Methods in enzymology最新文献_第10页

Northern blotting for human pre-tRNA and tRNA-derived RNAs. 人类前trna和trna衍生rna的Northern印迹。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-30 DOI: 10.1016/bs.mie.2024.11.013

Yoshika Takenaka, Katsuki Aoyama, Yasutoshi Akiyama

引用次数: 0

Profiling rare C-to-U editing events via direct RNA sequencing. 通过直接RNA测序分析罕见的C-to-U编辑事件。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-21 DOI: 10.1016/bs.mie.2024.11.040

Adriano Fonzino, Pietro Luca Mazzacuva, Graziano Pesole, Ernesto Picardi

{"title":"Profiling rare C-to-U editing events via direct RNA sequencing.","authors":"Adriano Fonzino, Pietro Luca Mazzacuva, Graziano Pesole, Ernesto Picardi","doi":"10.1016/bs.mie.2024.11.040","DOIUrl":"https://doi.org/10.1016/bs.mie.2024.11.040","url":null,"abstract":"In mammals, RNA editing involves the hydrolytic deamination of adenosine (A) to inosine (I) or of cytosine (C) to uracil (U) by the ADAR and APOBEC families of enzymes, respectively. Direct RNA (dRNA) sequencing by Oxford Nanopore Technology (ONT) allows the detection of Us and, thus, facilitates the unveiling of edited Cs avoiding Reverse Transcription and PCR amplification steps. However, dRNA data are noisy, and very rare events such as C-to-U conversions cannot be easily distinguished from background noise or mutation errors. To overcome this issue, we developed a novel machine-learning strategy based on the Isolation Forest (iForest) algorithm to denoise the signal deriving from dRNA highly-informative ONT data. Here we present a step-by-step protocol illustrating the usage of the C-to-U-Classifier package and how to apply its pretrained iForest models for ameliorating the detection of C-to-U events in mammalian transcriptomes. As an example, we show here the whole pipeline in action on data deriving from wild-type (WT) and APOBEC1 knock-out (KO) macrophagic cell lines. Additionally, the polishing power of our algorithm is proved through a synthetic in-vitro transcribed (IVT) sample in which C-to-U events are not present.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"713 ","pages":"221-254"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144004059","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Preface. 前言。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 DOI: 10.1016/S0076-6879(25)00055-2

Peter A Beal

引用次数: 0

Immobilisation for a stable biocatalyst application. 固定化稳定的生物催化剂应用。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-03-10 DOI: 10.1016/bs.mie.2025.01.018

Karolina Labus, Aleksandra Lambarska, Katarzyna Czyżewska, Katarzyna Szymańska

引用次数: 0

Functional metaproteomics for enzyme discovery. 酶发现的功能宏蛋白质组学。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-01-25 DOI: 10.1016/bs.mie.2025.01.029

Marina Prisacar, Lars I Leichert

{"title":"Functional metaproteomics for enzyme discovery.","authors":"Marina Prisacar, Lars I Leichert","doi":"10.1016/bs.mie.2025.01.029","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.029","url":null,"abstract":"Discovery of microbial biocatalysts traditionally relied on activity screening of isolated bacterial strains. However, since most microorganisms cannot be cultivated in the lab, such an approach leaves the majority of the microbial enzyme diversity untapped. Metagenomic approaches, in which the DNA from a microbial community is directly isolated and then used either for the creation of an expression library or for sequencing and metagenome annotation have alleviated this shortcoming to an extent, but have their own limitations: the generation of large expression libraries is time-consuming and their screening is costly, while metagenome annotation can infer biocatalytic function only from prior knowledge. We have thus developed a functional metaproteomic approach, which combines the immediacy of traditional activity screening with the comprehensiveness of a meta-omics approach. Briefly, the whole metaproteome of an environmental sample is separated on a 2-D gel, biocatalytically active proteins are visualized in-gel through zymography, and those candidate biocatalysts are then identified through mass spectrometry, searching against a metagenome-derived database obtained from the very same environmental sample. Here we explain the process in detail, with a focus on esterases, and give guidelines on how to develop a functional metaproteomic workflow for enzyme discovery.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"714 ","pages":"61-82"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144002886","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Peroxygenase-catalysed oxyfunctionalisation reactions. 过氧酶催化的氧化官能化反应。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-06 DOI: 10.1016/bs.mie.2025.01.041

Thomas Hilberath, Frank Hollmann, Florian Tieves, Wuyuan Zhang

{"title":"Peroxygenase-catalysed oxyfunctionalisation reactions.","authors":"Thomas Hilberath, Frank Hollmann, Florian Tieves, Wuyuan Zhang","doi":"10.1016/bs.mie.2025.01.041","DOIUrl":"10.1016/bs.mie.2025.01.041","url":null,"abstract":"Peroxygenases represent a class of versatile heme-thiolate enzymes capable of catalysing highly selective oxyfunctionalisation reactions, particularly the hydroxylation of non-activated C-H bonds. This transformation, which poses substantial challenges in conventional organic synthesis, underscores the potential of peroxygenases in green chemistry applications. While cytochrome P450 monooxygenases have long been the primary focus for such biocatalytic transformations, their industrial adoption has been limited due to complex electron transfer chains and cofactor requirements. In contrast, peroxygenases bypass these limitations by directly utilising hydrogen peroxide (H2O2) to activate the catalytic heme site, thereby circumventing the oxygen dilemma typically encountered in P450 catalysis. Key milestones in peroxygenase research include the identification of chloroperoxidase from Caldariomyces fumago and the subsequent discovery of unspecific peroxygenases, such as those from Agrocybe aegerita, which exhibit broad substrate specificity and high catalytic efficiency. Here, we explore the mechanistic pathway of peroxygenase-catalysed reactions, emphasising the formation and decay of Compound I and the catalytic cycle's various functional outcomes. Critical aspects such as in situ H2O2 generation to mitigate enzyme inactivation, substrate loading strategies for practical applications, and the role of enzyme and reaction engineering in enhancing regio- and stereoselectivity are examined. Additionally, we address challenges in reaction scalability and operational stability for preparative-scale applications, offering insights into innovative protocols involving immobilised enzymes and non-aqueous reaction media. This review highlights recent advancements in the peroxygenase field and underscores the enzyme's promising role in sustainable oxyfunctionalisation reactions.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"714 ","pages":"425-443"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144029415","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Isolation of Hafnia paralvei FB315, a polyamine-high-producing bacterium, from aged cheese. 从陈化奶酪中分离多胺高产菌Hafnia parvei FB315。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-19 DOI: 10.1016/bs.mie.2025.02.004

Yuta Ami, Emi Sugiwaka, Shin Kurihara

引用次数: 0

An enhanced method for the determination of polyamine content in biological samples by dansyl chloride derivatization and HPLC. 氯丹酰衍生化-高效液相色谱法测定生物样品中多胺含量。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-13 DOI: 10.1016/bs.mie.2025.01.066

Ashley Nwafor, Tracy Murray Stewart, Robert A Casero

{"title":"An enhanced method for the determination of polyamine content in biological samples by dansyl chloride derivatization and HPLC.","authors":"Ashley Nwafor, Tracy Murray Stewart, Robert A Casero","doi":"10.1016/bs.mie.2025.01.066","DOIUrl":"https://doi.org/10.1016/bs.mie.2025.01.066","url":null,"abstract":"The polycationic alkylamines known as polyamines are ubiquitous organic cations found in all forms of life and are involved in multiple biological processes including RNA and DNA replications and conformational changes, transcription, translation, regulation of ion channels, free radical scavenging, and cell proliferation. Therefore, polyamine homeostasis is critical to normal cellular function and health. In cancer and other hyperproliferative diseases, polyamines and their metabolism are frequently dysregulated due to the increased requirement for polyamines under conditions of rapid, or continued, uncontrolled growth. Consequently, polyamine function and metabolism are of considerable interest as possible targets for antiproliferative therapies. As the awareness of the importance of polyamines in health and disease has increased, so has the need for rapid and precise measurements of polyamines in biological samples, including cells, blood, serum, urine, and other biological fluids. Here we present an improvement over one of the standard high performance liquid chromatography methods that is faster, uses fewer reagents, and is at least as precise as previously published methods.","PeriodicalId":18662,"journal":{"name":"Methods in enzymology","volume":"715 ","pages":"323-335"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144094280","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Analysis of translational regulation using polysome profiling and puromycin incorporation. 用多体分析和嘌呤霉素掺入分析翻译调控。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-18 DOI: 10.1016/bs.mie.2025.01.035

Quinton W Wood, Teresa L Mastracci

引用次数: 0

Alterations in polyamine metabolism induced by the pathogen Helicobacter pylori: Implications for gastric inflammation and carcinogenesis. 幽门螺杆菌诱导的多胺代谢的改变：对胃炎症和癌变的影响。

4区生物学

Methods in enzymology Pub Date : 2025-01-01 Epub Date: 2025-02-20 DOI: 10.1016/bs.mie.2025.01.037

Alain P Gobert, Caroline V Hawkins, Kara M McNamara, Keith T Wilson

引用次数: 0