bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural evidence for two-stage binding of mitochondrial ferredoxin 2 to the core iron-sulfur cluster assembly complex
Authors: Steinhilper Ralf , Freibert Sven-A. , Kaltwasser Susann , Lill Roland , Murphy Bonnie J. 
Published: February 2024

AbstractIron-sulfur (FeS) clusters are ubiquitous metallocofactors that are essential for life. In eukaryotes, FeS cluster biosynthesis begins with thede novoassembly of a [2Fe-2S] cluster by the core iron-sulfur cluster assembly (ISC) complex in the mitochondrial matrix. This complex comprises the scaffold protein ISCU2, the cysteine desulfurase subcomplex NFS1-ISD11-ACP1, the allosteric activator frataxin (FXN) and the electron donor ferredoxin 2 (FDX2). The interaction of FDX2 with the complex remains unclear. Here, we present cryo-EM structures of the FDX2-bound core ISC complex and show that FDX2 and FXN compete for overlapping binding sites during [2Fe-2S] cluster biosynthesis. FDX2 binds in two conformations; in the ‘distal’ conformation, helix F of FDX2 shows loose electrostatic interaction with an arginine patch of NFS1, while in the ‘proximal’ conformation this interaction tightens and the FDX2-specific C terminus forms contacts with NFS1; in this conformation, the [2Fe-2S] cluster of FDX2 is close enough to the ISCU2 FeS cluster assembly site for rapid electron transfer.
Computational analysis of the effect of a binding protein (RbpA) on the dynamics ofMycobacterium tuberculosisRNA polymerase assembly
Authors: Bheemireddy Sneha , Sowdhamini Ramanathan , Srinivasan Narayanaswamy 
Published: February 2024

AbstractRNA polymerase-binding protein A (RbpA) is an actinomycetes-specific protein crucial for the growth and survival of the pathogenMycobacterium tuberculosis. Its role is essential and influences the transcription and antibiotic responses. However, the regulatory mechanisms underlying RbpA-mediated transcription remain unknown. In this study, we employed various computational techniques to investigate the role of RbpA in the formation and dynamics of the RNA polymerase complex.Our analysis reveals significant structural rearrangements in RNA polymerase happen upon interaction with RbpA. Hotspot residues, crucial amino acids in the RbpA-mediated transcriptional regulation, were identified through our examination. The study elucidates the dynamic behavior within the complex, providing insights into the flexibility and functional dynamics of the RbpA-RNA polymerase interaction. Notably, potential allosteric mechanisms, involving the interface of subunitsα1 andα2 were uncovered, shedding light on how RbpA modulates transcriptional activity.Finally, potential ligands meant for theα1-α2 binding site were identified through virtual screening. The outcomes of our computational study serve as a foundation for experimental investigations into inhibitors targeting the RbpA-regulated dynamics in RNA polymerase. Overall, this research contributes valuable information for understanding the intricate regulatory networks of RbpA in the context of transcription and suggests potential avenues for the development of RbpA-targeted therapeutics.Author SummaryInfection studies byMycobacterium tuberculosis(Mtb) acquires primary importance due to its severe infection and antibiotic resistance. There is an open need for highly effective drugs and one needs to employ novel approaches such as detailed structural analysis and the possibility to focus on allosteric inhibitors. We have exploited the availability of cryo-EM structures of RNA polymerase of Mtb, with and without its transcription-activator protein namely RNA polymerase-binding protein A (RbpA). In this study, we employed various computational techniques to investigate the role of RbpA in the formation and dynamics of the RNA polymerase complex. The assemblies were subject to molecular dynamics and perturbation scanning, followed by structural comparisons and measurement of subunit interface strength. These analyses could clearly show that α subunits, which are far away from the RbpA binding site, undergo differential structural changes. Hence, we focused on the αα’ site to recognize potential small molecule inhibitors using virtual screening. These analyses demonstrate that it is possible to perform comparative structural analysis of different forms of assemblies, which can be useful towards drug design.
Maturing neutrophils of lower density associate with thrombocytopenia in Puumala orthohantavirus-caused hemorrhagic fever with renal syndrome
Authors: Cabrera Luz E. , Tietäväinen Johanna , Jokiranta Suvi T. , Mäkelä Satu , Vaheri Antti , Mustonen Jukka , Vapalahti Olli , Kanerva Mari , Strandin Tomas 
Published: February 2024

AbstractPuumala orthohantavirus-caused hemorrhagic fever with renal syndrome (PUUV-HFRS) is characterized by strong neutrophil activation. Neutrophils are the most abundant immune cell type in the circulation and are specially equipped to rapidly respond to infections. They are more heterogenous than previously appreciated, with specific neutrophil subsets recently implicated in inflammation and immunosuppression. Furthermore, neutrophils can be divided based on their density to either low-density granulocytes (LDGs) or “normal density” polymorphonuclear cell (PMN) fractions. In the current study we aimed to identify and characterize the different neutrophil subsets in the circulation of PUUV-HFRS patients. PMNs exhibited an activation of antiviral pathways, while circulating LDGs were increased in frequency following acute PUUV-HFRS. Furthermore, cell surface marker expression analysis revealed that PUUV-associated LDGs are primarily immature and most likely reflect an increased neutrophil production from the bone marrow. Interestingly, both the frequency of LDGs and the presence of a “left shift” in blood associated with the extent of thrombocytopenia, one of the hallmarks of severe HFRS, suggesting that immature neutrophils could play a role in disease pathogenesis. These results imply that elevated circulating LDGs might be a general finding in acute viral infections. However, in contrast to the COVID-19 associated LDGs described previously, the secretome of PUUV LDGs did not show significant immunosuppressive ability, which suggests inherent biological differences in the LDG responses that can be dependent on the causative virus or differing infection kinetics.
Schwann cells have a limited window of time in which to initiate myelination program during early migrationin vivo
Authors: El-Hage Océane , Mikdache Aya , Boueid Marie-José , Degerny Cindy , Tawk Marcel 
Published: February 2024

ABSTRACTThe temporal control of mitotic exit of individual Schwann cells (SCs) is essential for radial sorting and peripheral myelination. However, it remains unknown when, during their multiple rounds of division, SCs initiate myelin signalingin vivo. By manipulating SC division during development, we report that when SCs skip their division during migration, but not during radial sorting, they fail to myelinate peripheral axons. This coincides with a sharp decrease in Laminin expression within the posterior lateral line nerve. Interestingly, elevating cAMP levels or forcing Laminin 2 expression within individual SCs restore their ability to myelinate, despite missing mitosis during migration. Our results demonstrate a limited time window during which migrating SCs initiate Laminin expression to activate the Laminin/Gpr126/cAMP signaling required for radial sorting and myelination at later stagesin vivo.
Finimizers: Variable-length bounded-frequency minimizers fork-mer sets
Authors: Alanko Jarno N. , Biagi Elena , Puglisi Simon J. 
Published: February 2024

AbstractThe minimizer of ak-mer is the smallestm-mer inside thek-mer according to some order relation<of them-mers. Minimizers are often used as keys in hash tables in indexing tasks in metagenomics and pangenomics. The main weakness of minimizer-based indexing is the possibility of very frequently occurring minimzers, which can slow query times down significantly. Popular minimizer alignment tools employ various and often wild heuristics as workarounds, typically by ignoring frequent minimizers or blacklisting commonly occurring patterns, to the detriment of other metrics (e.g., alignment recall, space usage, or code complexity).In this paper, we introducefrequency-bounded minimizers, which we callfinimizers, for indexing sets ofk-mers. The idea is to use an order relation<for minimizer comparison that depends on the frequency of the minimizers within the indexedk-mers. With finimizers, the lengthmof them-mers is not fixed, but is allowed to vary depending on the context, so that the length can increase to bring the frequency down below a user-specified thresholdt. Setting a maximum frequency solves the issue of very frequent minimizers and gives us a worstcase guarantee for the query time. We show how to implement a particular finimizer scheme efficiently using the Spectral Burrows-Wheeler Transform (SBWT) (Alanko et al., Proc. SIAM ACDA, 2023) augmented with longest common suffix information. In experiments, we explore in detail the special case in which we sett= 1. This choice simplifies the index structure and makes the scheme completely parameter-free apart from the choice ofk. A prototype implementation of this scheme exhibitsk-mer localization times close to, and often faster than, stateof-the-art minimizer-based schemes. The code is available athttps://github.com/ElenaBiagi/Finito.
Annotation and visualisation of parasite, fungi and arthropod genomes with Companion
Authors: Haese-Hill William , Crouch Kathryn , Otto Thomas D. 
Published: February 2024

ABSTRACTAlthough sequencing genomes has become increasingly popular, there is still a bottleneck for the annotation of the resulting assemblies. Structural and functional annotation is still challenging as it includes finding the correct gene sequences, annotating other elements such as RNA and being able to submit those data to databases to share it with the community. We developed the Companion web server to allow non-experts to annotate their genome using a reference-based method, enabling them to analyse their results before submitting to public databases. In this update paper, we describe how we included novel methods for gene finding and made the server more efficient to annotate genomes of up to 1 GB in size. The reference set was increased to genomes from the fungi and arthropod kingdoms. We show that Companion outperforms existing comparable tools.GRAPHICAL ABSTRACT
Impact of Triplet State Population on GFP-type Fluorescence and Photobleaching
Authors: Byrdin Martin , Byrdina Svetlana 
Published: February 2024

AbstractBased on recently published parameters (Rane et al. 2023, JPCB 127, 5046-5054) for (rs)EGFP triplet state formation and decay rates and yields, we consider the power density dependence of triplet state population dynamics and its consequences for the application of green fluorescent proteins in biological single molecule fluorescence microscopy. We find that under certain conditions, the photon budget of GFP type fluorescent proteins can be linearly dependent on power density and we propose a possible explanation for such a non-Hirschfeld photobleaching behavior. Moreover, illumination with ms pulses at sub-kHz rates is shown to improve photostability. We stipulate that a judicious choice of excitation wavelength should take into account the triplet state absorption spectrum along with the singlet state absorption spectrum. Formulas are given for the estimation of the effects of such choice as function of the experimental parameters.Abstract FigureHirschfeld predicted that photobleaching rates should scale with fluorescence brightness, which is obviously not the case for fluorescent proteins found in thefpbase.org. We investigate a modified theory considering the triplet state as precursor for photobleaching.
Enhanced Protein-Protein Interaction Discovery via AlphaFold-Multimer
Authors: Kim Ah-Ram , Hu Yanhui , Comjean Aram , Rodiger Jonathan , Mohr Stephanie E. , Perrimon Norbert 
Published: February 2024

AbstractAccurately mapping protein-protein interactions (PPIs) is critical for elucidating cellular functions and has significant implications for health and disease. Conventional experimental approaches, while foundational, often fall short in capturing direct, dynamic interactions, especially those with transient or small interfaces. Our study leverages AlphaFold-Multimer (AFM) to re-evaluate high-confidence PPI datasets fromDrosophilaand human. Our analysis uncovers a significant limitation of the AFM-derived interface pTM (ipTM) metric, which, while reflective of structural integrity, can miss physiologically relevant interactions at small interfaces or within flexible regions. To bridge this gap, we introduce the Local Interaction Score (LIS), derived from AFM’s Predicted Aligned Error (PAE), focusing on areas with low PAE values, indicative of the high confidence in interaction predictions. The LIS method demonstrates enhanced sensitivity in detecting PPIs, particularly among those that involve flexible and small interfaces. By applying LIS to large-scaleDrosophiladatasets, we enhance the detection of direct interactions. Moreover, we present FlyPredictome, an online platform that integrates our AFM-based predictions with additional information such as gene expression correlations and subcellular localization predictions. This study not only improves upon AFM’s utility in PPI prediction but also highlights the potential of computational methods to complement and enhance experimental approaches in the identification of PPI networks.
Lysis buffer selection guidance for mass spectrometry-based global proteomics including studies on the intersection of signal transduction and metabolism
Authors: Helm Barbara , Hansen Pauline , Lai Li , Schwarzmüller Luisa , Clas Simone M. , Richter Annika , Ruwolt Max , Liu Fan , Frey Dario , D’Alessandro Lorenza A. , Lehmann Wolf-Dieter , Schilling Marcel , Helm Dominic , Fiedler Dorothea , Klingmüller Ursula 
Published: February 2024

AbstractPrerequisite for a successful proteomics experiment is a high-quality lysis of the sample of interest, resulting in a large number of identified proteins as well as a high coverage of protein sequences. Therefore, the choice of suitable lysis conditions is crucial. Many buffers were previously employed in proteomics studies, yet a comprehensive comparison of lysate preparation conditions was so far missing. In this study, we compared the efficiency of four commonly used lysis buffers, containing the agents NP40, SDS, urea or GdnHCl, in four different types of biological samples (suspension and adherent cell lines, primary mouse cells and mouse liver tissue). After liquid chromatography-mass spectrometry (LC-MS) measurement and MaxQuant analysis, we compared chromatograms, intensities, number of identified proteins and the localization of the identified proteins. Overall, SDS emerged as the most reliable reagent, ensuring stable performance and reproducibility across diverse samples. Furthermore, our data advocated for a dual-sample lysis approach, including that the resulting pellet is lysed again after the initial lysis with a urea lysis buffer and subsequently both lysates are combined for a single LC-MS run to maximize the proteome coverage. However, none of the investigated lysis buffers proved to be superior in every category, indicating that the lysis buffer of choice depends on the proteins of interest and on the biological question. Further, we demonstrated with our systematic studies the establishment of conditions that allows to perform global proteomics and affinity purification-based interactome characterization from the same lysate. In sum our results provide guidance for the best-suited lysis buffer for mass spectrometry-based proteomics depending on the question of interest.
morphoHeart: a novel quantitative tool to perform integrated 3D morphometric analyses of heart and ECM morphology during embryonic development
Authors: Sánchez-Posada Juliana , Noël Emily S 
Published: February 2024

SummaryHeart development involves the complex structural remodelling of a linear heart tube into an asymmetrically looped and ballooned organ. Previous studies have associated regional expansion of extracellular matrix (ECM) space with tissue morphogenesis during development. We have developedmorphoHeart, an 3D image tissue segmentation and morphometry software which delivers the first integrated 3D visualisation and multiparametric analysis of both heart and ECM morphology in live embryos.morphoHeartreveals that the ECM undergoes regional dynamic expansion and reduction during cardiac development, concomitant with chamber-specific morphological maturation. We usemorphoHeartto demonstrate that regionalised ECM expansion driven by the ECM crosslinker Hapln1a promotes atrial lumen expansion during heart development. Finally, we have developed a GUI that allows the morphometric analysis tools ofmorphoHeartto be applied toz-stack images of any fluorescently-labelled tissue.
Prevalence and genotypes ofChlamydia psittaciin pet birds of Hong Kong
Authors: Ko Jackie Cheuk Kei , Choi Yannes Wai Yan , Poon Emily Shui Kei , Wyre Nicole , Go Jennifer Le Lin , Poon Leo Lit Man , Sin Simon Yung Wa 
Published: February 2024

AbstractPsittacosis, or parrot fever, is a zoonotic disease caused byChlamydiaspecies associated with birds. One of the causative agents of the disease isChlamydia psittaci, which is often carried by psittacine and can be highly pathogenic and virulent to humans. In Hong Kong, a city with high population density, psittacosis is a notifiable disease with over 60% of cases in the last decade resulting in hospitalization. However, the source of transmission ofC. psittaciand its prevalence in pet birds in Hong Kong are currently unknown. To evaluate the risks of psittacosis transmission through pet birds, we tested the presence ofC. psittaciand determined its genotypes in samples obtained from 516 captive birds from households, pet shops, and a veterinary hospital in Hong Kong. Results revealed that five samples (0.97%), collected from budgerigars and cockatiels, wereC. psittaci-positive, while 80% of them were obtained from pet shops. The identified strains belonged to genotype A and were closely related to the strain SP15 that was previously discovered in mainland China. Our study highlights the need for genotypingChlamydiaspecies in psittacosis patients to understand their source(s) of origin.
Assessment of the Histone Mark-based Epigenomic Landscape in Human Myometrium at Term Pregnancy
Authors: Wu San-Pin , Quiroz Elvis , Wang Tianyuan , Redecke Skylar Montague , Xu Xin , Lin Lin , Anderson Matthew L. , DeMayo Francesco J. 
Published: February 2024

AbstractThe myometrium plays a critical role during pregnancy as it is responsible for both the structural integrity of the uterus and force generation at term. Emerging studies in mice indicate a dynamic change of the myometrial epigenome and transcriptome during pregnancy to ready the contractile machinery for parturition. However, the regulatory systems underlying myometrial gene expression patterns throughout gestation remain largely unknown. Here we investigated human term pregnant nonlabor myometrial biopsies for transcriptome, enhancer histone mark cistrome, and chromatin conformation pattern mapping. More than thirty-thousand putative enhancers with H3K27ac and H3K4me1 double positive marks were identified in the myometrium. Enriched transcription factor binding motifs include known myometrial regulators AP-1, STAT, NFkB, and PGR among others. Putative myometrial super enhancers are mostly colocalized with progesterone receptor occupying sites and preferentially associated with highly expressing genes, suggesting a conserved role of PGR in regulating the myometrial transcriptome between species. In human myometrial specimens, inferred PGR activities are positively correlated with phospholipase C like 2 (PLCL2)mRNA levels, supporting that PGR may act through this genomic region to promotePLCL2expression. PGR overexpression facilitatedPLCL2gene expression in myometrial cells. Using CRISPR activation, we assessed the functionality of a PGR putative enhancer 35-kilobases upstream of the contractile-restrictive genePLCL2. In summary, results of this study serve as a resource to study gene regulatory mechanisms in the human myometrium at the term pregnancy stage for further advancing women’s health research.
Directed evolution of a bacterial leucyl tRNA in mammalian cells for enhanced noncanonical amino acid mutagenesis
Authors: Huang Rachel L. , Jewel Delilah , Kelemen Rachel E. , Pham Quan , Wang Shu , Roy Soumya Jyoti Singha , Huang Zeyi , Levinson Samantha D. , Sundaresh Bharathi , Miranda Suyen Espinoza , Van Opijnen Tim , Chatterjee Abhishek 
Published: February 2024

AbstractTheE. colileucyl-tRNA synthetase (EcLeuRS)/tRNAEcLeupair has been engineered to genetically encode a structurally diverse group of enabling noncanonical amino acids (ncAAs) in eukaryotes, including those with bioconjugation handles, environment-sensitive fluorophores, photocaged amino acids, and native post-translational modifications. However, the scope of this toolbox in mammalian cells is limited by the poor activity of tRNAEcLeu. Here, we overcome this limitation by evolving tRNAEcLeudirectly in mammalian cells using a virus-assisted selection scheme. This directed evolution platform was optimized for higher throughput such that the entire acceptor stem of tRNAEcLeucould be simultaneously engineered, which resulted in the identification of several variants with remarkably improved efficiency for incorporating a wide range of ncAAs. The advantage of the evolved leucyl tRNAs was demonstrated by expressing ncAA mutants in mammalian cells that were challenging to express before using the wild-type tRNAEcLeu, by creating viral vectors that facilitated ncAA mutagenesis at a significantly lower dose, and by creating more efficient mammalian cell lines stably expressing the ncAA-incorporation machinery.
Inhibition of Indirect Pathway Activity Causes Abnormal Decision-Making In a Mouse Model of Impulse Control Disorder in Parkinson’s Disease
Authors: Zhuang Xiaowen , Lemak Julia , Sridhar Sadhana , Nelson Alexandra B. 
Published: February 2024

ABSTRACTHealthy action selection relies on the coordinated activity of striatal direct and indirect pathway neurons. In Parkinson’s disease (PD), in which loss of midbrain dopamine neurons is associated with progressive motor and cognitive deficits, this coordination is disrupted. Dopamine replacement therapy can remediate motor symptoms, but can also cause impulse control disorder (ICD), which is characterized by pathological gambling, hypersexuality, and/or compulsive shopping. The cellular and circuit mechanisms of ICD remain unknown. Here we developed a mouse model of PD/ICD, in which ICD-like behavior was assayed with a delay discounting task. We found that in parkinsonian mice, the dopamine agonist pramipexole drove more pronounced delay discounting, as well as disrupted firing in both direct and indirect pathway neurons. We found that chemogenetic inhibition of indirect pathway neurons in parkinsonian mice drove similar phenotypes. Together, these findings provide a new mouse model and insights into ICD pathophysiology.