bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cas9 RNP Physiochemical Analysis for Enhanced CRISPR-AuNP Assembly and Function
Authors: Lane Daniel D. , Gottimukkala Karthikeya S.V. , Cunningham Rachel A. , Jwa Shirley , Cassidy Molly E. , Castelli Jack M.P. , Adair Jennifer E. 
Published: April 2024

AbstractCRISPR therapy for hematological disease has proven effective for transplant dependent beta thalassemia and sickle cell anemia, with additional disease targets in sight. The success of these therapies relies on high rates of CRISPR-induced double strand DNA breaks in hematopoietic stem and progenitor cells (HSPC). To achieve these levels, CRISPR complexes are typically delivered by electroporation ex vivo which is toxic to HSPCs. HSPCs are then cultured in stimulating conditions that promote error-prone DNA repair, requiring conditioning with chemotherapy to facilitate engraftment after reinfusion. In vivo delivery by nanocarriers of CRISPR gene editing tools has the potential to mitigate this complexity and toxicity and make this revolutionary therapy globally available. To achieve in vivo delivery, the inherent restriction factors against oligonucleotide delivery into HSPCs, that make ex vivo manipulation including electroporation and stimulation essential, must be overcome. To this end, our group developed a CRISPR carrying gold nanoparticle (CRISPR-AuNP) capable of delivering either Cas9 or Cas12a CRISPRs as ribonucleoprotein complexes (RNP) without compromising HSPC fitness. However, the most commonly used CRISPR, Cas9, demonstrated inconsistent activity in this delivery system, with lower activity relative to Cas12a. Investigation of Cas9 RNP biophysics relative to Cas12a revealed duplex RNA instability during the initial loading onto Au cores, resulting in undetectable Cas9 loading to the particle surface. Here we demonstrate preformation of RNP before loading, coupled with optimization of the loading chemistry and conditions, resulted in 39.6 ± 7.0 Cas9 RNP/AuNP without compromising RNP activity in both in vitro assays and primary human HSPC. The same alterations improved Cas12a RNP/AuNP loading 10-fold over previously reported levels. To achieve particle stability, the reported polyethyleneimine outer coating was altered to include PEGylation and the resulting 2ndgeneration CRISPR-AuNP demonstrates favorable nanoformulation characteristics for in vivo administration, with a hydrophilic, more neutral nanoparticle surface. Direct treatment of HSPC in vitro showed 72.5 ± 7.37% uptake of 2ndgeneration CRISPR-AuNP in primary human HSPC, but with endosomal accumulation and low rates of gene editing consistent with low levels of endosomal escape.
Serum from patients with Idiopathic inflammatory myopathy induces skeletal muscle weakness
Authors: Leijding Cecilia , Andreasson Kristofer M. , Horuluoglu Begum , Galindo-Feria Angeles Shunashy , Van Gompel Eveline , Dastmalchi Maryam , Gastaldello Stefano , Alexanderson Helene , Lundberg Ingrid E. , Andersson Daniel C. 
Published: April 2024

AbstractIdiopathic inflammatory myopathies (IIM) are a group of systemic autoimmune inflammatory disorders that primarily affect striated muscles leading to weakness and accelerated fatigue. The disseminated muscle phenotype points to systemic humoral factors as mediators of the disease. Autoantibodies are important biomarkers for disease classification; it is not known if they play a direct role in IIM disease development. This study aims to investigate if IIM patient serum or isolated IgG could directly impair contractile function in muscle.Isolated flexor digitorum brevis (FDB) muscles from healthy mice were exposed to serum (10-50%) from healthy controls or patients with recent onset IIM. Some muscles were exposed to isolated total IgG (50 or 150μg/ml) from patients with IIM. Muscle force in whole muscles was measured before and after exposure to sera or IgG. Muscle force and intracellular [Ca2+] in single muscle fibers were measured after exposure to serum.FDB muscles exposed to serum from patients with IIM displayed a marked reduction in force production in both 10% and 50% serum. Moreover, single myofibers dissected from FDB muscles exposed to IIM sera displayed lower force but unaffected Ca2+release during contractions, which indicates myofibrillar dysfunction, but not intracellular Ca2+release, as the cause to weakness. FDB muscles exposed to total IgG from IIM patients did not display any reduction in muscle force.In conclusion IIM patient serum, but not total IgG, impairs force production in skeletal muscle fibers. As the experiments were performed in isolated muscle, our results cannot be explained by infiltrating immune cells, impaired neuronal or vascular functions. This suggests that humoral factors play a direct role in the pathogenesis of muscle weakness in recent onset IIM.
Fibroblast-like synoviocytes orchestrate daily rhythmic inflammation in arthritis
Authors: Downton Polly , Dickson Suzanna H. , Ray David W. , Bechtold David A. , Gibbs Julie E. 
Published: April 2024

SummaryRheumatoid arthritis is a chronic inflammatory disease that shows characteristic diurnal variation in symptom severity, where joint resident fibroblast-like synoviocytes (FLS) act as important mediators of arthritis pathology. We investigate the role of FLS circadian clock function in directing rhythmic joint inflammation in a murine model of inflammatory arthritis. We demonstrate FLS time of day-dependent gene expression is attenuated in arthritic joints, except for a subset of disease-modifying genes. Deletion of essential clock geneBmal1in FLS reduced susceptibility to collagen-induced arthritis (CIA), but did not impact symptomatic severity in affected mice. Notably, FLSBmal1deletion resulted in loss of diurnal expression of disease-modulating genes across the joint, and elevated production of MMP3, a prognostic marker of joint damage in inflammatory arthritis. This work identifies the FLS circadian clock as an influential driver of daily oscillations in joint inflammation, and a potential regulator of destructive pathology in chronic inflammatory arthritis.
Airway injury induces alveolar epithelial and mesenchymal responses mediated by macrophages
Authors: Wong Irene G. , Stark Jillian , Ya VanNashlee , Moye Aaron L. , Vazquez Alan Baez , Dang Susanna M. , Shehaj Andrea , Rouhani Maral J. , Bronson Roderick , Janes Sam M. , Rowbotham Samuel P. , Paschini Margherita , Franklin Ruth A. , Kim Carla F. 
Published: April 2024

SummaryAcute injury in the airways or the lung activates local progenitors and stimulates changes in cell-cell interactions to restore homeostasis, but it is not appreciated how more distant niches are impacted. We utilized mouse models of airway-specific epithelial injury to examine secondary tissue-wide alveolar, immune, and mesenchymal responses. Single-cell transcriptomics andin vivovalidation revealed transient, tissue-wide proliferation of alveolar type 2 (AT2) progenitor cells after club cell-specific ablation. The AT2 cell proliferative response was reliant on alveolar macrophages (AMs) via upregulation ofSpp1which encodes the secreted factor Osteopontin. A previously uncharacterized mesenchymal population we termed Mesenchymal Airway/Adventitial Niche Cell 2 (MANC2) also exhibited dynamic changes in abundance and a pro-fibrotic transcriptional signature after club cell ablation in an AM-dependent manner. Overall, these results demonstrate that acute airway damage can trigger distal lung responses including altered cell-cell interactions that may contribute to potential vulnerabilities for further dysregulation and disease.Highlights-Airway epithelial cell injury causes proliferation of AT2 cells, the alveolar epithelial progenitor cells-Alveolar macrophages promote AT2 cell proliferation after airway injury via upregulation ofSpp1-Alveolar macrophages have a pro-fibrotic effect on lung mesenchyme after airway injury-Damage to one epithelial compartment can elicit altered epithelial- immune-mesenchymal cell crosstalk in another compartment of the same tissue
Multilayered interaction between CRISPR-Cas subtype I-A and adjacently encoded Acrs of virus SIRV2
Authors: Bhoobalan-Chitty Yuvaraj , Dwiputra Nicodemus , Mayo-Muñoz David , Baadsgaard Karen , Skafte Detlefsen Mette Rehtse Kvistrup , Peng Xu 
Published: April 2024

AbstractAmong the >100 anti-CRISPRs (Acrs) identified to date, the vast majority inhibit CRISPR-Cas immunity on its own. Here we report a multilayered interaction between CRISPR-Cas subtype I-A immunity and two Acrs encoded adjacently in the genome of Saccharolobus virus SIRV2, gp47 (AcrIA3) and gp48 (previously known as AcrIIIB1, hence termed AcrIIIB1/AcrIA4). The host subtype I-A CRISPR-Cas interference module was shown previously to be up-regulated upon SIRV2 infection, through the release of transcriptional repressor Csa3b from the promoter. We demonstrate that AcrIIIB1/AcrIA4 on its own increases viral infectivity 4-5 orders of magnitude in the presence of the host subtype I-A CRISPR-Cas immunity. This Acr is able to completely inhibit the subtype I-A CRISPR-Cas immunity when the transcriptional activation of the latter is artificially removed, suggesting that Acrs might be one of the driving forces for the evolution of CRISPR-Cas up-regulation. Interestingly, AcrIA3 cooperates with AcrIIIB1/AcrIA4 by inhibiting transcriptional activation of the host subtype I-A CRISPR-Cas interference module through interaction with the promoter of the latter. Taken together, our data shed light on how virus-host arms race shaped the evolution of CRISPR-Cas and Acrs.
Evolutionary insights into the emergence of virulentLeptospiraspirochetes
Authors: Giraud-Gatineau Alexandre , Nieves Cecilia , Harrison Luke B. , Benaroudj Nadia , Veyrier Frédéric J. , Picardeau Mathieu 
Published: April 2024

ABSTRACTPathogenicLeptospiraare spirochete bacteria which cause leptospirosis, a re-emerging zoonotic disease of global importance. Here, we use a recently described lineage of environmental-adapted leptospires, which are evolutionarily the closest relatives of the highly virulentLeptospiraspecies, to explore the key phenotypic traits and genetic determinants ofLeptospiravirulence. Through a comprehensive approach integrating phylogenomic comparisons within vitroandin vivophenotyping studies, we show that the evolution towards pathogenicity is associated with both a decrease of the ability to survive in the environment and the acquisition of strategies that enable successful host colonization. This includes the evasion of the human complement system and the adaptations to avoid activation of the innate immune cells. Moreover, our analysis reveals specific genetic determinants that have undergone positive selection during the course of evolution inLeptospira, contributing directly to virulence and host adaptation as demonstrated by gain-of-function and knock-down studies. Taken together, our findings define a new vision onLeptospirapathogenicity, identifying virulence attributes associated with clinically relevant species, and provide insights into the evolution and emergence of these life-threatening pathogens.AUTHOR SUMMARYLeptospirais a highly heterogeneous bacterial genus and leptospires are ubiquitous bacteria found as free-living saprophytes or as pathogens that can cause disseminated infections, from asymptomatic carriage in rats to lethal acute infection in both humans and animals. Leptospirosis is thus causing over one million cases and nearly 60,000 deaths annually. Despite leptospirosis being a re-emerging zoonosis, little is known about the ability of the etiologic agent to adapt to different hosts and cause disease. Here, combining genome analysis and phenotyping studies of representative species and mutant strains, we show that only a small group of species have the ability to evade the host immune system and cause disease. In addition, our findings provide key insight into the emergence of pathogens from a saprophytic ancestor through events of gene gain and genome reduction.
Agrochemical control of gene expression using evolved split RNA polymerase. II
Authors: Yuan Yuan , Miao Jin 
Published: April 2024

AbstractAgrochemical inducible gene expression system provides cost-effective and orthogonal control of energy and information flow in bacterial cells. However, the previous version of Mandipropamid inducible gene expression system (Mandi-T7) became constitutively active at room temperature. We moved the split site of the eRNAP from position LYS179 to position ILE109. This new eRNAP showed proximity dependence at 23°C, but not at 37°C. We built Mandi-T7-v2 system based on the new eRNAP and it worked in bothEscherichia coli and Agrobacteria tumefaciens. The modified eRNAP when combined with the leucine zipper-based dimerization system, behaved as a cold inducible gene expression system. Our new system provides a means to broaden the application of agrochemicals for both research and agricultural application.
Functional activation of dorsal striatum astrocytes improves movement deficits in hemi-parkinsonian mice
Authors: Evans Wesley R. , Baskar Sindhuja S. , Ana Raquel Castro E Costa , Ravoori Sanya , Arigbe Abimbola , Huda Rafiq 
Published: April 2024

SummaryParkinson’s disease (PD) is characterized by the degeneration of dopaminergic nigrostriatal inputs, which causes striatal network dysfunction and leads to pronounced motor deficits. Recent evidence highlights astrocytes as a potential local source of striatal network modulation. However, it remains unknown how dopamine loss affects striatal astrocyte activity and whether astrocyte activity regulates behavioral deficits in PD. We addressed these questions by performing astrocyte-specific calcium recordings and manipulations usingin vivofiber photometry and chemogenetics. We find that locomotion elicits astrocyte calcium activity over a slower timescale than neurons. Unilateral dopamine depletion reduced locomotion-related astrocyte responses. Chemogenetic activation facilitated astrocyte activity, and improved asymmetrical motor deficits and open field exploratory behavior in dopamine lesioned mice. Together, our results establish a novel role for functional striatal astrocyte signaling in modulating motor function in PD and highlight non-neuronal targets for potential PD therapeutics.
Chromosome-level genome assembly of the primitive loach goby,Rhyacichthys aspro, reveals mechanisms underlying Gobioidei diversification
Authors: Wang Tzi-Yuan , Lu Hao-Jun , Wu Yu-Wei , Liao Te-Yu , Huang Shih-Pin , Wang Feng-Yu , Ting Chau-Ti , Chaw Shu-Miaw , Wang Hurng-Yi 
Published: April 2024

ABSTRACTThe percomorph fish clade Gobioidei are a suborder that comprises over 2,200 species distributed in nearly all aquatic habitats. To understand the genetics underlying their diversification, we sequenced and annotated the genome of the loach goby,Rhyacichthys aspro, the basal most group, and compared it with nine additional Gobioidei species. Within Gobioidei, the loach goby possesses the smallest genome at 607 Mb, and a rise in species diversity from basal to derived lineages is mirrored by enlarged genomes and a higher presence of repeat elements (REs), particularly DNA transposons. These transposons are enriched in coding and regulatory regions and their copy number increase is strongly correlated with mutation rate, suggesting that DNA repair after transposon excision/insertion leads to nearby mutations. Consequently, the proliferation of DNA transposons might be the crucial driver of Gobioidei diversification and adaptability. The loach goby genome also points to mechanisms of ecological adaptation. It contains relatively few genes for lateral line development but an over representation of synaptic function genes, with genes putatively under selection linked to synapse organization and calcium signaling, suggesting a sensory system distinct from other Gobioidei species. We also see an overabundance of genes involved in neurocranium development and renal function, adaptations likely connected to its flat morphology suited for strong currents and an amphidromous life cycle. Comparative analyses with hill-stream loaches and the European eel reveal convergent adaptations in body shape and saltwater balance. These findings shed light on the loach goby’s survival mechanisms and the broader evolutionary trends within Gobioidei.
Estimating essential phenotypic and molecular traits from integrative biodiversity data
Authors: Peters Kristian , Ziegler Jörg , Neumann Steffen 
Published: April 2024

AbstractIn the context of biodiversity, only few functional traits and mechanisms are known from underrepresented groups such as mosses (bryophytes). Here, we use 16 field samples of complex thallose liverworts (order Marchantiales) collected from biological soil crusts as reference data for the reusable computational framework iESTIMATE that integrates and extracts phenotypic and molecular traits; and estimates Essential Molecular Variables (EMV). Our reference data involves (1) bioimaging, (2) metabolomics, and (3) DNA marker sequencing. These data are used to demonstrate the systematic and standardized extraction of phenotypic and molecular traits. To demonstrate the reusability of our framework, we propose naming schemes, apply Random Forest to estimate EMVs, phylogenetic dendrograms and partitioning around medoids to connect evolutionary relationships with ecological hypotheses and to document knowledge gains across domains. With this work we want to encourage the combined assessment, reuse and integration of phenotypic and molecular traits into functional ecology, biodiversity and related disciplines.
Combination of oncolytic Maraba virus with immune checkpoint blockade overcomes therapy resistance in an immunologically cold model of advanced melanoma with dysfunctional T cell receptor signalling
Authors: Armstrong Edward , Chiu Matthew K.L. , Foo Shane , Appleton Lizzie , Nenclares Pablo , Patrikeev Anton , Mohan Nitya , Mclaughlin Martin , Bozhanova Galabina , Hoebart Julia , Roulstone Victoria , Patin Emmanuel C , Pedersen Malin , Kyula Joan , Errington-Mais Fiona , Bell John C. , Harrington Kevin J. , Melcher Alan A. , Jennings Victoria A. 
Published: April 2024

AbstractBackgroundOver the past decade, cancer immunotherapies have revolutionised the treatment of melanoma; however, responses vary across patient populations. Recently, baseline tumour size has been identified as an independent prognostic factor for overall survival in melanoma patients receiving immune checkpoint inhibitors (ICIs). MG1 is a novel oncolytic agent with broad tumour tropism that has recently entered early phase clinical trials. The aim of this study was to characterise T cell responses in human and mouse melanoma models following MG1 treatment and to establish if features of the tumour immune microenvironment (TIME) at two distinct tumour burdens would impact the efficacy of oncolytic virotherapy.MethodsHuman 3Din vitropriming assays were performed to measure anti-tumour and anti-viral T cell responses following MG1 infection. TCR sequencing, T2 killing assay, and peptide recall assays were used to assess the evolution of the TCR repertoire, and measure specific T cell responses, respectively.In vivo, subcutaneous 4434 melanomas were characterised using RNAseq, immunohistochemistry (IHC), and flow cytometry. The effectiveness of intra-tumoural MG1 was assessed in advancing 4434 tumours and the generation of anti-tumour and anti-viral T cells measured by splenocyte recall assays. Finally, combination MG1 and α-PD-1 therapy was investigated in advanced 4434 tumours.ResultsMG1 effectively primed functional cytotoxic T cells (CTLs) against tumour associated antigens (TAA) as well as virus-derived peptides, as assessed using peptide recall and T2 killing assays, respectively. TCR sequencing revealed that MG1-primed CTL comprised larger clusters of similar CDR3 amino acid sequences compared to controls.In vivotesting of MG1 demonstrated that MG1 monotherapy was highly effective at treating early disease, resulting in 90% cures; however, the efficacy of MG1 reduced as the disease burden (local tumour size) increased, and the addition of α-PD-1 was required to overcome resistance in more advanced disease. Differential gene expression profiles revealed that increased tumour burden was associated with an immunologically colder TIME. Furthermore, analysis of TCR signalling in advancing tumours demonstrated a different dynamic of TCR engagement compared to smaller tumours, in particular a shift in antigen recognition by CD4+ cells, from conventional to regulatory subset.ConclusionCombination of MG1 with αPD-1 overcomes therapy resistance in an immunologically ‘cold’ model of advanced melanoma.
Transposable element products, functions, and regulatory networks in Arabidopsis
Authors: Borredá Carles , Leduque Basile , Colot Vincent , Quadrana Leandro 
Published: April 2024

ABSTRACTTransposable elements (TEs) are DNA sequences with the ability to propagate themselves within genomes. Their mobilization is catalyzed by self-encoded factors, yet these factors have been poorly investigated. Here, we leveraged extensive long-and short-read transcriptome data, structural predictions, and regulatory networks analyses, to construct a comprehensive atlas of TE transcripts and their encoded products in the model organismArabidopsis thaliana. We uncovered hundreds of transcriptionally competent TEs, each potentially encoding multiple proteins either through distinct genes, alternative splicing, or post-translational processing. Structural-based protein analyses revealed hitherto unidentified domains, uncovering proteins with multimerization and DNA binding domains forming macromolecular complexes. Furthermore, we demonstrate that TE expression is highly intertwined with the transcriptional network of cellular genes, and identified transcription factors and cis-regulatory elements associated with their coordinated expression during development or in response to environmental cues. This comprehensive functional atlas provides a valuable resource for studying the mechanisms involved in transposition and their consequences for genome and organismal function.
Cheap, Robust and Versatile Two-Dimensional Chromatography system for Proteomics of Nanogram Scale Samples
Authors: Kitano Eduardo S. , Nisbet Gareth , Demyanenko Yana , Kowalczyk Katarzyna M , Iselin Louisa , Cross Stephen , Castello Alfredo , Mohammed Shabaz 
Published: April 2024

ABSTRACTIn this work we describe a low loss fractionation system comprised of a reconfigured Evosep One LC system for the first dimension and a repurposed 3D-printer as a fraction collector. The setup operates as a high-pH fractionation system capable of effectively working with nanogram scales of lysate digests. The 2D RP-RP system demonstrated superior proteome coverage over single-shot data-dependent acquisition (DDA) analysis using only 5 ng of human cell lysate digest with performance increasing with increasing amounts of material. We found that the fractionation system allowed over 70% signal recovery at the peptide level and, more importantly, we observed over 30% increase on protein level intensity which indicates the complexity reduction afforded by the system outweighs the sample losses endured. The application of data-independent acquisition (DIA) and wide window acquisition (WWA) to fractionated samples allowed more than 8,000 proteins to be identified from 50 ng of material. The utility of the 2D system was further investigated for phosphoproteomics (>21,000 phosphosites from 50 μg starting material) and pull-down type experiments and showed substantial improvements over single-shot experiments. We show that the 2D RP-RP system is highly versatile and powerful tool for many proteomics workflows.