bluuebunny/crossref_metadata_2025_split huggingface dataset:
Robust CRISPR/dCas13 RNA blockers specifically perturb miRNA-target interactions and rescue type 1 myotonic dystrophy pathology
Authors: Hanifi Muhammad , Ates-Kalkan Perihan Seda , Wen Sean , Fischer Mathieu , Kroesen Amanda , Yu Zulin , Wood Matthew , Thongjuea Supat , Mead Adam , Fulga Tudor Alexandru , Rinaldi Carlo , Sauka-Spengler Tatjana 
Published: September 2024

AbstractWhile RNA-targeting strategies are powerful tools for disease therapy, challenges, including low target engagement and off-target collateral effects, currently limit their efficacy. Here, we report the engineering and optimisation of a CRISPR/dCas13 RNA steric blocker (CRISPR-Lock) that prevents mRNA translation, shields mRNAs from miRNA-mediated silencing, and blocks RNA-protein interactions. By tuning the spatial resolution and mismatch tolerance of CRISPR-Lock, we develop a high-resolution perturbation approach that employs genetically encoded CRISPR-Lock as a miRNA target protector. This system enables precise spatiotemporal control of miRNA:mRNA interactions, offering broader applicability compared to phosphorodiamidate mor-pholino (PMO) target protectors. Moreover, we demonstrate the potential therapeutic application of CRISPR-Lock for blocking pathological RNA-protein interactions in type 1 myotonic dystro-phy (DM1). Optimising CRISPR-Lock to target expanded repeat RNAs corrects approximately 85% of clinically relevant splicing biomarkers in patient-derived myotubes and significantly out-performs third-generation PMO antisense oligonucleotides. Finally, by delivering a miniaturised AAV-encoded CRISPR-Lock system into an established DM1 mouse model, we demonstrate the dose-dependent correction of intranuclear foci and splicing dysregulation, underscoring the potential therapeutic application of this technology.
Optimal control of immune checkpoint inhibitor therapy in a heart-tumour model
Authors: van der Vegt Solveig A. , Baker Ruth E. , Waters Sarah L. 
Published: September 2024

AbstractAutoimmune myocarditis, or cardiac muscle inflammation, is a rare but frequently fatal side–effect of immune checkpoint inhibitors (ICIs), a class of cancer therapies. Despite the dangers that side-effects such as these pose to patients, they are rarely, if ever, included explicitly when mechanistic mathematical modelling of cancer therapy is used for optimization of treatment. In this paper, we develop a two-compartment mathematical model which incorporates the impact of ICIs on both the heart and the tumour. Such a model can be used to inform the conditions under which autoimmune myocarditis may develop as a consequence of treatment. We use this model in an optimal control framework to design optimized dosing schedules for three types of ICI therapy that balance the positive and negative effects of treatment. We show that including the negative side-effects of ICI treatment explicitly within the mathematical framework significantly impacts the predictions for the optimized dosing schedule, thus stressing the importance of a holistic approach to optimizing cancer therapy regimens.
Orchestrated long-distance gene activation by a ParB-like BisD-CTP DNA clamp in low-frequency transfer competence development inPseudomonas putida
Authors: Antar Hammam , Carraro Nicolas , Gruber Stephan , van der Meer Jan Roelof 
Published: September 2024

SummaryIntegrative conjugative elements (ICEs) are mobile DNA that remain integrated within the host bacterial genome until they activate their excision and transfer to recipient cells via conjugation. ICE transfer is initiated in a small subpopulation of cells that undergo a hierarchical gene expression cascade leading to transfer competence formation. In this study, we demonstrate that transfer competence formation of the ICEclcelement in stationary phasePseudomonas putidacells is regulated by the coordinated activity of a two-component transcriptional activator, BisC and BisD. Chromatin immunoprecipitation of tagged BisC or BisD followed by high throughput sequencing showed that both proteins accumulate at similar sites around ICEclctransfer competence promoters inP. putidacells. Genetic dissection and single cell microscopy showed that BisD is a dual domain protein, with a C-terminal gene activator domain and an N-terminal ParB-like domain, forming dimeric DNA clamps that self-load at distant sites and reach target promoters after extensive one-dimensional DNA sliding. Expressed mCherry-BisD inP. putidaICEclccells form discrete fluorescent foci, dependent onparS-like sequences on the ICE. This focus formation is similar as what is seen with canonical ParB proteins accumulating on chromosomal DNA, but in case of BisD corresponds to both chromosomal and excised ICE-molecules. Sliding of BisD over ∼50 kb of ICE-DNA is asymmetric and follows the direction of ICE gene transcription. This may help to establish a temporal order of activation of transfer competence formation, optimizing ICE transfer. Given that ICEclcis activated in stationary phase cells, we hypothesize that BisD is not involved in segregating excised ICE-DNA among daughter cells, but rather in actively directing ICE-DNA molecules towards the (multiple) conjugative systems that are produced in transfer competent cells. BisD thus serves as a twin function protein, integrating gene activation and DNA segregation functions.
Functional analyses and integrated mechanisms of cellular destruction by L-amino acid oxidase
Authors: Prak Krisna , Luft Christin , Tsefou Eliona , Chávez-Olórtegui Carlos , Kriston-Vizi Janos , Kettler Robin , Braga Vania M. M. 
Published: September 2024

SUMMARYSnakebite accidents are prevalent worldwide and cause a spectrum of severe clinical manifestations and reduction of patient quality of life and economic income. L-amino acid oxidase (LAAO) is a highly toxic enzyme present in various venoms that causes tissue necrosis, oedema, coagulopathies, and organ failure. Here we investigate the mechanisms of LAAO cytotoxicity preceding cell death using recombinant LAAO and a catalytic inactive LAAO mutant. Wild-type LAAO uptake by cells leads to a decrease in lysosome number and size and inhibition of autophagy flux. Mitochondria function is impaired by severe proton leakage and mitochondrial fission is stimulated. Despite engulfment by autophagosomes, mitochondrial clearance is prevented by the lysosomal defects. The coordinate multi-organelle dysfunction strongly perturbs energy production, cell metabolism and clearance of defective organelles by autophagy, thereby triggering an irreversible destructive path. Considering the fast organelle impairment, strategies to reduce multi-organelle injury after LAAO exposure may be effective to maintain critical cell functions and strengthen adaptive responses against cytotoxicity.
Rebalance the inhibitory system in the elderly brain: Influence of balance learning on GABAergic inhibition and functional connectivity
Authors: Liu Xinyu , Scherrer Selin , Egger Sven , Lim Song-I , Lauber Benedikt , Jelescu Ileana , Griffa Alessandra , Gambarota Giulio , Taube Wolfgang , Xin Lijing 
Published: September 2024

AbstractAging involves complex processes that impact the structure, function, and metabolism of the human brain. Declines in both structural and functional integrity along with reduced local inhibitory tone in the motor areas, as indicated by reduced γ-Aminobutyric acid (GABA) levels, are often associated with compromised motor performance in elderly adults. Using multi-modal neuroimaging techniques including magnetic resonance spectroscopy (MRS), diffusion magnetic resonance imaging (MRI), functional MRI as well as transcranial magnetic stimulation to assess short interval intracortical inhibition (SICI), this study explores whether these age-related changes can be mitigated by motor learning. The investigation focused on the effects of long-term balance learning (3 months) on intracortical inhibition, metabolism, structural and functional connectivity in the cortical sensorimotor network among an elderly cohort. We found that after three months of balance learning, subjects significantly improved balance performance, upregulated sensorimotor cortical GABA levels and ventral sensorimotor network functional connectivity (VSN-FC) compared to a passive control group. Furthermore, correlation analysis suggested a positive association between baseline VSN-FC and balance performance, between baseline VSN-FC and SICI, and between improvements in balance performance and upregulation in SICI in the training group, though these correlations did not survive the false discovery rate correction. These findings demonstrate that balance learning has the potential to counteract aging-related decline in functional connectivity and cortical inhibition on the ‘tonic’ (MRS) and ‘functional’ (SICI) level and shed new light on the close interplay between the GABAergic system, functional connectivity, and behavior.
Expansion microscopy allows quantitative characterisation of structural organisation of platelet aggregates
Authors: Faulkner Emma L. , Pike Jeremy A. , Garlick Evelyn , Neely Robert K. , Styles Iain B. , Watson Stephen P. , Poulter Natalie S. , Thomas Steven G. 
Published: September 2024

1AbstractCurrent microscopy approaches applied to platelet aggregates in both haemostatic and thrombotic settings indicate their structure has important implications in efficient haemostasis and in clinical treatment of thrombosis. However, current fluorescence microscopy approaches are not amenable to volumetric imaging of platelet aggregate structures. This is largely due to the small size of individual platelets and the tight packing of platelets within aggregates, resulting in optical opacity.Here we demonstrate that expansion microscopy, applied to platelet aggregates, can reveal multi-scale information about the structure of platelet aggregates. We produced volumetric images at nanoscale resolution of >700 platelet aggregates under normal and perturbed conditions, stained for cytoskeletal and membrane components. We demonstrate our custom analysis workflow provides quantitative description of platelet numbers, volumes and morphology within entire platelet aggregates. Additionally, we quantitatively describe subcellular organisation of F-actin. By comparing these measurements following treatment with the actin inhibitors, cytochalasin D and latrunculin A, we can robustly detect structural disruptions in platelet aggregates. Together these data provide a workflow to qualitatively and quantitatively describe the architecture of platelet aggregates at a range of scales (whole aggregates down to sub-cellular features within individual platelets).
Complete Genomic Characterization of Global Pathogens, Respiratory Syncytial Virus (RSV), and Human Norovirus (HuNoV) Using Probe-based Capture Enrichment
Authors: Bhamidipati Sravya V , Surathu Anil , Chao Hsu , Agustinho Daniel P , Xiang Qin , Kottapalli Kavya , Santhanam Abirami , Momin Zeineen , Walker Kimberly , Menon Vipin K , Weissenberger George , Emerick Nathanael , Mahjabeen Faria , Meng Qingchang , Hu Jianhong , Sucgang Richard , Henke David , Sedlazeck Fritz J , Khan Ziad , Metcalf Ginger A , Avadhanula Vasanthi , Piedra Pedro A , Ramani Sasirekha , Atmar Robert L , Estes Mary K , Petrosino Joseph F , Gibbs Richard A , Muzny Donna M , Cregeen Sara Javornik , Doddapaneni Harsha 
Published: September 2024

ABSTRACTRespiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, while human noroviruses (HuNoV) are a leading cause of epidemic and sporadic acute gastroenteritis. Generating full-length genome sequences for these viruses is crucial for understanding viral diversity and tracking emerging variants. However, obtaining high-quality sequencing data is often challenging due to viral strain variability, quality, and low titers. Here, we present a set of comprehensive oligonucleotide probe sets designed from 1,570 RSV and 1,376 HuNoV isolate sequences in GenBank. Using these probe sets and a capture enrichment sequencing workflow, 85 RSV positive nasal swab samples and 55 (49 stool and six human intestinal enteroids) HuNoV positive samples encompassing major subtypes and genotypes were characterized. The Ct values of these samples ranged from 17.0-29.9 for RSV, and from 20.2-34.8 for HuNoV, with some HuNoV having below the detection limit. The mean percentage of post-processing reads mapped to viral genomes was 85.1% for RSV and 40.8% for HuNoV post-capture, compared to 0.08% and 1.15% in pre-capture libraries, respectively. Full-length genomes were>99% complete in all RSV positive samples and >96% complete in 47/55 HuNoV positive samples—a significant improvement over genome recovery from pre-capture libraries. RSV transcriptome (subgenomic mRNAs) sequences were also characterized from this data. Probe-based capture enrichment offers a comprehensive approach for RSV and HuNoV genome sequencing and monitoring emerging variants.IMPORTANCERespiratory syncytial virus (RSV) and human noroviruses (HuNoV) are NIAID category C and category B priority pathogens, respectively, that inflict significant health consequences on children, adults, immunocompromised patients, and the elderly. Due to the high strain diversity of RSV and HuNoV genomes, obtaining complete genomes to monitor viral evolution and pathogenesis is challenging. In this paper, we present the design, optimization, and benchmarking of a comprehensive oligonucleotide target capture method for these pathogens. All 85 RSV samples and 49/55 HuNoV samples were patient-derived with six human intestinal enteroids. The methodology described here results has a higher success rate in obtaining full-length RSV and HuNoV genomes, enhancing the efficiency of studying these viruses and mutations directly from patient-derived samples.
Epigenomic analysis of hepatocellular carcinoma reveals aberrantcis-regulatory changes and dysregulated retrotransposons with prognostic potentials
Authors: Cheng Clooney C. Y. , Cheung Ming Fung , Lee Ah Young , Wu Qiong , Chow Savio Ho-Chit , Ang Julie Y. J. , Medina Ignacio Riquelme , Lo Grace , Wu Haoran , Yang Weiqin , Lai Paul B. S. , Yip Kevin Y. , Cheng Alfred S. , Leung Danny 
Published: September 2024

ABSTRACTHepatocellular carcinoma (HCC) exhibits widespread epigenetic alterations, yet their impact oncis-regulatory elements (CREs) and retrotransposons remains poorly understood. Here, we present an integrated epigenomic and transcriptomic analysis of HCC tumors and matched tumor-adjacent normal tissues. We identified extensive DNA hypomethylation coupled with changes in histone modifications at partially methylated domains, CREs, and retrotransposons. These epigenetic aberrations were associated with dysregulated expression of genes involved in cell cycle regulation, immune response, and extracellular matrix organization. Notably, our findings revealed a novel mechanism for the transcriptional dysregulation ofGPC3, a key HCC biomarker and immunotherapeutic target. We observed thatGPC3upregulation is driven by both the reactivation of a fetal liver super enhancer and hypomethylation ofGPC3-associated CpG islands. Moreover, we found that DNA hypomethylation-driven aberrant expression of retrotransposons carries prognostic significance in HCC. Patients with high expression of a long non-coding RNA driven by a HERVE-int element exhibited more aggressive tumors, poorer clinical outcomes, and molecular features associated with favorable immunotherapy response. Together, our study provides a comprehensive resource for understanding the role of epigenetic dysregulation in HCC and identifies retrotransposon-associated transcripts as potential biomarkers.
Peripheral opioid receptor antagonism alleviates fentanyl-induced cardiorespiratory depression and is devoid of aversive behavior
Authors: Ruyle Brian C. , Masud Sarah , Kesaraju Rohith , Tahirkheli Mubariz , Modh Juhi , Roth Caroline G. , Angulo-Lopera Sofia , Lintz Tania , Higginbotham Jessica A. , Massaly Nicolas , Moron Jose A. 
Published: September 2024

ABSTRACTMillions of Americans suffering from Opioid Use Disorders (OUD) face a high risk of fatal overdose due to opioid-induced respiratory depression (OIRD). Fentanyl, a powerful synthetic opioid, is a major contributor to the rising rates of overdose deaths. Reversing fentanyl overdoses has proved challenging due to its high potency and the rapid onset of OIRD. We assessed the contributions of central and peripheral mu opioid receptors (MORs) in mediating fentanyl-induced physiological responses. The peripherally restricted MOR antagonist naloxone methiodide (NLXM) both prevented and reversed OIRD to a degree comparable to that of naloxone (NLX), indicating substantial involvement of peripheral MORs to OIRD. Interestingly, NLXM-mediated OIRD reversal did not produce aversive behaviors observed after NLX. We show that neurons in the nucleus of the solitary tract (nTS), the first central synapse of peripheral afferents, exhibit a biphasic activity profile following fentanyl exposure. NLXM pretreatment attenuates this activity, suggesting that these responses are mediated by peripheral MORs. Together, these findings establish a critical role for peripheral MORs, including ascending inputs to the nTS, as sites of dysfunction during OIRD. Furthermore, selective peripheral MOR antagonism could be a promising therapeutic strategy for managing OIRD by sparing CNS-driven acute opioid-associated withdrawal and aversion observed after NLX.Significance StatementIn this study, we compare the central versus peripheral components underlying fentanyl-induced cardiorespiratory depression to prevent overdose deaths. Our data indicate that these effects are, at least partially, due to the activation of mu opioid receptors present in peripheral sites. These findings provide insight into peripheral contributions to fentanyl-induced overdoses and could potentially lead to the development of treatments selectively targeting the peripheral system, sparing individuals from the CNS-driven acute opioid withdrawal generally observed with the use of naloxone.
The Pattern and Staging of Brain Atrophy in Spinocerebellar Ataxia Type 2 (SCA2): MRI Volumetrics from ENIGMA-Ataxia
Authors: Robertson Jason W. , Adanyeguh Isaac , Bender Benjamin , Boesch Sylvia , Brunetti Arturo , Cocozza Sirio , Coutinho Léo , Deistung Andreas , Diciotti Stefano , Dogan Imis , Durr Alexandra , Fernandez-Ruiz Juan , Göricke Sophia L. , Grisoli Marina , Han Shuo , Mariotti Caterina , Marzi Chiara , Mascalchi Mario , Mochel Fanny , Nachbauer Wolfgang , Nanetti Lorenzo , Nigri Anna , Ono Sergio E. , Onyike Chiadi U. , Prince Jerry L. , Reetz Kathrin , Romanzetti Sandro , Saccà Francesco , Synofzik Matthis , Teive Hélio A. Ghizoni , Thomopoulos Sophia I. , Thompson Paul M. , Timmann Dagmar , Ying Sarah H. , Harding Ian H. , Hernandez-Castillo Carlos R. 
Published: September 2024

AbstractObjectiveSpinocerebellar ataxia type 2 (SCA2) is a rare, inherited neurodegenerative disease characterised by progressive deterioration in both motor coordination and cognitive function. Atrophy of the cerebellum, brainstem, and spinal cord are core features of SCA2, however the evolution and pattern of whole-brain atrophy in SCA2 remain unclear. We undertook a multi-site, structural magnetic resonance imaging (MRI) study to comprehensively characterize the neurodegeneration profile of SCA2.MethodsVoxel-based morphometry analyses of 110 participants with SCA2 and 128 controls were undertaken to assess groupwise differences in whole-brain volume. Correlations with clinical severity and genotype, and cross-sectional profiling of atrophy patterns at different disease stages, were also performed.ResultsAtrophy in SCA2 relative to controls was greatest (Cohen’sd>2.5) in the cerebellar white matter (WM), middle cerebellar peduncle, pons, and corticospinal tract. Very large effects (d>1.5) were also evident in the superior cerebellar, inferior cerebellar, and cerebral peduncles. In cerebellar grey matter (GM), large effects (d>0.8) mapped to areas related to both motor coordination and cognitive tasks. Strong correlations (|r|>0.4) between volume and disease severity largely mirrored these groupwise outcomes. Stratification by disease severity showed a degeneration pattern beginning in cerebellar and pontine WM in pre-clinical subjects; spreading to the cerebellar GM and cerebro-cerebellar/corticospinal WM tracts; then finally involving the thalamus, striatum, and cortex in severe stages.InterpretationThe magnitude and pattern of brain atrophy evolves over the course of SCA2, with widespread, non-uniform involvement across the brainstem, cerebellar tracts, and cerebellar cortex; and late involvement of the cerebral cortex and striatum.
NR2F2 regulation of interstitial to fetal Leydig cell differentiation in the testis: insights into differences of sex development
Authors: Estermann Martín Andrés , Grimm Sara , Kitakule Abigail , Rodriguez Karina , Brown Paula , McClelland Kathryn , Amato Ciro , Yao Humphrey Hung-Chang 
Published: September 2024

SummaryTesticular fetal Leydig cells are a specialized cell type responsible for embryo masculinization. Fetal Leydig cells produce androgens, that induce the differentiation of male reproductive system and sexual characteristics. Deficiencies in Leydig cell differentiation leads to various disorders of sex development and male reproductive defects such as ambiguous genitalia, hypospadias, cryptorchidism, and infertility. Fetal Leydig cells are thought to originate from proliferating progenitor cells in the testis interstitium, marked by genes likeArx,Pdgfra,Tcf21andWnt5a. However, the precise mechanisms governing the transition from interstitial cells to fetal Leydig cells remain elusive. Through integrated approaches involving mouse models and single-nucleus multiomic analyses, we discovered that fetal Leydig cells originate from aNr2f2-positive non-steroidogenic interstitial cell population. Embryonic deletion ofNr2f2in mouse testes resulted in disorders of sex development, including dysgenic testes, Leydig cell hypoplasia, cryptorchidism, and hypospadias. We found that NR2F2 promotes the progenitor cell fate while suppresses Leydig cell differentiation by directly and indirectly controlling a cohort of transcription factors and downstream genes. Bioinformatic analyses of single-nucleus ATAC-seq and NR2F2 ChIP-seq data revealed putative transcription factors co-regulating the process of interstitial to Leydig cell differentiation. Collectively, our findings not only highlight the critical role ofNr2f2in orchestrating the transition from interstitial cells to fetal Leydig cells, but also provide molecular insight into the disorders of sex development as a result ofNr2f2mutations.
Biological volume EM with focused Ga ion beam depends on formation of radiation-resistant Ga-rich layer at block face
Authors: Yang Zijie , Kim Joshua , Zhang Guofeng , Aronova Maria A. , Leapman Richard D. 
Published: September 2024

AbstractVolume electron microscopy (vEM) enables biologists to visualize nanoscale 3D ultrastructure of entire eukaryotic cells and tissues prepared by heavy atom staining and plastic embedding. The highest resolution vEM technique is focused ion-beam scanning electron microscopy (FIB-SEM), which provides nearly isotropic (~5-10 nm) spatial resolution at fluences of > 10,000 e−/nm2. However, it is not clear how such high resolution is achievable because serial block-face (SBF) SEM, which incorporates an in-situ ultramicrotome instead of a Ga+FIB beam, results in radiation-induced collapse of similar specimen blocks at fluences of only ~20 e−/nm2. Moreover, the z-resolution (perpendicular to block-face) in FIB-SEM is substantially better than predicted by Monte Carlo modeling of SBF-SEM samples. Here, we show that FIB-SEM implants a thin highly concentrated layer of Ga+ions at the block surface, which greatly reduces the effect of beam-damage, reduces the depth from which backscattered electrons are detected, and prevents specimen charging and collapse.