Bio-protocol最新文献

{"title":"Identification of Neurons Containing Calcium-Permeable AMPA and Kainate Receptors Using Ca²⁺ Imaging.","authors":"Sergei G Gaidin, Artem M Kosenkov, Valery P Zinchenko, Bakytzhan K Kairat, Arailim E Malibayeva, Sultan T Tuleukhanov","doi":"10.21769/BioProtoc.5199","DOIUrl":"10.21769/BioProtoc.5199","url":null,"abstract":"<p><p>Calcium-permeable AMPA receptors (CP-AMPARs) and kainate receptors (CP-KARs) play crucial roles in synaptic plasticity and are implicated in various neurological processes. Current methods for identifying neurons expressing these receptors, such as electrophysiological recordings and immunostaining, have limitations in throughput or inability to distinguish functional receptors. This protocol describes a novel approach for the vital identification of neurons containing CP-AMPARs and CP-KARs using calcium imaging. The method involves loading neurons with Fura-2 AM, a calcium-sensitive fluorescent probe, KCl application to identify all neurons, and further addition of specific AMPAR agonists (e.g., 5-fluorowillardiine) in the presence of voltage-gated calcium channel blockers and NMDAR/KAR antagonists to identify CP-AMPAR-containing neurons. CP-KAR-containing neurons are identified using domoic acid applications in the presence and absence of NASPM (a CP-AMPAR antagonist). This technique offers several advantages over existing methods, including the ability to assess large neuronal populations simultaneously, distinguish between different receptor types, and provide functional information about CP-AMPAR and CP-KAR expression in living neurons, making it a valuable tool for studying synaptic plasticity and neurological disorders. Key features • The described protocol allows vital identification of neurons containing calcium-permeable AMPA (CP-AMPARs) and kainate receptors (CP-KARs). • This approach can be combined with other methods, such as electrophysiological recordings or immunostaining. • The method is fast, reproducible, and allows non-invasive simultaneous identification of numerous CP-AMPAR-/CP-KAR-containing neurons. • The described protocol can be used for pharmacological screening of different drugs, including neuroprotectors, or investigation of features of CP-AMPAR-/CP-KAR-containing neurons in health and disease.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5199"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825306/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434540","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":"Cell-Sonar, an Easy and Low-cost Method to Track a Target Protein by Expression Changes of Specific Protein Markers.","authors":"Sabrina Brockmöller, Lara Maria Molitor, Franz Worek, Simone Rothmiller","doi":"10.21769/BioProtoc.5206","DOIUrl":"10.21769/BioProtoc.5206","url":null,"abstract":"<p><p>Different research methods aim to clarify the intracellular trafficking of target proteins or unknown pathways. Currently, existing methods are mostly complex and expensive, requiring expert knowledge. Detailed microscopy for protein co-localization detection or omic technologies, which provide holistic network data, are elaborate, mostly complex, and expensive to apply. Our protocol illustrates a method to track a target protein by detecting expression changes of user-selected marker proteins that directly or indirectly interact with the target. Modulation of protein expression indicates interactions between the target and marker protein. Even without co-localization analysis, the results of the protein expression change are the first insights into the target's fate. Moreover, the use of the cell-sonar is straightforward and affordable, and the results are rapidly available. Furthermore, this method could also be used to determine if and how pathways are affected by compounds added to the cells. In conclusion, our method is adaptable to a wide range of proteins, easy to apply, inexpensive, and expandable with substances that affect proteins. Key features • Easy-to-implement method to track intracellular proteins. • Marker protein expression change demonstrates protein interaction. • Combined data of all marker proteins is used to give an indirect overview of protein localization. • This method is also applicable to different compounds and thus provides information about protein induction or influence on pathways.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5206"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825303/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434597","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":"High-resolution Cryo-EM Structure Determination of a-Synuclein-A Prototypical Amyloid Fibril.","authors":"Juan C Sanchez, Joshua A Pierson, Collin G Borcik, Chad M Rienstra, Elizabeth R Wright","doi":"10.21769/BioProtoc.5171","DOIUrl":"10.21769/BioProtoc.5171","url":null,"abstract":"<p><p>The physiological role of a-synuclein (a-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, a-syn will self-assemble into β-sheet-rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient-derived brain tissues have concluded that these inclusions are associated with Parkinson's disease, the second most common neurodegenerative disorder, and other synuclein-related diseases called synucleinopathies. In addition, repetitions of specific mutations to the SNCA gene, the gene that encodes a-syn, result in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of a-syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 Å. Here, we provide a protocol for a-syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step-by-step protocol for high-resolution cryo-EM structure determination of a-syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high-resolution cryo-EM map. This workflow resulted in a 2.04 Å structure of a-syn fibrils with excellent resolution of residues 36-97 and an additional island of density for residues 15-22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of a-syn and other amyloid fibrils. Key features • In vitro fibril amplification method yielding twisting fibrils that span several micrometers in length and are suitable for cryo-EM structure determination. • High-throughput cryo-EM data collection of neurodegenerative fibrils, such as alpha-synuclein. • Use of RELION implementations of helical reconstruction algorithms to generate high-resolution 3D structures of a-synuclein fibrils. • Brief demonstration of the use of ChimeraX, Coot, and Phenix for molecular model building and refinement.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5171"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825309/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434528","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":"Quantitative, Dynamic Detection of Neuronal Na⁺ Transients Using Multi-photon Excitation and Fluorescence Lifetime Imaging (FLIM) in Acute Mouse Brain Slices.","authors":"Sara Eitelmann, Karl W Kafitz, Christine R Rose, Jan Meyer","doi":"10.21769/BioProtoc.5175","DOIUrl":"10.21769/BioProtoc.5175","url":null,"abstract":"<p><p>Fluorescence lifetime imaging microscopy (FLIM) is a highly valuable technique in the fluorescence microscopy toolbox because it is essentially independent of indicator concentrations. Conventional fluorescence microscopy analyzes changes in emission intensity. In contrast, FLIM assesses the fluorescence lifetime, which is defined as the time a fluorophore remains in an excited state before emitting a photon. This principle is advantageous in experiments where fluorophore concentrations are expected to change, e.g., due to changes in cell volume. FLIM, however, requires collecting a substantial number of photons to accurately fit distribution plots, which constrains its ability for dynamic imaging. This limitation has recently been overcome by rapidFLIM, which utilizes ultra-low dead-time photodetectors in conjunction with sophisticated rapid electronics. The resulting reduction in dead-time to the picosecond range greatly enhances the potential for achieving high spatio-temporal resolution. Here, we demonstrate the use of multi-photon-based rapidFLIM with the sodium indicator ION NaTRIUM Green-2 (ING-2) for the quantitative, dynamic determination of Na⁺ concentrations in neurons in acute rodent brain tissue slices. We describe the loading of the dye into neurons and present a procedure for its calibration in situ. We show that rapidFLIM not only allows the unbiased determination of baseline Na⁺ concentrations but also allows dynamic imaging of changes in intracellular Na⁺, e.g., induced by inhibition of cellular ATP production. Overall, rapidFLIM, with its greatly improved signal-to-noise ratio and higher spatio-temporal resolution, will also facilitate dynamic measurements using other FLIM probes, particularly those with a low quantum yield. Key features • RapidFLIM of the sodium indicator ING-2 enables the intensity-independent recording of neuronal Na⁺ transients at unparalleled full frame rates of 0.5-1 Hz. • RapidFLIM is essentially independent of dye concentrations and therefore not affected by dye bleaching. • Full in situ calibrations enable the quantification of intracellular Na⁺ changes at high spatio-temporal resolution. • RapidFLIM of ING-2 allows unbiased determination of cellular Na⁺ loading also in conditions of strong cell swelling.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5175"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825299/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434566","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":"Determination of Dissociation Constants for the Interaction of Myosin-5a with its Cargo Protein Using Microscale Thermophoresis (MST).","authors":"Rui Zhou, Jiabin Pan, Xiang-Dong Li","doi":"10.21769/BioProtoc.5176","DOIUrl":"10.21769/BioProtoc.5176","url":null,"abstract":"<p><p>Myosin-5a (Myo5a) is an actin-dependent molecular motor that recognizes a diverse range of cargo proteins through its tail domain, playing a crucial role in the transport and localization of various organelles within the cell. We have identified a new interaction between Myo5a and its cargo protein melanophilin (Mlph), i.e., the interaction between the middle tail domain of Myo5a (Myo5a-MTD) and the actin-binding domain of Mlph (Mlph-ABD), by GST pulldown assay. We then intend to obtain the dissociation constant between Myo5a-MTD and Mlph-ABD using isothermal titration calorimetry (ITC) or microscale thermophoresis (MST), both of which are two commonly used methods for determining quantitative data on protein interactions. The advantages of MST over ITC include less protein usage, shorter operation time, and higher sensitivity. In this protocol, we present a method for using MST to determine the dissociation constants of Myo5a-MTD and Mlph-ABD, which were purified through overexpression in bacteria using affinity chromatography. The dissociation constant values obtained directly reflect the binding strength between these two proteins and provide a foundation for the isolation and purification of the complex in the future. Key features • A protocol for determining the dissociation constants between two purified proteins using microscale thermophoresis (MST). • Detailed procedures for purification of recombinant proteins expressed in <i>E. coli</i>.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5176"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825297/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434521","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":"Real-time IncuCyte^® Assay for the Dynamic Assessment of Live and Dead Cells in 2D Cultures.","authors":"Arlene K Gidda, Suganthi Chittaranjan, Sharon M Gorski","doi":"10.21769/BioProtoc.5210","DOIUrl":"10.21769/BioProtoc.5210","url":null,"abstract":"<p><p>Cell viability and cytotoxicity assays are commonly used to investigate protein function and to evaluate drug efficacy in cancer and other disease models. Cytotoxicity is the measure of dead or damaged cells and is often quantified using assays based on cellular characteristics such as membrane integrity or mitochondrial metabolism. However, these assays are typically limited to endpoint analysis and lack emulation of physiological conditions. The IncuCyte Live and Dead Cell assay described here leverages common cell permeability methodologies but uses fluorescence microscopy channels to image both live and dead cells over time and phase microscopy channels to measure confluency. Cytotox green reagent is a cell membrane-impermeable dye that can only be taken up by cells with poor cell membrane integrity. NucLight rapid red dye is a cell membrane-permeable nuclear dye that can be taken up by all cells. Based on dye uptake and fluorescence intensity, the IncuCyte software can be used to analyze images for live and dead cell detection and quantification. Phase microscopy is used to determine confluency and can be further quantified using the IncuCyte software. We provide an application of this assay, using it to calculate IC₅₀ and EC₅₀ values for the assessment of drug efficacy. Key features • Quantify live and dead cells over time. • Determine drug IC₅₀ and/or EC₅₀ in 2D cell cultures. • This protocol requires the instrument IncuCyte^® S3 (or SX5) Live-Cell Analysis system and corresponding software.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5209"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825301/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434665","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":"Using the <i>Sleeping Beauty</i> Transposon System for Doxycycline-inducible Gene Expression in RAW264.7 Macrophage Cells to Study Phagocytosis.","authors":"Parsa Kamali, Gregory D Fairn","doi":"10.21769/BioProtoc.5178","DOIUrl":"10.21769/BioProtoc.5178","url":null,"abstract":"<p><p>Macrophages are known for engulfing and digesting pathogens and dead cells through a specialized form of endocytosis called phagocytosis. Unfortunately, many macrophage cell lines are refractory to most reagents used for transient transfections. Alternative transient approaches, such as electroporation or transduction with lentiviral vectors, typically cause cell death (electroporation) or can be time-consuming to generate numerous lentivirus when using different genes of interest. Therefore, we use the <i>Sleeping Beauty</i> system to generate stably transfected cells. The system uses a \"resurrected\" transposase gene named <i>Sleeping Beauty</i> found in salmonid fish. Experimentally, the system introduces two plasmids: one carrying the <i>Sleeping Beauty</i> transposase and the other with an integration cassette carrying the gene of interest, a reverse-doxycycline controlled repressor gene, and an antibiotic resistance gene. The construct used in this protocol provides puromycin resistance. Stable integrations are selected by culturing the cells in the presence of puromycin, and further enrichment can be obtained using fluorescence-activated cell sorting (FACS). In this protocol, we use the <i>Sleeping Beauty</i> transposon system to generate RAW264.7 cells with doxycycline-inducible inositol polyphosphate 4-phosphatase B containing a C-terminal CaaX motif (INPP4B-CaaX). INPP4B-CaaX dephosphorylates the D-4 position of phosphatidylinositol 3,4-<i>bis</i>phosphate and inhibits phagocytosis. One benefit is that generating stable cell lines is substantially faster than selecting for random integrations. Without FACS, the method typically gives ~50% of the cells that are transfected; with sorting, this approaches 100%. This makes phagocytosis experiments easier since more cells can be analyzed per experiment, allowing for population-based measurements where a ~10% transient transfection rate is insufficient. Finally, using the doxycycline-promoter allows for low near endogenous expression of proteins or robust overexpression. Key features • This protocol builds on the protocols and reagents developed by Kowarz et al. [1] and extends it to using RAW macrophages. • Allows for the rapid generation of stably induced cell lines. • This protocol also determines the phagocytic index and efficiency.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5178"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825295/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434667","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":"Versatile Click Chemistry-based Approaches to Illuminate DNA and RNA G-Quadruplexes in Human Cells.","authors":"Angélique Pipier, David Monchaud","doi":"10.21769/BioProtoc.5209","DOIUrl":"10.21769/BioProtoc.5209","url":null,"abstract":"<p><p>The existence and functional relevance of DNA and RNA G-quadruplexes (G4s) in human cells is now beyond debate, but how did we reach such a level of confidence? Thanks to a panoply of molecular tools and techniques that are now routinely implemented in wet labs. Among them, G4 imaging ranks high because of its reliability and practical convenience, which now makes cellular G4 detection quick and easy; also, because this technique is sensitive and responsive to any G4 modulations in cells, which thus allows gaining precious insights into G4 biology. Herein, we briefly explain what a G4 is and how they can be visualized in human cells; then, we present the strategy we have been developing for several years now for in situ click G4 imaging, which relies on the use of biomimetic G4 ligands referred to as TASQs (for template-assembled synthetic G-quartets) and is far more straightforward and modular than classically used immunodetection methods. We thus show why and how to illuminate G4s with TASQs and provide a detailed, step-by-step methodology (including the preparation of the materials, the methodology per se, and a series of notes to address any possible pitfalls that may arise during the experiments) to make G4 imaging ever easier to operate. Key features • MultiTASQs are clickable probes usable for the detection of cellular DNA and RNA G-quadruplex (G4). • In situ click chemistry relies on the labeling of G4 by clickable probes once in their cellular binding sites. • Experiments can be performed by incubating the clickable probe either in live cells or in fixed cells. • The published but unoptimized protocol is now totally revised to allow for reliable G4 detection in human cells.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5209"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825307/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434686","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":"Detection of Amylin-β-amyloid Hetero-Oligomers by Enzyme-Linked Immunosorbent Assay.","authors":"Noah S Leibold, Deepak Kotiya, Nirmal Verma, Florin Despa","doi":"10.21769/BioProtoc.5179","DOIUrl":"10.21769/BioProtoc.5179","url":null,"abstract":"<p><p>Amylin is an amyloidogenic neuroendocrine hormone co-synthesized and co-secreted with insulin from the pancreas. It readily crosses the blood-brain barrier and synergistically forms mixed amyloid plaques with β-amyloid (Aβ) in brain parenchyma. Parenchymal amylin-Aβ plaques are found in both sporadic and early-onset familial Alzheimer's disease (AD), yet their (patho)physiological role remains elusive, particularly due to a lack of detection modalities for these mixed plaques. Previously, we developed an enzyme-linked immunosorbent assay (ELISA) capable of detecting amylin-Aβ hetero-oligomers in brain lysate and blood using a polyclonal anti-amylin antibody to capture hetero-oligomers and a monoclonal anti-Aβ mid-domain detection antibody combination. This combination allows for the recognition of distinct amylin epitopes, which remain accessible after amylin-Aβ oligomerization has begun, and precise detection of Aβ epitopes available after oligomer formation. The utility of this assay is evidenced in our previous report, wherein differences in hetero-oligomer content in brain tissue from patients with and without AD and patients with and without diabetes were distinguished. Additionally, using AD model rats, we provided evidence that our assay can be employed for the detection of amylin-Aβ in blood. This assay and protocol are important innovations in the field of AD research because they meet an unmet need to detect mixed amyloid plaques that, if targeted therapeutically, could reduce AD progression and severity. Key features • Detects amylin-Aβ hetero-oligomers in blood from patients with Alzheimer's disease. • Enables simultaneous, high-throughput analysis of hetero-oligomer content of brain and blood tissue. • Allows exploration into the amylin-Aβ interaction during AD pathogenesis, potentially leading to novel treatment mechanisms by controlling the amylin-Aβ interaction.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5179"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825304/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434599","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":"Model Architecture Analysis and Implementation of TENET for Cell-Cell Interaction Network Reconstruction Using Spatial Transcriptomics Data.","authors":"Ziyang Wang, Yujian Lee, Yongqi Xu, Peng Gao, Chuckel Yu, Jiaxing Chen","doi":"10.21769/BioProtoc.5205","DOIUrl":"10.21769/BioProtoc.5205","url":null,"abstract":"<p><p>Cellular communication relies on the intricate interplay of signaling molecules, which come together to form the cell-cell interaction (CCI) network that orchestrates tissue behavior. Researchers have shown that shallow neural networks can effectively reconstruct the CCI from the abundant molecular data captured in spatial transcriptomics (ST). However, in scenarios characterized by sparse connections and excessive noise within the CCI, shallow networks are often susceptible to inaccuracies, leading to suboptimal reconstruction outcomes. To achieve a more comprehensive and precise CCI reconstruction, we propose a novel method called triple-enhancement-based graph neural network (TENET). The TENET framework has been implemented and evaluated on both real and synthetic ST datasets. This protocol primarily introduces our network architecture and its implementation. Key features • Cell-cell reconstruction network using ST data. • To facilitate the implementation of a more holistic CCI, we incorporate diverse CCI modalities into consideration. • To further enrich the input information, the downstream gene regulatory network (GRN) is also incorporated as an input to the network. • The network architecture considers global and local cellular and genetic features rather than solely leveraging the graph neural network (GNN) to model such information.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5205"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11833462/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143451339","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}