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Quantification of Infectious Rhinovirus A and B Serotypes by Plaque Assay. 用空斑法定量测定传染性鼻病毒A、B血清型。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4410-2_4
Mohsen Tabasi, Haleh Ganjian, Umadevi Sajjan
{"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":"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}
引用次数: 0
Evaluation of Constitutive or Induced Activation State of the VLA-4 Integrin in Human and Murine Samples. hla -4整合素在人、鼠体内本构激活或诱导激活状态的评价。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4442-3_4
Erika Tissino, Antonella Zucchetto, Federico Pozzo, Tanja Nicole Hartmann, Valter Gattei
{"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":"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}
引用次数: 0
Cryopreservation of Small Ruminant Sperm and Detection of Sperm Cryocapacitation. 小型反刍动物精子的低温冷冻和精子冷冻能力检测。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4406-5_12
Patricia Peris-Frau, Silvia Gimeno-Martos
{"title":"Cryopreservation of Small Ruminant Sperm and Detection of Sperm Cryocapacitation.","authors":"Patricia Peris-Frau, Silvia Gimeno-Martos","doi":"10.1007/978-1-0716-4406-5_12","DOIUrl":"10.1007/978-1-0716-4406-5_12","url":null,"abstract":"<p><p>Sperm cryopreservation is an indispensable tool for preserving the genetic and epigenetic material of valuable males or endangered species. Here, we describe the protocols most frequently used for conventional cryopreservation of small ruminant sperm, considering differences between the origin of the sperm sample (epididymal vs. ejaculated sperm) and species. In addition, since one of the major drawbacks of sperm cryopreservation is the reduction of viability and fertility due to capacitation-like changes in a considerable amount of sperm, different methods to detect sperm cryocapacitation in small ruminants are explained.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2897 ","pages":"165-182"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143811757","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}
引用次数: 0
A Deep Learning and PSSM Profile Approach for Accurate SNARE Protein Prediction. 基于深度学习和PSSM谱的陷阱蛋白准确预测方法。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4314-3_5
Quang Hien Kha, Huu Phuc Lam Nguyen, Nguyen Quoc Khanh Le
{"title":"A Deep Learning and PSSM Profile Approach for Accurate SNARE Protein Prediction.","authors":"Quang Hien Kha, Huu Phuc Lam Nguyen, Nguyen Quoc Khanh Le","doi":"10.1007/978-1-0716-4314-3_5","DOIUrl":"10.1007/978-1-0716-4314-3_5","url":null,"abstract":"<p><p>SNARE proteins play a pivotal role in membrane fusion and various cellular processes. Accurate identification of SNARE proteins is crucial for elucidating their functions in both health and disease contexts. This chapter presents a novel approach employing multiscan convolutional neural networks (CNNs) combined with position-specific scoring matrix (PSSM) profiles to accurately recognize SNARE proteins. By leveraging deep learning techniques, our method significantly enhances the accuracy and efficacy of SNARE protein classification. We detail the step-by-step methodology, including dataset preparation, feature extraction using PSI-BLAST, and the design of the multiscan CNN architecture. Our results demonstrate that this approach outperforms existing methods, providing a robust and reliable tool for bioinformatics research.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2887 ","pages":"79-89"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142979129","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}
引用次数: 0
Measuring Cellular Adenine Nucleotides by Liquid Chromatography-Coupled Mass Spectrometry. 液相色谱-耦合质谱法测定细胞腺嘌呤核苷酸。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4284-9_1
Ashley J Ovens, Dingyi Yu, Toby A Dite, Bruce E Kemp, Jonathan S Oakhill
{"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":"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}
引用次数: 0
Picrotoxin-Induced Epileptogenic Hippocampal Organotypic Slice Cultures (hOTCs). picrotoxin诱导的癫痫性海马器官型切片培养（hotc）。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4386-0_23
Ane Rodríguez-Bodero, Paolo Bonifazi, Jan Tønnesen, Juan Manuel Encinas-Pérez
{"title":"Picrotoxin-Induced Epileptogenic Hippocampal Organotypic Slice Cultures (hOTCs).","authors":"Ane Rodríguez-Bodero, Paolo Bonifazi, Jan Tønnesen, Juan Manuel Encinas-Pérez","doi":"10.1007/978-1-0716-4386-0_23","DOIUrl":"10.1007/978-1-0716-4386-0_23","url":null,"abstract":"<p><p>Cultured organotypic hippocampal slices (hOTCs) have become increasingly popular as a model for studying brain function. This model offers significant advantages over traditional in vitro methods, as they allow the examination of mid to long-term manipulations while preserving the structure of the dentate gyrus (DG) in the hippocampus. In this chapter, we focus on a protocol based on hOTCs of mouse entorhinal cortex and hippocampus, which by integrating techniques such as retroviral injections, immunohistochemistry, and microscopy imaging, physiological or pathological processes can be easily investigated.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2899 ","pages":"367-388"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143605499","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}
引用次数: 0
Engineered Hydrogels for 3D Cell Culture and Bioprinting of Human Induced Pluripotent Stem Cell-Derived Neuroepithelial Stem Cells. 人类诱导多能干细胞衍生神经上皮干细胞的3D细胞培养和生物打印工程水凝胶。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4530-7_16
Daniel Aili, Anna Herland
{"title":"Engineered Hydrogels for 3D Cell Culture and Bioprinting of Human Induced Pluripotent Stem Cell-Derived Neuroepithelial Stem Cells.","authors":"Daniel Aili, Anna Herland","doi":"10.1007/978-1-0716-4530-7_16","DOIUrl":"https://doi.org/10.1007/978-1-0716-4530-7_16","url":null,"abstract":"<p><p>This protocol outlines the synthesis and use of engineered hyaluronan-based hydrogels for 3D cell culture and bioprinting of human induced pluripotent stem cell (hiPSC)-derived neuroepithelial stem cells (lt-NES). Key steps include hydrogel formation using bioorthogonal chemistries, cell encapsulation, and 3D bioprinting with a Cellink BioX printer, enabling the creation of complex tissue models. The protocol ensures high cell viability and supports differentiation, essential for neuroscience research and drug development.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2924 ","pages":"223-233"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144022332","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}
引用次数: 0
Using Zymography to Assess Circulating Matrix Metalloproteinase (MMP)-2 and MMP-9 in Clinical Samples. 应用酶谱法评估临床样品中循环基质金属蛋白酶(MMP)-2和MMP-9。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4482-9_14
Sandra O Conde-Tella, Raquel F Gerlach, Jose E Tanus-Santos
{"title":"Using Zymography to Assess Circulating Matrix Metalloproteinase (MMP)-2 and MMP-9 in Clinical Samples.","authors":"Sandra O Conde-Tella, Raquel F Gerlach, Jose E Tanus-Santos","doi":"10.1007/978-1-0716-4482-9_14","DOIUrl":"https://doi.org/10.1007/978-1-0716-4482-9_14","url":null,"abstract":"<p><p>Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that cleave a variety of different substrates and play key roles in both physiological and pathological processes. They are involved in the pathophysiology of many disease conditions, and for this reason, many clinical studies have been carried out to examine whether circulating MMP concentrations reflect the severity of disease conditions. This article presents detailed information that is critical to assess circulating MMP-2 and MMP-9 activity in clinical samples by using zymography, which is a powerful technique that has been used for more than three decades to detect picogram quantities of MMPs. Important methodological issues have been addressed in the past regarding this technique, and some analytical and preanalytical details may severely affect the results and thus hamper the diagnostic and prognostic value of such measurements. While fasting or storing plasma samples for 1 month at -20 °C or at -70 °C does not significantly affect plasma MMP-2 or MMP-9 activity, repeating freeze-thaw cycles of plasma samples decrease MMP-9 activity after seven cycles. Importantly, the appropriate volume to assess MMP-2 is significantly lower than the appropriate volume to assess MMP-9 activity. Finally, special concerns exist with respect to circulating MMP-9 (but not MMP-2) measurements, which are found at artificially higher concentrations in serum than in plasma (EDTA, citrate or heparin) samples, even though significant correlations exist between MMP-9 levels assessed in plasma and in serum samples.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2918 ","pages":"177-186"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144026268","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}
引用次数: 0
Psoralen Crosslinking-Chromatin Endogenous Cleavage Assay to Examine Histone DNA Interactions of Active and Inactive rRNA Genes. 补骨脂素交联-染色质内源性切割试验检测活性和非活性rRNA基因的组蛋白DNA相互作用。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4486-7_8
Alexia Muguet, Thomas Gardrat, Antonio Conconi, Audrey Paillé
{"title":"Psoralen Crosslinking-Chromatin Endogenous Cleavage Assay to Examine Histone DNA Interactions of Active and Inactive rRNA Genes.","authors":"Alexia Muguet, Thomas Gardrat, Antonio Conconi, Audrey Paillé","doi":"10.1007/978-1-0716-4486-7_8","DOIUrl":"https://doi.org/10.1007/978-1-0716-4486-7_8","url":null,"abstract":"<p><p>In the nucleoli of eukaryotic cells, the multiple copies of ribosomal RNA genes (rRNA genes) coexist in two different forms that have distinct characteristics: transcribed (active) and non-transcribed (inactive) units. \"Active\" rRNA genes are loaded with RNA polymerase I and are largely depleted of nucleosomes, whereas \"inactive\" rRNA genes are covered with two copies of the four histone proteins that are folded in nucleosomes. A third form of chromatin is observed in Saccharomyces cerevisiae (here called as yeast) arrested in the G1 phase of the cell cycle. In yeast synchronized before DNA replication, nucleosomes are also absent in the non-transcribed rRNA genes, which are described as \"open\" units.The presence of two distinct groups of rRNA genes compromises the interpretation of standard biochemical assays that are employed to study the structure of chromatin during DNA transcription, DNA replication, and DNA repair. This chapter describes protocols to investigate the association of histone proteins with rRNA genes in yeast. In addition, it provides a comprehensive list of studies that applied psoralen photo-crosslinking to follow the structure of rRNA gene chromatin in a variety of high eukaryotic cells.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2919 ","pages":"133-154"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144026281","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}
引用次数: 0
Kinetics of Protease Thermal Inactivation. 蛋白酶热失活动力学。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4478-2_21
Natalija Andrejević, Natalija Polović, Jelica Milošević
{"title":"Kinetics of Protease Thermal Inactivation.","authors":"Natalija Andrejević, Natalija Polović, Jelica Milošević","doi":"10.1007/978-1-0716-4478-2_21","DOIUrl":"https://doi.org/10.1007/978-1-0716-4478-2_21","url":null,"abstract":"<p><p>Proteolytic enzymes have various applications in biomedicine, biotechnology, pharmaceuticals, and the food industry. Some of these applications require incubation at extreme temperatures, high pressure, different pH of the solution, and the presence of various additives that might destabilize enzyme structure and function. Structural features of proteases define the mechanism of denaturation, which is reflected in different activation energies for the process. Understanding the activation energy gives clues about the kinetic inertness of the enzyme and its resistance to destabilizing factors. Monitoring the kinetics of enzyme thermal inactivation by measuring activity loss upon heating at various temperatures enables the determination of activation energy for the inactivation process using the Arrhenius plot. This way, measuring the activity loss as a result of structural perturbations gives direct information on the enzyme stability in the relevant conditions for applicative processes.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2917 ","pages":"247-258"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144017400","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}
引用次数: 0
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