往期刊物2023
卷册: 13, 期号: 13
生物化学
Human-rabbit Hybrid Translation System to Explore the Function of Modified Ribosomes
探索修饰核糖体功能的人兔混合翻译系统
In vitro translation systems are a useful biochemical tool to research translational regulation. Although the preparation of translation-competent cell extracts from mammals has often been a challenge, the commercially available rabbit reticulocyte lysate (RRL) is an exception. However, its valid use, investigating the mechanism of translation machinery such as ribosomes in RRL, presents an analytic hurdle. To overcome this issue, the hybrid translation system, which is based on the supplementation of purified human ribosomes into ribosome-depleted RRL, has been developed. Here, we describe the step-by-step protocol of this system to study translation driven by ribosomes lacking post-translational modifications of the ribosomal protein. Moreover, we combined this approach with a previously developed reporter mRNA to assess the processivity of translation elongation. This protocol could be used to study the potency of heterologous ribosomes.
Cuticular Hydrocarbon Profiling by Fractionation and GC-MS in Socially Parasitic Ants
通过分馏和气相色谱-质谱分析对社会性寄生蚂蚁的表皮碳氢化合物进行分析
Ants use cuticular hydrocarbon (CHC) as a semiochemical for recognizing their nestmates. For socially parasitic ants, deceiving the CHC is an important survival strategy. Profiling and quantifying CHC is a potent approach to understanding such nestmate discrimination behavior. Thus, a highly efficient, stable, and reproducible extraction method for CHC is essential for this purpose. This paper describes a method for socially parasitic ants to disguise the host species’ CHC profile under laboratory conditions, as well as the extraction and measurement of CHC from ants (from a previous study). First, the artificial isotopic substance is applied to the host worker; then, the socially parasitic ant disguises the host-like CHC profile against the above host worker. Next, the CHC is extracted and fractionated from a socially parasitic ant using hexane and silica gel. After concentrating the fractionated product, this product is then used for measurement by gas chromatographymass spectrometry (GC-MS). The CHC extraction protocol described in this paper may be used for various ant species.
生物工程
In vitro Selection and in vivo Testing of Riboswitch-inspired Aptamers
受核开关启发的适体的体外筛选和体内测试
Engineered aptamers for new compounds are typically produced by using in vitro selection methods. However, aptamers that are developed in vitro might not function as expected when introduced into complex cellular environments. One approach that addresses this concern is the design of initial RNA pools for selection that contain structural scaffolds from naturally occurring riboswitch aptamers. Here, we provide guidance on design and experimental principles for developing riboswitch-inspired aptamers for new ligands. The in vitro selection protocol (based on Capture-SELEX) is generalizable to diverse RNA scaffold types and amenable to multiplexing of ligand candidates. We discuss strategies to avoid propagation of selfish sequences that can easily dominate the selection. We also detail the identification of aptamer candidates using next-generation sequencing and bioinformatics, and subsequent biochemical validation of aptamer candidates. Finally, we describe functional testing of aptamer candidates in bacterial cell culture.Key features• Develop riboswitch-inspired aptamers for new ligands using in vitro selection.• Ligand candidates can be multiplexed to conserve time and resources.• Test aptamer candidates in bacterial cells by grafting the aptamer back onto its expression platform.Graphical overview
生物物理学
Dual-color Colocalization in Single-molecule Localization Microscopy to Determine the Oligomeric State of Proteins in the Plasma Membrane
单分子定位显微镜双色共定位法测定质膜中蛋白质的寡聚状态
Determining the oligomeric state of membrane proteins is critical for understanding their function. However, traditional ex situ methods like clear native gel electrophoresis can disrupt protein subunit interactions during sample preparation. In situ methods such as stepwise photobleaching have limitations due to high expression levels and limitations of optical resolution in microscopy. Super-resolution microscopy techniques such as single-molecule localization microscopy (SMLM) have the potential to overcome these limitations, but the stochastic nature of signals can lead to miscounting due to over-expression, background noise, and temporal separation of signals. Additionally, this technique has limited application due to the limited selection of fluorescent labels and the demanding control of laser power. To address these issues, we developed a dual color colocalization (DCC) strategy that offers higher tolerance to background noise and simplifies data acquisition and processing for high-throughput and reliable counting. The DCC strategy was used to determine the oligomeric states of membrane proteins of the SLC17 and SLC26 family with SMLM, providing a robust and efficient method for studying protein interactions.Graphical overview (A) Illustration of the principle for determining the oligomeric state of protein complexes with dual color colocalization–single-molecule localization microscopy (DCC-SMLM). In the inset, as an example, a dimeric protein (brown) is labeled with a marker (M) and an indicator fluorescent protein (F) on each of its two subunits. The overall probability of detecting the dimer with SMLM, as denoted by R, the colocalization ratio, is equal to the ratio of the number of colocalized marker and indicator clusters (NMF) to that of the marker clusters (NM). The plot shows the linear relationship of the oligomeric state (n) vs. the natural logarithm of 1 subtracted by the colocalization ratio, supplemented by the equation of the fitting curve, in which p denotes the recall rate of the indicator fluorescent protein (F). (B) The workflow diagram shows the procedures of DCC-SMLM (Locs: localizations; COM: coefficient of mismatch; LCA: lateral chromatic aberration).
癌症生物学
mRNA Delivery Platform Based on Bacterial Outer Membrane Vesicles for Tumor Vaccine
基于细菌外膜囊泡的mRNA传递平台用于肿瘤疫苗
The rapid display and delivery method for customized tumor mRNA vaccines is limited. Herein, bacteria-derived outer membrane vesicles (OMVs) are employed as an mRNA delivery platform by surface engineering of an RNA-binding protein, L7Ae. OMV-L7Ae can rapidly adsorb boxC/D sequence-labeled mRNA antigens through L7Ae-boxC/D binding and deliver them into HEK-293T and dendritic cells. This platform provides an mRNA delivery technology distinct from lipid nanoparticles (LNPs) for personalized mRNA tumor vaccination and with a Plug-and-Display strategy suitable for rapid preparation of the personalized mRNA tumor vaccine against varied tumor antigens.Key features• OMVs are employed as an mRNA delivery platform through L7Ae-boxC/D binding.Graphical overview
3D Ultrastructural Visualization of Mitosis Fidelity in Human Cells Using Serial Block Face Scanning Electron Microscopy (SBF-SEM)
利用连续块面扫描电子显微镜（SBF-SEM）对人细胞有丝分裂保真度的三维超微结构可视化
Errors in chromosome segregation during mitosis lead to chromosome instability, resulting in an unbalanced number of chromosomes in the daughter cells. Light microscopy has been used extensively to study chromosome missegregation by visualizing errors of the mitotic spindle. However, less attention has been paid to understanding spindle function in the broader context of intracellular structures and organelles during mitosis. Here, we outline a protocol to visualize chromosomes and endomembranes in mitosis, combining light microscopy and 3D volume electron microscopy, serial block-face scanning electron microscopy (SBF-SEM). SBF-SEM provides high-resolution imaging of large volumes and subcellular structures, followed by image analysis and 3D reconstruction. This protocol allows scientists to visualize the whole subcellular context of the spindle during mitosis.
细胞生物学
Iterative Indirect Immunofluorescence Imaging (4i) on Adherent Cells and Tissue Sections
粘附细胞和组织切片的迭代间接免疫荧光成像（4i）
Highly multiplexed protein measurements from multiple spatial scales using fluorescence microscopy recently emerged as a powerful way to investigate tumor microenvironments in biomedicine and the multivariate nature of complex systems’ interactions. A range of methods for this exist, which either rely on directly labeling the primary antibody with oligonucleotides/rare metals or employing methods to remove fluorescence for cyclic acquisition. Here, we describe a protocol that uses off-the-shelf primary and secondary antibodies without further need for modification and only commonly available chemical reagents. The method harnesses the observation that antibodies can crosslink to bound epitopes during light exposure, thus preventing elution. By utilizing a simple oxygen radical scavenging buffer during imaging and by blocking free sulfhydryl groups before antibody incubation, the presented method can employ comparably mild conditions to remove bound antibodies from epitopes, which preserves sample integrity. Thus, with the stated minor modifications, it allows for a standard immunofluorescence imaging protocol in cyclic fashion, currently permitting staining of up to ~80 unique epitopes.Graphical overview
Visualizing NBD-lipid Uptake in Mammalian Cells by Confocal Microscopy
共聚焦显微镜观察哺乳动物细胞对NBD脂质的摄取
Eukaryotic cells use a series of membrane transporters to control the movement of lipids across their plasma membrane. Several tools and techniques have been developed to analyze the activity of these transporters in the plasma membrane of mammalian cells. Among them, assays based on fluorescence microscopy in combination with fluorescent lipid probes are particularly suitable, allowing visualization of lipid internalization in living cells. Here, we provide a step-by-step protocol for mammalian cell culture, lipid probe preparation, cell labeling, and confocal imaging to monitor lipid internalization by lipid flippases at the plasma membrane based on lipid probes carrying a fluorophore at a short-chain fatty acid. The protocol allows studying a wide range of mammalian cell lines, to test the impact of gene knockouts on lipid internalization at the plasma membrane and changes in lipid uptake during cell differentiation.Key features• Visualization and quantification of lipid internalization by lipid flippases at the plasma membrane based on confocal microscopy.• Assay is performed on living adherent mammalian cells in culture.• The protocol can be easily modified to a wide variety of mammalian cell lines.Graphical overviewAnalysis of NBD-lipid uptake in adherent mammalian cells by confocal microscopy. Scale bar, 30 μm.
发育生物学
Visualization of Actin Cytoskeleton in Cellular Protrusions in Medaka Embryos
青鳉胚胎中细胞突起的肌动蛋白细胞骨架的可视化研究
Cellular protrusions are fundamental structures for a wide variety of cellular behaviors, such as cell migration, cell–cell interaction, and signal reception. Visualization of cellular protrusions in living cells can be achieved by labeling of cytoskeletal actin with genetically encoded fluorescent probes. Here, we describe a detailed experimental procedure to visualize cellular protrusions in medaka embryos, which consists of the following steps: preparation of Actin-Chromobody-GFP and α-bungarotoxin mRNAs for actin labeling and immobilization of the embryo, respectively; microinjection of the mRNAs into embryos in a mosaic fashion to sparsely label individual cells; removal of the hard chorion, which hampers observation; and visualization of cellular protrusions in the embryo with a confocal microscope. Overall, our protocol provides a simple method to reveal cellular protrusions in vivo by confocal microscopy.
Systematic Analysis of Smooth Muscle and Cartilage Ring Formation during Mouse Tracheal Tubulogenesis
小鼠气管插管过程中平滑肌和软骨环形成的系统分析
The trachea tube is the exclusive route to allow gas exchange between the external environment and the lungs. Recent studies have shown the critical role of mesenchymal cells in tracheal tubulogenesis. Improved methods for studying the dynamics of the tracheal mesenchyme development are needed to investigate the cellular and molecular mechanisms during tracheal tubulogenesis. Here, we describe a detailed protocol for a systematic analysis of tracheal tube development to enable observing tracheal smooth muscle (SM) and cartilage ring formation. We describe immunostaining, confocal and stereomicroscopy imaging, and quantitative methods to study the process of tracheal SM and cartilage ring development, including SM cell alignment, polarization, and changes in cell shape as well as mesenchymal condensation. The technologies and approaches described here not only improve analysis of the patterning of the developing trachea but also help uncover the mechanisms underlying airway disease. This protocol also provides a useful technique to analyze cell organization, polarity, and nuclear shape in other organ systems.
免疫学
Measuring Myeloperoxidase Activity as a Marker of Inflammation in Gut Tissue Samples of Mice and Rat
测量小鼠和大鼠肠道组织样本中作为炎症标志物的骨髓过氧化物酶活性
Myeloperoxidase (MPO) is an enzyme contained in lysosomal azurophilic granules of neutrophils. MPO activity has been shown to correlate with the number of neutrophils in histological sections of the gastrointestinal tract and is therefore accepted as a biomarker of neutrophil invasion in the gut. This protocol describes an easy, cost-effective kinetic colorimetric assay to quantify myeloperoxidase activity in intestinal tissue samples. It is explained using tissue collected in mice but can also be used for other laboratory animals. In a first step, tissue specimens are homogenized using a phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (HTAB), which extracts MPO from neutrophils. The obtained supernatant is added to a reagent solution containing o-dianisidine dihydrochloride, which is a peroxidase substrate. Finally, the change in absorption is measured via spectrophotometry and converted to a standardized unit of enzyme activity. The assay is illustrated and compared to a commercially available enzyme-linked immunoassay (ELISA), demonstrating that MPO activity does not necessarily correlate with MPO protein expression in tissue samples.Key features• Optimized for use in mice and rats but can also be used for samples of other species.• Measures enzymatic activity instead of mRNA or protein expression.• Requires a spectrophotometer.• Can be performed in duplo using 10 mg of (dry-blotted) gut tissue or more.Graphical overview
Primary Mouse Invariant Natural Killer T (iNKT) Cell Purification and Transduction
原代小鼠恒定自然杀伤T（iNKT）细胞的纯化和转导
Invariant natural killer T (iNKT) cells are a non-conventional T-cell population expressing a conserved semi-invariant T-cell receptor (TCR) that reacts to lipid antigens, such as α-galactosyl ceramide (α-GalCer), presented by the monomorphic molecule CD1d. iNKT cells play a central role in tumor immunosurveillance and represent a powerful tool for anti-cancer treatment, notably because they can be efficiently redirected against hematological or solid malignancies by engineering with tumor-specific chimeric antigen receptors (CARs) or TCRs. However, iNKT cells are rare and require specific ex vivo pre-selection and substantial in vitro expansion to be exploited for adoptive cell therapy (ACT). This protocol describes a robust method to obtain a large number of mouse iNKT cells that can be effectually engineered by retroviral (RV) transduction. A major advantage of this protocol is that it requires neither particular instrumentation nor a high number of mice. iNKT cells are enriched from the spleens of iVα14-Jα18 transgenic mice; the rapid purification protocol yields a highly enriched iNKT cell population that is activated by anti-CD3/CD28 beads, which is more reproducible and less time consuming than using bone marrow–derived dendritic cells loaded with α-GalCer, without risks of expanding contaminant T cells. Forty-eight hours after activation, iNKT cells are transduced with the selected RV by spin inoculation. This protocol allows to obtain, in 15 days, millions of ready-to-use, highly pure, and stably transduced iNKT cells that might be exploited for in vitro assays and ACT experiments in preclinical studies.
医学
Ex vivo Culture and Contractile Force Measurements of Non-human Primate Heart Slices
非人灵长类动物心脏切片的离体培养和收缩力测定
Cardiovascular diseases are the leading cause of death and morbidity worldwide. Patient mortality has been successfully reduced by nearly half in the last four decades, mainly due to advances in minimally invasive surgery techniques and interventional cardiology methods. However, a major hurdle is still the translational gap between preclinical findings and the conversion into effective therapies, which is partly due to the use of model systems that fail to recapitulate key aspects of human physiology and disease. Large animal models such as pigs and non-human primates are highly valuable because they closely resemble humans but are costly and time intensive. Here, we provide a method for long-term ex vivo culture of non-human primate (NHP) myocardial tissue that offers a powerful alternative for a wide range of applications including electrophysiology studies, drug screening, and gene function analyses.Graphical overview
微生物学
Qualitative and Quantitative Methods to Measure Antibacterial Activity Resulting from Bacterial Competition
测定细菌竞争产生的抗菌活性的定性和定量方法
In the environment, bacteria compete for niche occupancy and resources; they have, therefore, evolved a broad variety of antibacterial weapons to destroy competitors. Current laboratory techniques to evaluate antibacterial activity are usually labor intensive, low throughput, costly, and time consuming. Typical assays rely on the outgrowth of colonies of prey cells on selective solid media after competition. Here, we present fast, inexpensive, and complementary optimized protocols to qualitatively and quantitively measure antibacterial activity. The first method is based on the degradation of a cell-impermeable chromogenic substrate of the β-galactosidase, a cytoplasmic enzyme released during lysis of the attacked reporter strain. The second method relies on the lag time required for the attacked cells to reach a defined optical density after the competition, which is directly dependent on the initial number of surviving cells.Key features• First method utilizes the release of β-galactosidase as a proxy for bacterial lysis.• Second method is based on the growth timing of surviving cells.• Combination of two methods discriminates between cell death and lysis, cell death without lysis, or survival to quasi-lysis.• Methods optimized to various bacterial species such as Escherichia coli, Pseudomonas aeruginosa, and Myxococcus xanthus.Graphical overview
Large-scale Purification of Type III Toxin-antitoxin Ribonucleoprotein Complex and its Components from Escherichia coli for Biophysical Studies
从大肠杆菌中大规模纯化III型毒素-抗毒素核糖蛋白复合物及其成分，用于生物物理学研究
Toxin–antitoxin (TA) systems are widespread bacterial immune systems that confer protection against various environmental stresses. TA systems have been classified into eight types (I–VIII) based on the nature and mechanism of action of the antitoxin. Type III TA systems consist of a noncoding RNA antitoxin and a protein toxin, forming a ribonucleoprotein (RNP) TA complex that plays crucial roles in phage defence in bacteria. Type III TA systems are present in the human gut microbiome and several pathogenic bacteria and, therefore, could be exploited for a novel antibacterial strategy. Due to the inherent toxicity of the toxin for E. coli, it is challenging to overexpress and purify free toxins from E. coli expression systems. Therefore, protein toxin is typically co-expressed and co-purified with antitoxin RNA as an RNP complex from E. coli for structural and biophysical studies. Here, we have optimized the co-expression and purification method for ToxIN type III TA complexes from E. coli that results in the purification of TA RNP complex and, often, free antitoxin RNA and free active toxin in quantities required for the biophysical and structural studies. This protocol can also be adapted to purify isotopically labelled (e.g., uniformly 15N- or 13C-labelled) free toxin proteins, free antitoxin RNAs, and TA RNPs, which can be studied using multidimensional nuclear magnetic resonance (NMR) spectroscopy methods.Key features• Detailed protocol for the large-scale purification of ToxIN type III toxin–antitoxin complexes from E. coli.• The optimized protocol results in obtaining milligrams of TA RNP complex, free toxin, and free antitoxin RNA.• Commercially available plasmid vectors and chemicals are used to complete the protocol in five days after obtaining the required DNA clones.• The purified TA complex, toxin protein, and antitoxin RNA are used for biophysical experiments such as NMR, ITC, and X-ray crystallography.Graphical overview
easyPACId, a Simple Method for Induced Production, Isolation, Identification, and Testing of Natural Products from Proteobacteria
easyPACId，一种诱导生产、分离、鉴定和测试变形杆菌天然产物的简单方法
The easyPACId (easy Promoter Activation and Compound Identification) approach is focused on the targeted activation of natural product biosynthetic gene clusters (BGCs) encoding non-ribosomal peptide synthetases (NRPS), polyketide synthases (PKS), NRPS-PKS hybrids, or other BGC classes. It was applied to entomopathogenic bacteria of the genera Xenorhabdus and Photorhabdus by exchanging the natural promoter of desired BGCs against the L-arabinose inducible PBAD promoter in ∆hfq mutants of the respective strains. The crude (culture) extracts of the cultivated easyPACId mutants are enriched with the single compound or compound class and can be tested directly against various target organisms without further purification of the produced natural products. Furthermore, isolation and identification of compounds from these mutants is simplified due to the reduced background in the ∆hfq strains. The approach avoids problems often encountered in heterologous expression hosts, chemical synthesis, or tedious extraction of desired compounds from wild-type crude extracts. This protocol describes easyPACId for Xenorhabdus and Photorhabdus, but it was also successfully adapted to Pseudomonas entomophila and might be suitable for other proteobacteria that carry hfq.
A Simple and Reproducible Stereomicroscopic Method to Assess Fungal Biofilms: Application to Antifungal Susceptibility Testing
评估真菌生物膜的简单和可重复的立体显微镜方法： 在抗真菌药敏试验中的应用
Candida albicans, a well-known opportunistic pathogen, is a major cause of human fungal infections. Biofilm formation is considered an important pathogenesis factor. Biofilms are less sensitive to antibiotics and immune responses, allowing them to colonize and persist in host niches. Biofilm screening is important in the identification of anti-biofilm drugs. However, developing nations, with limited financial resources, often do not have access to advanced scientific equipment. Here, we describe an in vitro, protocol using common materials and simple equipment to evaluate static microbial biofilms.
分子生物学
In vivo Electroporation of Skeletal Muscle Fibers in Mice
小鼠骨骼肌纤维的体内电穿孔
In vitro models are essential for investigating the molecular, biochemical, and cell-biological aspects of skeletal muscle. Still, models that utilize cell lines or embryonic cells do not fully recapitulate mature muscle fibers in vivo. Protein function is best studied in mature differentiated tissue, where biological context is maintained, but this is often difficult when reliable detection reagents, such as antibodies, are not commercially available. Exogenous expression of tagged proteins in vivo solves some of these problems, but this approach can be technically challenging because either a mouse must be engineered for each protein of interest or viral vectors are required for adequate levels of expression. While viral vectors can infect target cells following local administration, they carry the risk of genome integration that may interfere with downstream analyses. Plasmids are another accessible expression system, but they require ancillary means of cell penetration; electroporation is a simple physical method for this purpose that requires minimal training or specialized equipment. Here, we describe a method for in vivo plasmid expression in a foot muscle following electroporation.Graphical overview
植物科学
Autolysin Production from Chlamydomonas reinhardtii
莱茵衣藻自溶素的生产
Chlamydomonas reinhardtii is a model organism for various processes, from photosynthesis to cilia biogenesis, and a great chassis to learn more about biofuel production. This is due to the width of molecular tools available, which have recently expanded with the development of a modular cloning system but, most importantly, with CRISPR/Cas9 editing now being possible. This technique has proven to be more efficient in the absence of a cell wall by using specific mutants or by digesting Chlamydomonas cell wall using the mating-specific metalloprotease autolysin (also called gametolysin). Multiple protocols have been used and shared for autolysin production from Chlamydomonas cells; however, they provide very inconsistent results, which hinders the capacity to routinely perform CRISPR mutagenesis. Here, we propose a simple protocol for autolysin production requiring transfer of cells from plates into a dense liquid suspension, gametogenesis by overnight incubation before mixing of gametes, and enzyme harvesting after 2 h. This protocol has shown to be highly efficient for autolysin production regardless of precise control over cell density at any step. Requiring a minimal amount of labor, it will provide a simple, ready-to-go approach to produce an enzyme critical for the generation of targeted mutants. Graphical overview Workflow for autolysin production from Chlamydomonas reinhardtii
系统生物学
Lipidomics Workflow for Analyzing Lipid Profiles Using Multiple Reaction Monitoring (MRM) in Liver Homogenate of Mice with Non-alcoholic Steatohepatitis (NASH)
使用多重反应监测（MRM）分析非酒精性脂肪性肝炎（NASH）小鼠肝匀浆中脂质分布的脂质组学工作流程
Non-alcoholic steatohepatitis (NASH) is a condition characterized by inflammation and hepatic injury/fibrosis caused by the accumulation of ectopic fats in the liver. Recent advances in lipidomics have allowed the identification and characterization of lipid species and have revealed signature patterns of various diseases. Here, we describe a lipidomics workflow to assess the lipid profiles of liver homogenates taken from a NASH mouse model. The protocol described below was used to extract and analyze the metabolites from the livers of mice with NASH by liquid chromatography–mass spectrometry (LC-MS); however, it can be applied to other tissue homogenate samples. Using this method, over 1,000 species of lipids from five classes can be analyzed in a single run on the LC-MS. Also, partial elucidation of the identity of neutral lipid (triacylglycerides and diacylglycerides) aliphatic chains can be performed with this simple LC-MS setup.Key features• Over 1,000 lipid species (sphingolipids, cholesteryl esters, neutral lipids, phospholipids, fatty acids) are analyzed in one run.• Analysis of liver lipids in non-alcoholic steatohepatitis (NASH) mouse model.• Normal-phase chromatography coupled to a triple quadrupole mass spectrometer.Graphical overviewSchematic procedure for the homogenization and extraction of mouse liver tissue in preparation for LC-MS analysis (Created with BioRender.com)