Methods in molecular biology最新文献

{"title":"Concepts and Methods for Predicting Viral Evolution.","authors":"Matthijs Meijers, Denis Ruchnewitz, Jan Eberhardt, Malancha Karmakar, Marta Łuksza, Michael Lässig","doi":"10.1007/978-1-0716-4326-6_14","DOIUrl":"10.1007/978-1-0716-4326-6_14","url":null,"abstract":"<p><p>The seasonal human influenza virus undergoes rapid evolution, leading to significant changes in circulating viral strains from year to year. These changes are typically driven by adaptive mutations, particularly in the antigenic epitopes, the regions of the viral surface protein hemagglutinin targeted by human antibodies. Here, we describe a consistent set of methods for data-driven predictive analysis of viral evolution. Our pipeline integrates four types of data: (1) sequence data of viral isolates collected on a worldwide scale, (2) epidemiological data on incidences, (3) antigenic characterization of circulating viruses, and (4) intrinsic viral phenotypes. From the combined analysis of these data, we obtain estimates of relative fitness for circulating strains and predictions of clade frequencies for periods of up to 1 year. Furthermore, we obtain comparative estimates of protection against future viral populations for candidate vaccine strains, providing a basis for pre-emptive vaccine strain selection. Continuously updated predictions obtained from the prediction pipeline for influenza and SARS-CoV-2 are available at https://previr.app .</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2890 ","pages":"253-290"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143073650","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Discovering Novel Proteoforms Using Proteogenomic Workflows Within the Galaxy Bioinformatics Platform.","authors":"Praveen Kumar, James E Johnson, Thomas McGowan, Matthew C Chambers, Mohammad Heydarian, Subina Mehta, Caleb Easterly, Timothy J Griffin, Pratik D Jagtap","doi":"10.1007/978-1-0716-4152-1_7","DOIUrl":"10.1007/978-1-0716-4152-1_7","url":null,"abstract":"<p><p>Proteogenomics is a growing \"multi-omics\" research area that combines mass spectrometry-based proteomics and high-throughput nucleotide sequencing technologies. Proteogenomics has helped in genomic annotation for organisms whose complete genome sequences became available by using high-throughput DNA sequencing technologies. Apart from genome annotation, this multi-omics approach has also helped researchers confirm expression of variant proteins belonging to unique proteoforms that could have resulted from single-nucleotide polymorphism (SNP), insertion and deletions (Indels), splice isoforms, or other genome or transcriptome variations.A proteogenomic study depends on a multistep informatics workflow, requiring different software at each step. These integrated steps include creating an appropriate protein sequence database, matching spectral data against these sequences, and finally identifying peptide sequences corresponding to novel proteoforms followed by variant classification and functional analysis. The disparate software required for a proteogenomic study is difficult for most researchers to access and use, especially those lacking computational expertise. Furthermore, using them disjointedly can be error-prone as it requires setting up individual parameters for each software. Consequently, reproducibility suffers. Managing output files from each software is an additional challenge. One solution for these challenges in proteogenomics is the open-source Web-based computational platform Galaxy. Its capability to create and manage workflows comprised of disparate software while recording and saving all important parameters promotes both usability and reproducibility. Here, we describe a workflow that can perform proteogenomic analysis on a Galaxy-based platform. This Galaxy workflow facilitates matching of spectral data with a customized protein sequence database, identifying novel protein variants, assessing quality of results, and classifying variants along with visualization against the genome.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2859 ","pages":"109-128"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142469686","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantification of Infectious Rhinovirus A and B Serotypes by Plaque Assay.","authors":"Mohsen Tabasi, Haleh Ganjian, Umadevi Sajjan","doi":"10.1007/978-1-0716-4410-2_4","DOIUrl":"https://doi.org/10.1007/978-1-0716-4410-2_4","url":null,"abstract":"<p><p>Plaque assay is a quantitative assay that determines the number of infective virions in the viral stock or the infected cells. Plaques are essentially virus-infected cells that produce progeny virus and infect adjacent cells. The cells producing progeny virus die and detach from the dish, leaving an empty space in the confluent monolayer of cells. Unlike other viruses, rhinovirus does not form characteristic round plaques but shows small clear areas of different shapes surrounded by dying cells. Rhinoviruses form plaques only in highly susceptible H1HeLa cells but not in their primary target, airway epithelial cells. The plaque assay to determine the number of infective virions works only for rhinovirus species A and B, but not C, because the latter does not infect H1HeLa cells. Here we describe a method to quantify the infective virions by plaque assay for rhinovirus speciesA and B.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2903 ","pages":"31-38"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143523967","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Detection and Characterization of Antiviral Proteins Against Bunyaviruses in Mosquito-Derived Cells.","authors":"P Böhmer, M Reuter, M Altinli, E Schnettler","doi":"10.1007/978-1-0716-4338-9_7","DOIUrl":"10.1007/978-1-0716-4338-9_7","url":null,"abstract":"<p><p>Viruses of the class Bunyaviricetes are often transmitted by arthropods, including mosquitoes. The innate immune response in mosquitoes comprises several pathways, including sequence-specific degradation through RNA interference (RNAi). It is known that bunyavirus infections are targeted by the innate immune response in mosquitoes and derived cells; however, detailed information is often still missing. In this chapter, we describe a detailed protocol to determine if a protein of interest acts antiviral against bunyavirus infection in mosquito-derived cells. In line, we also describe a detailed protocol to investigate the effect of overexpression of a mosquito-encoded protein or analysis of mutant proteins on bunyavirus infection.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2893 ","pages":"73-84"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142818559","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Measuring Cellular Adenine Nucleotides by Liquid Chromatography-Coupled Mass Spectrometry.","authors":"Ashley J Ovens, Dingyi Yu, Toby A Dite, Bruce E Kemp, Jonathan S Oakhill","doi":"10.1007/978-1-0716-4284-9_1","DOIUrl":"https://doi.org/10.1007/978-1-0716-4284-9_1","url":null,"abstract":"<p><p>Adenine nucleotides (AXPs, also referred to as adenosines or adenylates) are a group of organic molecules including adenosine 5'- mono-, di-, and tri-phosphate (AMP, ADP, and ATP, respectively) that, combined, resembles an electrochemical storage cell to facilitate cellular energy storage and transfer. ATP, generated from ADP by photosynthesis, anaerobic respiration, and oxidative phosphorylation, powers many energy-requiring processes in the cell through hydrolysis of its terminal (γ) phosphate, whereas ADP is equilibrated with AMP and ATP by the adenylate kinase reaction. AXPs are major signaling molecules that regulate a wide range of anabolic and catabolic enzymes including AMP-activated protein kinase (AMPK), phosphofructokinase, and pyruvate dehydrogenase.Methods to determine concentrations of AXPs from cells and biological samples have historically relied on high-performance liquid chromatography (HPLC)/capillary electrophoresis techniques to measure [ATP] and [ADP]. However, due to its low basal concentrations, these techniques lack sufficient sensitivity to directly measure [AMP], which must be extrapolated using assumptions of adenylate kinase equilibrium that neglect AMP degradation and synthesis pathways. Here, we describe a detailed protocol to accurately measure [AXP] from cells by liquid chromatography-coupled mass spectrometry (LC/MS), applicable to a wide range of fields including our specific interest in AMPK-dependent metabolic regulation.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2882 ","pages":"3-14"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143483644","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"FRET-Based Sensor for Measuring Adenine Nucleotide Binding to AMPK.","authors":"Roland Abi Nahed, Martin Pelosse, Francesco Aulicino, Florine Cottaz, Imre Berger, Uwe Schlattner","doi":"10.1007/978-1-0716-4284-9_2","DOIUrl":"https://doi.org/10.1007/978-1-0716-4284-9_2","url":null,"abstract":"<p><p>AMP-activated protein kinase (AMPK) has evolved to detect a critical increase in cellular AMP/ATP and ADP/ATP concentration ratios as a signal for limiting energy supply. Such energy stress then leads to AMPK activation and downstream events that maintain cellular energy homeostasis. AMPK activation by AMP, ADP, or pharmacological activators involves a conformational switch within the AMPK heterotrimeric complex. We have engineered an AMPK-based sensor, AMPfret, which translates the activating conformational switch into a fluorescence signal, based on increased fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores. Here we describe how this sensor can be used to analyze direct AMPK activation by small molecules in vitro using a fluorimeter, or to estimate changes in the energy state of cells using standard fluorescence or confocal microscopy.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2882 ","pages":"15-45"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143483693","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Computational Pipeline for Identifying Gene Regulatory Networks: A Case Study of Response to Exercise.","authors":"Nadia Moore, Jeffrey Page, William E Kraus, Kim M Huffman, Gordon Broderick","doi":"10.1007/978-1-0716-4200-9_13","DOIUrl":"10.1007/978-1-0716-4200-9_13","url":null,"abstract":"<p><p>Gene regulatory networks are foundational in the control of virtually all biological processes. These networks orchestrate a myriad of cell functions ranging from metabolic rate to the response to a drug or other intervention. The data required to accurately identify these control networks remains very cost and labor intensive typically leading to relatively sparse time course data that is largely incompatible with conventional data-driven model identification techniques. In this work, we combine empirical identification of gene-gene interactions with constraints describing the expected dynamic behavior of the network to infer regulatory dynamics from under-sampled data. We apply this to the identification of gene regulatory subnetworks recruited in groups of subjects participating in several different exercise interventions. Intervention-specific response networks are compared to one another and control actions driving differences are identified. We propose that this approach can extract statistically robust and biologically meaningful insights into gene regulatory dynamics from a dataset consisting of a small number of participants with very limited longitudinal sampling, for example pre- and post- intervention only.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2868 ","pages":"247-264"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142639339","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Enzymatically Ligated Nucleic Acid Nanocapsules for the Delivery of Therapeutic Nucleic Acids and Small Molecule Drugs.","authors":"Jenna N Cannata, Jessica L Rouge","doi":"10.1007/978-1-0716-4402-7_4","DOIUrl":"https://doi.org/10.1007/978-1-0716-4402-7_4","url":null,"abstract":"<p><p>Spherical nucleic acids (SNAs) offer intriguing properties for cellular uptake and stability. A novel SNA-like structure known as the nucleic acid nanocapsule (NAN) combines the benefits of SNAs with the added properties of nucleic acid functionalization and drug cargo release. Herein, we will discuss various ways NANs can be adapted to allow for gene targeting via the delivery of nucleic acids, as well as the delivery of small molecule drugs for combination therapies.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2902 ","pages":"55-67"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542583","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of Constitutive or Induced Activation State of the VLA-4 Integrin in Human and Murine Samples.","authors":"Erika Tissino, Antonella Zucchetto, Federico Pozzo, Tanja Nicole Hartmann, Valter Gattei","doi":"10.1007/978-1-0716-4442-3_4","DOIUrl":"https://doi.org/10.1007/978-1-0716-4442-3_4","url":null,"abstract":"<p><p>The integrin heterodimer CD49d/CD29 (a.k.a. Very Late Antigen-4, VLA-4) mediates cell-cell and cell-matrix interaction through binding to its specific ligands. VLA-4 can be present on the cell surface at different conformation states that affect the binding affinity for the ligands. In chronic lymphocytic leukemia (CLL), higher VLA-4 levels have been demonstrated to be associated with a worse prognosis both in the chemo-immunotherapy era and in the BCR inhibitor setting, in keeping with the role of VLA-4 as a key molecule favoring CLL cell localization in protective niches of bone marrow and lymph nodes. Here, we describe functional flow cytometry-based methods to assess the activation state of the VLA-4 integrin, applicable in both human and murine settings, as well as in fresh or thawed samples. A specific \"R\" script for analyzing flow cytometry data is also provided.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2909 ","pages":"45-60"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542642","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-Resolution Imaging of Intracellular Trafficking of B Cell Receptor Using Specific Hybridization Internalization Probe (SHIP).","authors":"Sara Hernández-Pérez, Pieta K Mattila","doi":"10.1007/978-1-0716-4442-3_6","DOIUrl":"https://doi.org/10.1007/978-1-0716-4442-3_6","url":null,"abstract":"<p><p>Recent advancements in microscopy have greatly expanded our understanding of intracellular traffic. Yet, due to the inherent characteristics of B cells, such as their small size and high receptor density on the plasma membrane, visualization of internalized cargo or receptors remains challenging. This challenge is particularly pronounced in the case of the B cell receptor (BCR), where accurate detection of internalized, antigen-bound BCR molecules can be strongly hindered by the signal from the plasma membrane-bound pool of the same molecules.To tackle this issue, we adapted the Specific Hybridization Internalization Probe (SHIP) assay, initially designed for flow cytometry studies, for the study of BCR internalization using microscopy. This assay utilizes a single-stranded DNA (ssDNA) fluorescence internalization probe (FIP) paired with a complementary ssDNA quenching probe that \"turns off\" the signal from the (extracellular) surface-bound BCRs, greatly facilitating the unambiguous identification of internalized (intracellular) receptors. Moreover, the assay is versatile and adaptable to a range of imaging modalities, including live-cell imaging and super-resolution microscopy. SHIP proves to be a valuable tool in the study of intracellular processes, offering enhanced imaging precision for the detection of internalized BCRs.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2909 ","pages":"73-82"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542645","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}