bioRxiv : the preprint server for biology最新文献

{"title":"Multiple lesion inductions intensify central sensitization driven by neuroinflammation in a mouse model of endometriosis.","authors":"Madeleine E Harvey, Mingxin Shi, Yeongseok Oh, Debra A Mitchell, Ov D Slayden, James A MacLean, Kanako Hayashi","doi":"10.1101/2025.01.23.634555","DOIUrl":"https://doi.org/10.1101/2025.01.23.634555","url":null,"abstract":"<p><strong>Introduction: </strong>Endometriosis is an inflammatory disease associated with chronic pelvic pain (CPP). Growing evidence indicates that endometriotic lesions are not the sole source of pain. Instead, central nervous system (CNS) dysfunction created by prolonged peripheral and central sensitization plays a role in developing endometriosis-associated CPP. This study investigated how CPP is established using a multiple lesion induction mouse model of endometriosis, as repeated retrograde menstruation is considered underlying endometriosis pathogenesis.</p><p><strong>Methods: </strong>We generated endometriosis-like lesions by injecting endometrial tissue fragments into the peritoneal cavity in mice. The mice received a single (1x) or multiple inductions (6x) to simulate recurrent retrograde menstruation. Lesion development, hyperalgesia by behavioral testing, signs of peripheral sensitization, chronic inflammation, and neuroinflammation were examined with lesions, peritoneal fluids, dorsal root ganglia (DRG), spinal codes, and brain.</p><p><strong>Results: </strong>Multiple lesion inductions increased lesion numbers and elevated abdominal and hind paw hypersensitivity compared to single induction mice. Elevated persistent glial cell activation across several brain regions and/or spinal cords was found in the multiple induction mice. Specifically, IBA1+ microglial soma size was increased in the hippocampus and thalamus. IBA1+ cells were abundant in the cortex, hippocampus, thalamus, and hypothalamus of the multiple induction mice. GFAP+ astrocytes were mainly elevated in the hippocampus. Elevated TRPV1, SP, and CGRP expressions in the DRG were persistent in the multiple induction mice. Furthermore, multiple inductions induced the severe disappearance of TIM4 ^hi MHCII ^lo residential macrophages and the influx of increased proinflammatory TIM4 ^lo MHCII ^hi macrophages in the peritoneal cavity. The single and multiple inductions elevated secreted TNFα, IL-1β, and IL-6 levels in the peritoneal cavity at 2 weeks. Elevated cytokine levels returned to the pre-induction levels in the single induction mice at 6 weeks; however, they remained elevated in the multiple induction mice.</p><p><strong>Conclusions: </strong>Our results indicate that the repeatedly occurring lesion inductions (=mimic retrograde menstruation) can be a peripheral stimulus that induces nociceptive pain and creates composite chronic inflammatory stimuli to cause neuroinflammation and sensitize the CNS. The circuits of neuroplasticity and stimulation of peripheral organs via a feedback loop of neuroinflammation may mediate widespread endometriosis-associated CPP.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11785222/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143083003","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Diet gel-based oral drug delivery system for controlled dosing of small molecules for microglia depletion and inducible Cre recombination in mice.","authors":"Joel Jovanovic, Megan L Stone, Samantha R Dooyema, Yuankai K Tao, Sabine Fuhrmann, Edward M Levine","doi":"10.1101/2025.01.23.634530","DOIUrl":"https://doi.org/10.1101/2025.01.23.634530","url":null,"abstract":"<p><p>Small molecules like PLX5622 for microglia depletion and Tamoxifen for inducible Cre recombination are commonly used in mouse research. Traditional application methods, such as chow or oral gavage and injections, have limitations, including uncontrolled dosage and risk of injury. To address this issue, we have developed an alternative oral drug delivery system using a gel-based rodent maintenance diet that allows for controlled consumption and adjustment of dosage and is suitable for water-insoluble small molecules. We tested DietGel ^® 93M (93M) infused with PLX5622 (0.8 mg/g and 2.0 mg/g) in the <i>Cx3cr1</i> ^<i>gfp/+</i> retinal microglia reporter mouse and Tamoxifen-infused 93M (0.3125 mg/g) in the Rlbp1-Cre ^ERT2 ; <i>Rosa</i> ^<i>ai14</i> mouse with an inducible tdTomato reporter in retinal Müller glia. Mice were single-caged and received daily batches of PLX5622-infused 93M over 14 days or Tamoxifen-infused 93M for one or three days followed by a 14-day observation period. Longitudinal scanning laser ophthalmoscopy <i>in vivo</i> and fixed tissue imaging were used to track GFP and tdTomato expression. Following evaluation of a suitable 93M consumption rate (g/d) to sustain body weight, the PLX5622-93M diet at both concentrations showed a 94% microglia depletion rate at 3 days and >99% after one and two weeks. The Tamoxifen-93M diet confirmed suitability for inducible Cre recombination, with significant treatment-time dependent efficacy and a positive correlation between total Tamoxifen dose and tdTomato expression. This study demonstrates that a diet gel-based drug delivery system offers a controllable and less invasive alternative to current drug application methods for PLX5622 and Tamoxifen.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11785137/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143083035","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Loss of Fanconi anemia proteins causes a reliance on lysosomal exocytosis.","authors":"Becky Xu Hua Fu, Albert Xu, Hua Li, Daniel E Johnson, Jennifer R Grandis, Luke A Gilbert","doi":"10.1101/2025.01.23.634631","DOIUrl":"https://doi.org/10.1101/2025.01.23.634631","url":null,"abstract":"<p><p>Mutations in the FA pathway lead to a rare genetic disease that increases risk of bone marrow failure, acute myeloid leukemia, and solid tumors. FA patients have a 500 to 800-fold increase in head and neck squamous cell carcinoma compared to the general population and the treatment for these malignancies are ineffective and limited due to the deficiency in DNA damage repair. Using unbiased CRISPR-interference screening, we found the loss of FA function renders cells dependent on key exocytosis genes such as SNAP23. Further investigation revealed that loss of FA pathway function induced deficiencies in lysosomal health, dysregulation of autophagy and increased lysosomal exocytosis. The compromised cellular state caused by the loss of FA genes is accompanied with decreased lysosome abundance and increased lysosomal membrane permeabilization in cells. We found these signatures <i>in vitro</i> across multiple cell types and cell lines and in clinically relevant FA patient cancers. Our findings are the first to connect the FA pathway to lysosomal exocytosis and thus expands our understanding of FA as a disease and of induced dependencies in FA mutant cancers.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11785144/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143083043","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Allosteric Modulation of Pathological Ataxin-3 Aggregation: A Path to Spinocerebellar Ataxia Type-3 Therapies.","authors":"Alexandra Silva, Sara Duarte-Silva, Pedro M Martins, Beatriz Rodrigues, Débora Serrenho, Daniela Vilasboas-Campos, Andreia Teixeira-Castro, Julio Vieyto-Nuñez, Joel Mieres-Perez, Francisco Figueiredo, Joana Fraga, James Noble, Carter Lantz, Niki Sepanj, Daniela Monteiro-Fernandes, Sara Guerreiro, Andreia Neves-Carvalho, Joana Pereira-Sousa, Frank-Gerrit Klärner, Thomas Schrader, Joseph A Loo, Annalisa Pastore, Elsa Sanchez-Garcia, Gal Bitan, Ana Luísa Carvalho, Patrícia Maciel, Sandra Macedo-Ribeiro","doi":"10.1101/2025.01.22.633970","DOIUrl":"https://doi.org/10.1101/2025.01.22.633970","url":null,"abstract":"<p><p>Spinocerebellar ataxia type 3 (SCA3) is a rare inherited neurodegenerative disease caused by the expansion of a polyglutamine repeat in the protease ataxin-3 (Atx3). Despite extensive knowledge of the downstream pathophysiology, no disease-modifying therapies are currently available to halt disease progression. The accumulation of protein inclusions enriched in the polyQ-expanded Atx3 in neurons suggests that inhibiting its self-assembly may yield targeted therapeutic approaches. Here it is shown that a supramolecular tweezer, CLR01, binds to a lysine residue on a positively charged surface patch of the Atx3 catalytic Josephin domain. At this site, the binding of CLR01 decreases the conformational fluctuations of the distal flexible hairpin. This results in reduced exposure of the nearby aggregation-prone region, which overlaps with the substrate ubiquitin binding site and primes Atx3 self-assembly, ultimately delaying Atx3 amyloid fibril formation and reducing the secondary nucleation rate, a process linked to fibril proliferation and toxicity. These effects translate into the reversal of synapse loss in a SCA3 cultured cortical neuron model, an improved locomotor function in a C. elegans SCA3 model, and a delay in disease onset, accompanied by reduced severity of motor symptoms in a SCA3 mouse model. This study provides critical insights into Atx3 self-assembly, revealing a novel allosteric site for designing CLR01-inspired therapies targeting pathological aggregation pathways while sparing essential functional sites. These findings emphasize that targeting allosteric sites in amyloid-forming proteins may offer unique opportunities to develop safe therapeutic strategies for various protein misfolding disorders.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11785186/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143082862","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Artificial Intelligence Apps for Medical Image Analysis using pyCERR and Cancer Genomics Cloud.","authors":"Aditya P Apte, Eve LoCastro, Aditi Iyer, Sharif Elguindi, Jue Jiang, Jung Hun Oh, Harini Veeraraghavan, Amita Shukla-Dave, Joseph O Deasy","doi":"10.1101/2025.01.19.633756","DOIUrl":"https://doi.org/10.1101/2025.01.19.633756","url":null,"abstract":"<p><p>This work introduces a user-friendly, cloud-based software framework for conducting Artificial Intelligence (AI) analyses of medical images. The framework allows users to deploy AI-based workflows by customizing software and hardware dependencies. The components of our software framework include the Python-native Computational Environment for Radiological Research (pyCERR) platform for radiological image processing, Cancer Genomics Cloud (CGC) for accessing hardware resources and user management utilities for accessing images from data repositories and installing AI models and their dependencies. GNU-GPL copyright pyCERR was ported to Python from MATLAB-based CERR to enable researchers to organize, access, and transform metadata from high dimensional, multi-modal datasets to build cloud-compatible workflows for AI modeling in radiation therapy and medical image analysis. pyCERR provides an extensible data structure to accommodate metadata from commonly used medical imaging file formats and a viewer to allow for multi-modal visualization. Analysis modules are provided to facilitate cloud-compatible AI-based workflows for image segmentation, radiomics, DCE MRI analysis, radiotherapy dose-volume histogram-based features, and normal tissue complication and tumor control models for radiotherapy. Image processing utilities are provided to help train and infer convolutional neural network-based models for image segmentation, registration and transformation. The framework allows for round-trip analysis of imaging data, enabling users to apply AI models to their images on CGC and retrieve and review results on their local machine without requiring local installation of specialized software or GPU hardware. The deployed AI models can be accessed using APIs provided by CGC, enabling their use in a variety of programming languages. In summary, the presented framework facilitates end-to-end radiological image analysis and reproducible research, including pulling data from sources, training or inferring from an AI model, utilities for data management, visualization, and simplified access to image metadata.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11785098/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143082898","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transposable Elements are Dysregulated in Brains of Individuals with Major Depressive Disorder.","authors":"Natalie L Truby, Corinne Smith, Peter J Hamilton","doi":"10.1101/2025.01.22.634143","DOIUrl":"https://doi.org/10.1101/2025.01.22.634143","url":null,"abstract":"<p><p>Transposable elements (TEs) are repetitive DNA sequences capable of being transcribed and reintegrated, or transposed, into distinct loci throughout the genome. While thought to be largely transcriptionally silenced in brain, TE transcription is increasingly recognized as dynamic and involved in human health and disease states, including in disorders of the brain. In this study, we annotated TE transcripts in publicly available RNA sequencing (RNAseq) of postmortem human brain tissue to investigate the expression profile of TE transcripts in individuals with Major Depressive Disorder (MDD) compared to healthy controls. Our findings reveal a robust and uniform downregulation of TE transcript expression in the brains of subjects with MDD relative to controls, this occurs most prominently in the orbitofrontal cortex (OFC) brain region, and MDD differentially impacts this TE expression by age and sex. This work points to the aberrant transcription of cortical TEs as a potentially overlooked molecular signature of MDD.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11785094/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143083073","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Old World alphaviruses use distinct mechanisms to infect brain microvascular endothelial cells for neuroinvasion.","authors":"Pablo A Alvarez, Ashley Tang, Declan M Winters, Prashant Kaushal, Angelica Medina, Karolina E Kaczor-Urbanowicz, Bryan Ramirez Reyes, Robyn M Kaake, Oliver I Fregoso, April D Pyle, Mehdi Bouhaddou, Hengli Tang, Melody M H Li","doi":"10.1101/2025.01.22.634395","DOIUrl":"https://doi.org/10.1101/2025.01.22.634395","url":null,"abstract":"<p><p>Several alphaviruses bypass the blood-brain barrier (BBB), causing debilitating or fatal encephalitis. Sindbis virus (SINV) has been extensively studied <i>in vivo</i> to understand alphavirus neuropathogenesis; yet the molecular details of neuroinvasion at the BBB remain poorly understood. We investigated alphavirus-BBB interactions by pairing a physiologically relevant, human pluripotent stem cell derived model of brain microvascular endothelial cells (BMECs) with SINV strains of opposite neuroinvasiveness. Our system demonstrates that SINV neuroinvasion correlates with robust infection of the BBB. Specifically, SINV genetic determinants of neuroinvasion enhance viral entry into BMECs. We also identify solute carrier family 2 member 3 (SLC2A3, also named GLUT3) as a potential BMEC-specific entry factor exploited for neuroinvasion. Strikingly, efficient BBB infection is a conserved phenotype that correlates with the neuroinvasive capacity of several Old World alphaviruses, including chikungunya virus. Here, we reveal BBB infection as a shared pathway for alphavirus neuroinvasion that can be targeted for preventing alphavirus-induced encephalitis.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11785202/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143083009","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A single dorsal vagal complex circuit mediates the aversive and anorectic responses to GLP1R agonists.","authors":"Warren T Yacawych, Yi Wang, Guoxiang Zhou, Shad Hassan, Stace Kernodle, Frederike Sass, Martin DeVaux, Iris Wu, Alan Rupp, Abigail J Tomlinson, Zitian Lin, Anna Secher, Kirsten Raun, Tune Pers, Randy J Seeley, Martin Myers, Weiwei Qiu","doi":"10.1101/2025.01.21.634167","DOIUrl":"https://doi.org/10.1101/2025.01.21.634167","url":null,"abstract":"<p><p>GLP-1 receptor agonists (GLP1RAs) effectively reduce feeding to treat obesity, although nausea and other aversive side effects of these drugs can limit their use. Brainstem circuits that promote satiation and that mediate the physiologic control of body weight can be distinguished from those that cause aversion. It remains unclear whether brainstem <i>Glp1r</i> neurons contribute to the normal regulation of energy balance and whether GLP1RAs control appetite via circuits distinct from those that mediate aversive responses, however. Hence, we defined roles for AP and NTS <i>Glp1r</i> -expressing neurons (AP ^Glp1r and NTS ^Glp1r neurons, respectively) in the physiologic control of body weight, the GLP1RA-dependent suppression of food intake, and the GLP1RA-mediated stimulation of aversive responses. While silencing non-aversive NTS ^Glp1r neurons interfered with the physiologic restraint of feeding and body weight, restoring NTS ^Glp1r neuron <i>Glp1r</i> expression on an otherwise <i>Glp1r</i> -null background failed to enable long-term body weight suppression by GLP1RAs. In contrast, selective <i>Glp1r</i> expression in AP ^Glp1r neurons restored both aversive responses and long-term body weight suppression by GLP1RAs. Thus, while non-aversive NTS ^Glp1r neurons control physiologic feeding, aversive AP ^Glp1r neurons mediate both the anorectic and weight loss effects of GLP1RAs, dictating the functional inseparability of these pharmacologic GLP1RA responses at a circuit level.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11785067/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143082726","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"DMEM and EMEM are suitable surrogate media to mimic host environment and expand leptospiral pathogenesis studies using <i>in vitro</i> tools.","authors":"Leandro E Garcia, Zitong Lin, Sophie Culos, M Catherine Muenker, Emily E Johnson, Zheng Wang, Francesc Lopez-Giraldez, Alexandre Giraud-Gatineau, Angela Jackson, Mathieu Picardeau, David R Goodlett, Jeffrey P Townsend, Helena Pětrošová, Elsio A Wunder","doi":"10.1101/2025.01.22.634353","DOIUrl":"https://doi.org/10.1101/2025.01.22.634353","url":null,"abstract":"<p><p>Pathogenic <i>Leptospira</i> species can survive and thrive in a wide range of environments. Distinct environments expose the bacteria to different temperatures, osmolarities, and amounts and sources of nutrition. However, leptospires are mostly cultured, in a laboratory setting under <i>in vitro</i> conditions that do not reflect natural environments. This constraint on laboratory cultures limits the applicability of <i>in vitro</i> studies to the understanding of even simple pathogenic processes. Here we report, investigate, and identify a medium and conditions that mimic the host environment during leptospirosis infection, expanding the available <i>in vitro</i> tools to evaluate leptospiral pathogenesis. We quantified genome-wide gene expression of pathogenic <i>Leptospira interrogans</i> cultured in different <i>in vitro</i> media compositions (EMJH, DMEM, EMEM, and HAN). Using EMJH as standard, we compared gene expression in these compositions to genome-wide gene expression gathered in a host environment: whole blood (WB) of hamsters after infection with pathogenic leptospires. Leptospires cultured in DMEM and EMEM media shared 40% and 47% of all differentially expressed genes (DEGs) of leptospires present within WB (FDR<0.01), while leptospires cultured in HAN media only shared 20% of DEGs with those from WB. Furthermore, gene and pathway expression of leptospires cultured on DMEM and EMEM media exhibited a better correlation with leptospires grown in WB, including promoting expression of a similar leptospiral lipid A profile to the one identified directly in host tissues. Taken together, these results indicate that commercial cell-culture media EMEM or DMEM are better surrogates for <i>in vivo</i> pathogenic studies than EMJH or HAN media in <i>Leptospira</i> . These alternative culture conditions, using media that are a standard supply worldwide, provide a reproducible and cost-effective approach that can accelerate research investigation and reduce the number of animal infections necessary for basic research of leptospirosis.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11785191/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143082777","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single-cell differential detergent fractionation for detection of cytokeratin 8 proteoforms.","authors":"Anna Fomitcheva Khartchenko, Trinh Lam, Amy E Herr","doi":"10.1101/2025.01.21.634008","DOIUrl":"https://doi.org/10.1101/2025.01.21.634008","url":null,"abstract":"<p><p>Simultaneous profiling of proteoforms and nucleic acids at the single-cell level, i.e., multi omics, directly links the central dogma. However, current single-cell approaches are limited in their ability to identify proteoforms while preserving the nucleus for further analysis. This limitation is especially pronounced in proteins where their proteoforms present diverse biological functions such as cytokeratin 8 (CK8), which, while commonly known for its structural role, is also involved in several diseases. Here, we present a single-cell western blot (scWB) integrated with differential detergent fractionation (DDF) to selectively solubilize and separate CK8 proteoforms while preserving nuclear integrity for subsequent nucleus-based assays. We report on assay development, including screening a panel of lysis buffers based on nonionic detergents and electrophoresis conditions to achieve a separation resolution between two proteoforms of up to 0.94 with an electric field of 30 V/cm, while preserving an intact nucleus. The cytoplasm-specific lysis approach (DDF buffer) demonstrated comparable solubilization efficiency to whole-cell solubilization (RIPA buffer), achieving proteoform solubilization in 14.3% and 10.3% of solubilized cells using DDF and RIPA buffers, respectively, while keeping the nucleus intact. To understand the broad applicability of the assay conditions, we scrutinized electrophoresis performance for resolving CK8 proteoforms across a panel of widely used breast cancer cell lines (MCF7, SKBR3, and MDA-MB-231), showing presence of proteoforms only in MCF7. Our approach allows for tailored solubilization, achieving reliable proteoform detection and nuclear retention across different cell types. Proteoform profiling at the single-cell level forms a basis for the exploration of the role of specific CK8 molecular forms in cellular processes.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11785136/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143083102","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}