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BioRxiv : the Preprint Server For... Jun 2024The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation....
The axon initial segment (AIS) constitutes not only the site of action potential initiation, but also a hub for activity-dependent modulation of output generation. Recent studies shedding light on AIS function used predominantly post-hoc approaches since no robust murine live reporters exist. Here, we introduce a reporter line in which the AIS is intrinsically labeled by an ankyrin-G-GFP fusion protein activated by Cre recombinase, tagging the native gene. Using confocal, superresolution, and two-photon microscopy as well as whole-cell patch-clamp recordings , and , we confirm that the subcellular scaffold of the AIS and electrophysiological parameters of labeled cells remain unchanged. We further uncover rapid AIS remodeling following increased network activity in this model system, as well as highly reproducible labeling of AIS over weeks. This novel reporter line allows longitudinal studies of AIS modulation and plasticity in real-time and thus provides a unique approach to study subcellular plasticity in a broad range of applications.
PubMed: 38948770
DOI: 10.1101/2023.02.01.525891 -
BioRxiv : the Preprint Server For... Jun 2024In this paper, we introduce a new, open-source software developed in Python for analyzing Auditory Brainstem Response (ABR) waveforms. ABRs are a far-field recording of...
In this paper, we introduce a new, open-source software developed in Python for analyzing Auditory Brainstem Response (ABR) waveforms. ABRs are a far-field recording of synchronous neural activity generated by the auditory fibers in the ear in response to sound, and used to study acoustic neural information traveling along the ascending auditory pathway. Common ABR data analysis practices are subject to human interpretation and are labor-intensive, requiring manual annotations and visual estimation of hearing thresholds. The proposed new Auditory Brainstem Response Analyzer (ABRA) software is designed to facilitate the analysis of ABRs by supporting batch data import/export, waveform visualization, and statistical analysis. Techniques implemented in this software include algorithmic peak finding, threshold estimation, latency estimation, time warping for curve alignment, and 3D plotting of ABR waveforms over stimulus frequencies and decibels. The excellent performance on a large dataset of ABR collected from three labs in the field of hearing research that use different experimental recording settings illustrates the efficacy, flexibility, and wide utility of ABRA.
PubMed: 38948763
DOI: 10.1101/2024.06.20.599815 -
BioRxiv : the Preprint Server For... Jun 2024Serine protease cascades regulate key innate immune responses. In mosquitoes, these cascades involve clip-domain serine proteases and their non-catalytic homologs...
Serine protease cascades regulate key innate immune responses. In mosquitoes, these cascades involve clip-domain serine proteases and their non-catalytic homologs (CLIPs), forming a complex network whose make-up and structural organization is not fully understood. This study assessed the impact of 85 CLIPs on humoral immunity in . By coupling RNAi with assays measuring antimicrobial activity and melanization, we identified 27 CLIPs as immunoregulators that together form two distinct subnetworks. CLIPs regulating antimicrobial activity were found to control infection resistance, as knockdowns reduced bacterial load and improved survival. Furthermore, our analysis of CLIP gene expression unveiled a novel immunoregulatory mechanism reliant on protease baseline co-expression rather than infection-induced upregulation. These findings underscore that despite its complexity mosquito immune regulation may be targeted for malaria interventions.
PubMed: 38948760
DOI: 10.1101/2024.06.18.599423 -
mutation induces tumor cell-intrinsic defects in enhancer landscape and resistance to immunotherapy.BioRxiv : the Preprint Server For... Jun 2024Cancer genomic studies have identified frequent alterations in components of the SWI/SNF (SWItch/Sucrose Non- Fermenting) chromatin remodeling complex including and ....
Cancer genomic studies have identified frequent alterations in components of the SWI/SNF (SWItch/Sucrose Non- Fermenting) chromatin remodeling complex including and . Importantly, clinical reports indicate that -mutant lung cancers respond poorly to immunotherapy and have dismal prognosis. However, the mechanistic basis of immunotherapy resistance is unknown. Here, we corroborated the clinical findings by using immune-humanized, syngeneic, and genetically engineered mouse models of lung cancer harboring deficiency. Specifically, we show that loss caused decreased response to anti-PD1 immunotherapy associated with significantly reduced infiltration of dendritic cells (DCs) and CD4+ T cells into the tumor microenvironment (TME). Mechanistically, we show that loss in tumor cells led to profound downregulation of and other components of the innate immune system as well as inflammatory cytokines that are required for efficient recruitment and activity of immune cells. We establish that this deregulation of gene expression is caused by cancer cell-intrinsic reprogramming of the enhancer landscape with marked loss of chromatin accessibility at enhancers of genes involved in innate immune response such as type I IFN and inflammatory cytokines. Interestingly, we observed that transcription factor NF-κB binding motif was highly enriched in enhancers that lose accessibility upon deficiency. Finally, we confirmed that SMARCA4 and NF-κB co-occupy the same genomic loci on enhancers associated with and indicating a functional interplay between SMARCA4 and NF-κB. Taken together, our findings provide the mechanistic basis for the poor response of -mutant tumors to anti-PD1 immunotherapy and establish a functional link between SMARCA4 and NF-κB on innate immune and inflammatory gene expression regulation.
PubMed: 38948751
DOI: 10.1101/2024.06.18.599431 -
BioRxiv : the Preprint Server For... Jun 2024Beckwith-Wiedemann Syndrome (BWS) is an epigenetic overgrowth syndrome caused by methylation changes in the human 11p15 chromosomal locus. Patients with BWS exhibit...
Beckwith-Wiedemann Syndrome (BWS) is an epigenetic overgrowth syndrome caused by methylation changes in the human 11p15 chromosomal locus. Patients with BWS exhibit tissue overgrowth, as well as an increased risk of childhood neoplasms in the liver and kidney. To understand the impact of these 11p15 changes, specifically in the liver, we performed single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) to generate paired, cell-type-specific transcriptional and chromatin accessibility profiles of both BWS-liver and nonBWS-liver nontumorous tissue. Our integrated RNA+ATACseq multiomic approach uncovered hepatocyte-specific enrichment and activation of the peroxisome proliferator-activated receptor α (PPARA) - a liver metabolic regulator. To confirm our findings, we utilized a BWS-induced pluripotent stem cell (iPSC) model, where cells were differentiated into hepatocytes. Our data demonstrates the dysregulation of lipid metabolism in BWS-liver, which coincided with observed upregulation of PPARA during hepatocyte differentiation. BWS liver cells exhibited decreased neutral lipids and increased fatty acid β-oxidation, relative to controls. We also observed increased reactive oxygen species (ROS) byproducts in the form of peroxidated lipids in BWS hepatocytes, which coincided with increased oxidative DNA damage. This study proposes a putative mechanism for overgrowth and cancer predisposition in BWS liver due to perturbed metabolism.
PubMed: 38948745
DOI: 10.1101/2024.06.14.599077 -
BioRxiv : the Preprint Server For... Jun 2024Hematopoietic transcription factor RUNX1 is expressed from proximal P2 and distal P1 promoter to yield isoforms RUNX1 B and C, respectively. The roles of these isoforms...
BACKGROUND
Hematopoietic transcription factor RUNX1 is expressed from proximal P2 and distal P1 promoter to yield isoforms RUNX1 B and C, respectively. The roles of these isoforms in RUNX1 autoregulation and downstream-gene regulation in megakaryocytes and platelets are unknown.
OBJECTIVES
To understand the regulation of RUNX1 and its target genes by RUNX1 isoforms.
METHODS
We performed studies on RUNX1 isoforms in megakaryocytic HEL cells and HeLa cells (lack endogenous RUNX1), in platelets from 85 healthy volunteers administered aspirin or ticagrelor, and on the association of RUNX1 target genes with acute events in 587 patients with cardiovascular disease (CVD).
RESULTS
In chromatin immunoprecipitation and luciferase promoter assays, RUNX1 isoforms B and C bound and regulated P1 and P2 promoters. In HeLa cells RUNX1B decreased and RUNX1C increased P1 and P2 activities, respectively. In HEL cells, RUNX1B overexpression decreased RUNX1C and RUNX1A expression; RUNX1C increased RUNX1B and RUNX1A. RUNX1B and RUNX1C regulated target genes ( and others) differentially in HEL cells. In platelets RUNX1B transcripts (by RNAseq) correlated negatively with RUNX1C and RUNX1A; RUNX1C correlated positively with RUNX1A. RUNX1B correlated positively with , and others, and negatively with . In our previous studies, RUNX1C transcripts in whole blood were protective against acute events in CVD patients. We found that higher expression of RUNX1 targets and associated with acute events.
CONCLUSIONS
RUNX1 isoforms B and C autoregulate RUNX1 and regulate downstream genes in a differential manner and this associates with acute events in CVD.
SCIENTIFIC CATEGORY
Platelets.
ESSENTIALS
RUNX1 is expressed from 2 promoters (P1 and P2) to yield isoforms RUNX1C and RUNX1B.RUNX1B and RUNX1C regulate RUNX1 and target genes differentially in megakaryocytes/platelets.In platelets RUNX1B and RUNX1C expression is inversely related and ticagrelor increases RUNX1C RUNX1 target gene ( ) expression in blood is associated with death or MI in cardiac disease.
PubMed: 38948740
DOI: 10.1101/2024.06.18.599563 -
BioRxiv : the Preprint Server For... Jun 2024Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact...
UNLABELLED
Bacteria find suitable locations for colonization by sensing and responding to surfaces. Complex signaling repertoires control surface colonization, and surface contact sensing by the flagellum plays a central role in activating colonization programs. adheres to surfaces using a polysaccharide adhesin called the holdfast. In , disruption of the flagellum through interactions with a surface or mutation of flagellar genes increases holdfast production. Our group previously identified several genes involved in flagellar surface sensing. One of these, called , codes for a protein with homology to the flagellar C-ring protein FliN. We show here that a fluorescently tagged FssF protein localizes to the flagellated pole of the cell and requires all components of the flagellar C-ring for proper localization, supporting the model that FssF associates with the C-ring. Deleting results in a severe motility defect that we show is due to a disruption of chemotaxis. Epistasis experiments demonstrate that promotes adhesion through a stator-dependent pathway when late-stage flagellar mutants are disrupted. Separately, we find that disruption of chemotaxis through deletion of or other chemotaxis genes results in a hyperadhesion phenotype. Key genes in the surface sensing network ( , , and ) contribute to both Δ dependent and Δ dependent hyperadhesion, but these genes affect adhesion differently in the two hyperadhesive backgrounds. Our results support a model in which the stator subunits of the flagella incorporate both mechanical and chemical signals to regulate adhesion.
IMPORTANCE
Biofilms pose a threat in clinical and industrial settings. Surface sensing is an early step in biofilm formation. Studying surface sensing can help develop strategies for combating harmful biofilms. Here, we use the freshwater bacterium to study surface sensing. We characterize a previously unstudied gene, , and find that it localizes to the cell pole in the presence of three proteins that make up a component of the flagellum called the C-ring. Additionally, we find that is required for chemotaxis but dispensable for swimming motility. Lastly, our results show that mutating and other genes required for chemotaxis causes a hyperadhesive phenotype. We propose that surface sensing requires chemotaxis for a robust response to a surface.
PubMed: 38948737
DOI: 10.1101/2024.06.20.599946 -
BioRxiv : the Preprint Server For... Jun 2024Syntax, the abstract structure of language, is a hallmark of human cognition. Despite its importance, its neural underpinnings remain obscured by inherent limitations of...
Syntax, the abstract structure of language, is a hallmark of human cognition. Despite its importance, its neural underpinnings remain obscured by inherent limitations of non-invasive brain measures and a near total focus on comprehension paradigms. Here, we address these limitations with high-resolution neurosurgical recordings (electrocorticography) and a controlled sentence production experiment. We uncover three syntactic networks that are broadly distributed across traditional language regions, but with focal concentrations in middle and inferior frontal gyri. In contrast to previous findings from comprehension studies, these networks process syntax mostly to the exclusion of words and meaning, supporting a cognitive architecture with a distinct syntactic system. Most strikingly, our data reveal an unexpected property of syntax: it is encoded independent of neural activity levels. We propose that this "low-activity coding" scheme represents a novel mechanism for encoding information, reserved for higher-order cognition more broadly.
PubMed: 38948730
DOI: 10.1101/2024.06.20.599931 -
BioRxiv : the Preprint Server For... Jun 2024Poor intervertebral disc (IVD) healing causes IVD degeneration (IVDD) and progression to herniation and back pain. This study identified distinct roles of...
UNLABELLED
Poor intervertebral disc (IVD) healing causes IVD degeneration (IVDD) and progression to herniation and back pain. This study identified distinct roles of TNFα-receptors (TNFRs) in contributing to poor healing in painful IVDD. We first isolated IVDD tissue of back pain subjects and determined the complex pro-inflammatory mixture contained many chemokines for recruiting inflammatory cells. Single-cell RNA-sequencing of human IVDD tissues revealed these pro- inflammatory cytokines were dominantly expressed by a small macrophage-population. Human annulus fibrosus (hAF) cells treated with IVDD-conditioned media (CM) underwent senescence with greatly reduced metabolic rates and limited inflammatory responses. TNFR1 inhibition partially restored hAF cell metabolism sufficiently to enable a robust chemokine and cytokine response to CM. We showed that the pro-reparative TNFR2 was very limited on hIVD cell membranes so that TNFR2 inhibition with blocking antibodies or activation using Atsttrin had no effect on hAF cells with CM challenge. However, TNFR2 was expressed in high levels on macrophages identified in scRNA-seq analyses, suggesting their role in repair responses. Results therefore point to therapeutic strategies for painful IVDD involving immunomodulation of TNFR1 signaling in IVD cells to enhance metabolism and enable a more robust inflammatory response including recruitment or delivery of TNFR2 expressing immune cells to enhance IVD repair.
SUMMARY STATEMENT
TNFR1 signaling drives cells towards senesce and muted inflammatory response in painful intervertebral disc degeneration, while limited TNFR2 signaling may limit disc cell repair responses.
PubMed: 38948728
DOI: 10.1101/2024.02.22.581620 -
BioRxiv : the Preprint Server For... Jun 2024Early diagnosis and biomarker discovery to bolster the therapeutic pipeline for Parkinson's disease (PD) are urgently needed. In this study, we leverage the large-scale...
UNLABELLED
Early diagnosis and biomarker discovery to bolster the therapeutic pipeline for Parkinson's disease (PD) are urgently needed. In this study, we leverage the large-scale whole-blood total RNA-seq dataset from the Accelerating Medicine Partnership in Parkinson's Disease (AMP PD) program to identify PD-associated RNAs, including both known genes and novel circular RNAs (circRNA) and enhancer RNAs (eRNAs). There were 1,111 significant marker RNAs, including 491 genes, 599 eRNAs, and 21 circRNAs, that were first discovered in the PPMI cohort (FDR < 0.05) and confirmed in the PDBP/BioFIND cohorts (nominal < 0.05). Functional enrichment analysis showed that the PD-associated genes are involved in neutrophil activation and degranulation, as well as the TNF-alpha signaling pathway. We further compare the PD-associated genes in blood with those in post-mortem brain dopamine neurons in our BRAINcode cohort. 44 genes show significant changes with the same direction in both PD brain neurons and PD blood, including neuroinflammation-associated genes , , and . Finally, we built a novel multi-omics machine learning model to predict PD diagnosis with high performance (AUC = 0.89), which was superior to previous studies and might aid the decision-making for PD diagnosis in clinical practice. In summary, this study delineates a wide spectrum of the known and novel RNAs linked to PD and are detectable in circulating blood cells in a harmonized, large-scale dataset. It provides a generally useful computational framework for further biomarker development and early disease prediction.
SIGNIFICANCE STATEMENT
Early and accurate diagnosis of Parkinson's disease (PD) is urgently needed. However, biomarkers for early detection of PD are still lacking. Also, the limit of sample size remains one of the main pitfalls of current PD biomarker studies. We employed an analysis of large-scale whole-blood RNA-seq data. By identifying 1,111 significant marker RNAs, we establish a robust foundation for early PD detection, which implicated in neutrophil activation, degranulation, and TNF-alpha signaling, offer unprecedented insights into PD pathogenesis. Our multi-omics machine learning model, boasting an AUC of 0.89, outperforms previous studies, promising a transformative tool for precise PD diagnosis in clinical settings. This study marks a pivotal step toward enhanced biomarker development and early disease prediction.
PubMed: 38948706
DOI: 10.1101/2024.06.18.599639