reconstructed 3D volumes of antibody-antigen complexes. We demonstrate that while map quality is a critical bottleneck, it is possible to sequence antibody variable domains from cryoEM reconstructions with accuracies of up to 80-90%. While the rate of errors exceeds the typical levels of somatic hypermutation, we show that the ModelAngelo-derived sequences can be used to assign the used V-genes. This provides a functional guide to assemblede novopeptides from LC-MS/MS data more accurately and improves the tolerance to a background of polyclonal antibody sequences. Following this proof-of-principle, we discuss the feasibility and future directions of this approach to characterize antigen-specific antibody repertoires.
Mono-allelic epigenetic regulation of bi-directional silencing of RNA Polymerase II polycistronic transcription initiation inTrypanosoma brucei
Authors: Kieft Rudo , Cliffe Laura , Yan Haidong , Schmitz Robert J. , Hajduk Stephen L. , Sabatini Robert 
Published: June 2024

ABSTRACTUnique for a eukaryote, protein-coding genes in trypanosomes are arranged in polycistronic units (PTUs). This genome arrangement has led to a model where Pol II transcription of PTUs is unregulated and that changes in gene expression are entirely post-transcriptional. Trypanosoma brucei brucei is unable to infect humans because of its susceptibility to an innate immune complex, trypanosome lytic factor (TLF) in the circulation of humans. The initial step in TLF mediated lysis ofT.b.bruceirequires high affinity haptoglobin/hemoglobin receptor (HpHbR) binding. Here we demonstrate that byin vitroselection with TLF, resistance is obtained in a stepwise process correlating with loss of HpHbR expression at an allelic level. RNA-seq, Pol II ChIP and run-on analysis indicate HpHbR silencing is at the transcriptional level, where loss of Pol II binding at the promoter region specifically shuts down transcription of the HpHbR containing gene cluster and the adjacent opposing gene cluster. Reversible transcriptional silencing of the divergent PTUs correlates with DNA base J modification of the shared promoter region. Therefore, we show that epigenetic mechanisms, including base J modification, are involved in regulating gene expression via Pol II transcription initiation of gene clusters in a mono-allelic fashion. These findings suggest epigenetic chromatin-based regulation of gene expression is deeply conserved among eukaryotes, including early divergent eukaryotes that rely on polycistronic transcription.IMPORTANCEThe single-cell parasiteTrypanosoma bruceicauses lethal diseases in both humans and livestock.T. bruceiundergoes multiple developmental changes to adapt in different environments during its digenetic life cycle. With protein-coding genes organized as polycistronic transcription and apparent absence of promoter-mediated regulation of transcription initiation, it is believed that developmental gene regulation in trypanosomes is essentially post-transcriptional. In this study, we found reversible Pol II transcriptional silencing of two adjacent polycistronic gene arrays that correlates with the novel DNA base J modification of the shared promoter region. Our findings support epigenetic regulation of Pol II transcription initiation as a viable mechanism of gene expression control inT. brucei. This has implications for our understanding how trypanosomes utilize polycistronic genome organization to regulate gene expression during its life cycle.
NMR structures of small molecules bound to a model of an RNA CUG repeat expansion
Authors: Chen Jonathan L. , Taghavi Amirhossein , Frank Alexander J. , Fountain Matthew A. , Choudhary Shruti , Roy Soma , Childs-Disney Jessica L. , Disney Matthew D. 
Published: June 2024

ABSTRACTTrinucleotide repeat expansions fold into long, stable hairpins and cause a variety of incurable RNA gain-of-function diseases such as Huntington’s disease, the myotonic dystrophies, and spinocerebellar ataxias. One approach for treating these diseases is to bind small molecules to the structured RNAs. Both Huntington’s disease-like 2 (HDL2) and myotonic dystrophy type 1 (DM1) are caused by a r(CUG) repeat expansion, or r(CUG)exp. The RNA folds into a hairpin structure with a periodic array of 1×1 nucleotide UU loops (5’CUG/3’GUC; where the underlined nucleotides indicate the Us in the internal loop) that sequester various RNA-binding proteins (RBP) and hence the source of its gain-of-function. Here, we report NMR-refined structures of single 5’CUG/3’GUC motifs in complex with three different small molecules, a diguandinobenzoate (1), a derivative of1where the guanidino groups have been exchanged for imidazole (2), and a quinoline with improved drug-like properties (3). These structures were determined using nuclear magnetic resonance (NMR) spectroscopy and simulated annealing with restrained molecular dynamics (MD). Compounds1,2, and3formed stacking and hydrogen bonding interactions with the 5’CUG/3’GUC motif. Compound3also formed van der Waals interactions with the internal loop. The global structure of each RNA-small molecule complexes retains an A-form conformation, while the internal loops are still dynamic but to a lesser extent compared to the unbound form. These results aid our understanding of ligand-RNA interactions and enable structure-based design of small molecules with improved binding affinity for and biological activity against r(CUG)exp. As the first ever reported structures of RNA r(CUG) repeats bound to ligands, these structures can enable virtual screening campaigns combined with machine learning assistedde novodesign.
Molecular Structure and Enzymatic Mechanism of the Human Collagen Hydroxylysine Galactosyltransferase GLT25D1/COLGALT1
Authors: De Marco Matteo , Rai Sristi Raj , Scietti Luigi , Mattoteia Daiana , Liberi Stefano , Pinnola Alberta , Forneris Federico 
Published: June 2024

ABSTRACTDuring biosynthesis, collagen lysine residues undergo extensive post-translational modifications essential for the stability and functions of collagen supramolecular assemblies. In the endoplasmic reticulum, two distinct metal ion dependent enzyme families (i.e., multifunctional lysyl hydroxylases-glucosyltransferases LH/PLODs and galactosyltransferases GLT25D/COLGALT) alternatively operate on collagen lysine side chains ultimately generating the highly conserved α-(1,2)-glucosyl-β-(1,O)-galactosyl-5-hydroxylysine pattern. Malfunctions in the collagen lysine post-translational modification machinery is linked to multiple developmental pathologies as well as extracellular matrix alterations causing enhanced cellular proliferation and invasiveness of several solid tumors, prompting for an in-depth characterization of LH/PLOD and GLT25D/COLGALT enzyme families. Here, we present an integrative molecular study of GLT25D1/COLGALT, highlighting an elongated head-to-head homodimeric assembly characterized by an N-terminal segment of each monomer wrapping around its dimerization partner. Each monomer encompasses two Rossman fold-type domains (GT1 and GT2) separated by an extended linker. Both domains were found capable of binding Mn2+cofactors and UDP-α-galactose donor substrates, resulting in four candidate catalytic sites per dimer. Site-directed mutagenesis and biochemical studies identify the C-terminal GT2 domain as the functional GLT25D1/COLGALT1 catalytic site, highlighting an unprecedented Glu-Asp-Asp motif critical for metal ion binding, and suggesting structural roles for the N-terminal GT1 essential for correct quaternary structure assembly. Conversely, dimerization was not a requirement for GLT25D1/COLGALT1 enzymatic activityin vitro, suggesting that the elongated enzyme homodimer assembly, resembling that of LH/PLOD binding partners, could represent a functional hallmark for correct recognition and successful processing of collagen lysine residues.
A genome resource for the marine annelidPlatynereis dumerilii
Authors: Mutemi Kevin Nzumbi , Simakov Oleg , Pan Leslie , Santangeli Luca , Null Ryan , Handberg-Thorsager Mette , Vellutini Bruno Cossermelli , Peterson Kevin J. , Fromm Bastian , Larsson Tomas , Savage Emily , Lopez Mireia Osuna , Hercog Rajna , Provaznik Jan , Ordoñez-Rueda Diana , Azevedo Nayara , Gazave Eve , Vervoort Michel , Tomancak Pavel , Tan Wenhua , Winkler Sylke , Benes Vladimir , Hui Jerome , Helm Conrad , Özpolat B. Duygu , Arendt Detlev 
Published: June 2024

AbstractThe marine annelidPlatynereis dumeriliiis a model organism used in many research areas including evolution and development, neurobiology, ecology and regeneration. Here we present the genomes ofP. dumerilii(laboratory culture reference and a single individual assembly) and of the closely relatedP. massiliensisandP. megalops(single individual assembly) to facilitate comparative genomic approaches and help explorePlatynereisbiology. We used long-read sequencing technology and chromosomal-conformation capture along with extensive transcriptomic resources to obtain and annotate a draft genome assembly of ∼1.47 Gbp forP. dumerilii, of which more than half represent repeat elements. We predict around 29,000 protein-coding genes, with relatively large intron sizes, over 38,000 non-coding genes, and 105 miRNA loci. We further explore the high genetic variation (∼3% heterozygosity) within thePlatynereisspecies complex. Gene ontology reveals the most variable loci to be associated with pigmentation, development and immunity. The current work sets the stage for further development ofPlatynereisgenomic resources.
VERICIGUAT RESCUES CYCLIC GUANOSINE MONOPHOSPHATE PRODUCTION IN HUMAN AORTIC VASCULAR SMOOTH MUSCLE CELLS AND AUGMENTS VASORELAXATION IN AORTIC RINGS EXPOSED TO HIGH GLUCOSE
Authors: Polhemus David , Almodiel Diego , Harb Tarek , Ziogos Efthymios , Amat-Codina Nuria , Ranek Mark , Santhanam Lakshmi , Gerstenblith Gary , Leucker Thorsten 
Published: June 2024

AbstractBackgroundNormal endothelial cell dependent vascular smooth muscle cell function is mediated by nitric oxide (NO), which stimulates soluble guanylyl cyclase (sGC) production of the second messenger, cyclic guanosine monophosphate (cGMP) leading to increased protein kinase G (PKG) activity and vascular smooth muscle relaxation. NO bioavailability is impaired in inflammatory settings, such as high glucose (HG). We examined whether the direct sGC sensitizer/stimulator vericiguat, augments cGMP production in human vascular smooth muscle cells (HVSMC) exposed to high glucose and explored its effect on vasorelaxation.MethodsAortic HVSMCs were exposed to HG for 24h. In the treatment group, cells also received 1uM vericiguat for 24h. After incubation, cGMP and PKG activity were measured. Additionally, thoracic murine aortas were exposed to HG or to normal glucose (NG) control. The rings were then placed in an organ chamber bath and dose response curves to increasing doses of acetylcholine (Ach) and sodium nitroprusside were constructed for three groups: control (normal glucose), HG alone, and HG + vericiguat.ResultsHVSMCs exposed to HG produced significantly less cGMP than those exposed to NG. cGMP production in the presence of HG was rescued when treated with 1uM vericiguat. Additionally, PKG activity was impaired in the presence of HG and enzyme activity was restored with vericiguat. In isolated mouse aortic rings, ACh mediated relaxation was impaired following treatment with HG, but was improved when a HG group was treated with vericiguat.ConclusionsThe sGC sensitizer/stimulator vericiguat restored cGMP production and PKG activity in the setting of HG. Vericiguat enhanced ACh-mediated vasorelaxation in the setting of HG. The findings suggest clinical studies are warranted to investigate the potential of sGC sensitization/stimulation as a therapeutic intervention to improve vascular endothelial-dependent function that is impaired in pro-inflammatory settings that are associated with the development of atherosclerotic disease.
In- & Out-Cloning: Plasmid toolboxes for scarless transcription unit and modular Golden Gate acceptor plasmid assembly
Authors: de Vries Stijn T. , Köbel Tania S. , Sanal Ahmet , Schindler Daniel 
Published: June 2024

AbstractGolden Gate cloning has become one of the most important DNA assembly strategies. The construction of standardized and reusable part libraries, their assembly into transcription units, and the subsequent assembly of multigene constructs is highly reliable and sustainable. Researchers can quickly construct derivatives of their assemblies or entire pathways, and importantly, the standardization of Golden Gate assemblies is compatible with laboratory automation. Most Golden Gate strategies rely on four nucleotide overhangs generated by commonly used Type IIS enzymes. However, reduction to three nucleotide overhangs allows the use of codons as fusion sites and reduces potential scar sequences. This is particularly important when studying biological functions, as additional nucleotides may alter the structure or stability of the transcribed RNA. To address this issue we use SapI, a Type IIS enzyme generating three nucleotide overhangs, for transcription unit assembly, allowing for codon-based fusion in coding sequences. We created a corresponding plasmid toolbox for basic part generation and transcription unit assembly, a workflow we termIn-Cloning. In-Cloningis downstream compatible with the Modular Cloning standard developed by Sylvestre Marillonnet’s group for standardized assembly of multigene constructs. However, the multigene construct plasmids may not be compatible for use with the model organism of choice. Therefore, we have developed a workflow calledOut-Cloningto rapidly generate Golden Gate acceptor plasmids.Out-Cloninguses standardized plasmid parts that are assembled into Golden Gate acceptor plasmids using flexible linkers. This allows the systematic construction of acceptor plasmids needed to transfer assembled DNA into the organism of interest.
Biogenesis of DNA-carrying extracellular vesicles by the dominant human gut methanogenic archaeon
Authors: Baquero Diana P. , Borrel Guillaume , Gazi Anastasia , Martin-Gallausiaux Camille , Cvirkaite-Krupovic Virginija , Commere Pierre-Henri , Pende Nika , Tachon Stéphane , Sartori-Rupp Anna , Douché Thibaut , Matondo Mariette , Gribaldo Simonetta , Krupovic Mart 
Published: June 2024

ABSTRACTExtracellular vesicles (EVs) are membrane-bound particles secreted by cells from all domains of life and implicated in a variety of important processes, from intercellular communication to pathogenesis. Here, we characterize EVs produced by the dominant human gut methanogen,Methanobrevibacter smithii, which, unlike most archaea, contains a peptidoglycan cell wall. Using quantitative proteomics, we show thatM. smithiiEVs are enriched in various proteins responsible for chromatin structure, including histones, and DNA repair. Consistently, theM. smithiiEVs carry DNA, with fragments covering the entire cellular chromosome. Notably, the EVs are strongly enriched in extrachromosomal circular DNA (eccDNA) molecules which originate from excision of a 2.9-kb chromosomal fragment and a proviral genome. The eccDNA encodes two of the key methanogenesis enzymes and could boost their expression inside the cells through the gene dosage effect. Furthermore, four of the top ten most abundant EV proteins are implicated in methanogenesis. Cryo-electron tomography (Cryo-ET) suggests that EVs are formed by budding from the cell membrane and are trapped under the cell wall prior to liberation through local disruptions in the cell wall. Collectively, our results reveal parallels with EV biogenesis in bacteria and suggest thatM. smithiiEVs facilitate the export of both cellular and viral DNA as well as key metabolic proteins in the gut environment, with potential impact on methane production.
Proteomic and Metabolomic Profiling of Archaeal Extracellular Vesicles from the Human Gut
Authors: Weinberger Viktoria , Darnhofer Barbara , Mertelj Polona , Stentz Regis , Thapa Himadri B , Jones Emily , Grabmann Gerlinde , Mohammadzadeh Rokhsareh , Shinde Tejus , Juodeikis Rokas , Pernitsch Dominique , Hingerl Kerstin , Zurabishvili Tamara , Kumpitsch Christina , Kuehnast Torben , Kolb Dagmar , Gotts Kathryn , Weichhart Thomas , Köcher Thomas , Köfeler Harald , Carding Simon R. , Schild Stefan , Moissl-Eichinger Christine 
Published: June 2024

AbstractOne potential mechanism for microbiome-host, and microbiome constituents’ interaction and communication involves extracellular vesicles (EVs). Here, for the first time, we report the capability of two M. smithii strains (ALI and GRAZ-2), Candidatus M. intestini, and Methanosphaera stadtmanae, as underrepresented components of the gut microbiome, to produce EVs. Interesting, size, morphology, and composition of AEVs were comparable to bacterial EVs, as indicated by ultrastructure, composition, proteomic and metabolomic analyses; however, EVs were substantially less prevalent in the studied Archaea. When looking at the proteomics more precisely, although AEVs from M. smithii ALI and M. intestini were found to be carrying unique proteins (n=135 and n=30, respectively), the shared proteins in AEVs within this genus (n=229), were mostly adhesins(/like) proteins, or proteins with IG-like domains. One remarkable observation was the uptake of AEVs obtained from Methanosphaera stadtmanae and the studied Methanobrevibacter species by human monocytes and the subsequent IL-8 secretion.