往期刊物2024
卷册: 14, 期号: 6
生物信息学与计算生物学
Classification of a Massive Number of Viral Genomes and Estimation of Time of Most Recent Common Ancestor (tMRCA) of SARS-CoV-2 Using Phylodynamic Analsysis
使用系统动力学对大量病毒基因组分类并预估SARS-CoV-2最近共同祖先年代（tMRCA）
Estimating the time of most recent common ancestor (tMRCA) is important to trace the origin of pathogenic viruses. This analysis is based on the genetic diversity accumulated in a certain time period. There have been thousands of mutant sites occurring in the genomes of SARS-CoV-2 since the COVID-19 pandemic started; six highly linked mutation sites occurred early before the start of the pandemic and can be used to classify the genomes into three main haplotypes. Tracing the origin of those three haplotypes may help to understand the origin of SARS-CoV-2. In this article, we present a complete protocol for the classification of SARS-CoV-2 genomes and calculating tMRCA using Bayesian phylodynamic method. This protocol may also be used in the analysis of other viral genomes.Key features• Filtering and alignment of a massive number of viral genomes using custom scripts and ViralMSA.• Classification of genomes based on highly linked sites using custom scripts.• Phylodynamic analysis of viral genomes using Bayesian evolutionary analysis sampling trees (BEAST).• Visualization of posterior distribution of tMRCA using Tracer.v1.7.2.• Optimized for the SARS-CoV-2.Graphical overviewGraphical workflow of time of most recent common ancestor (tMRCA) estimation process
生物工程
From Llama to Nanobody: A Streamlined Workflow for the Generation of Functionalised VHHs
从美洲驼到纳米体：生成功能化VHHs的简化工作流程
Nanobodies are recombinant antigen-specific single domain antibodies (VHHs) derived from the heavy chain–only subset of camelid immunoglobulins. Their small molecular size, facile expression, high affinity, and stability have combined to make them unique targeting reagents with numerous applications in the biomedical sciences. From our work in producing nanobodies to over sixty different proteins, we present a standardised workflow for nanobody discovery from llama immunisation, library building, panning, and small-scale expression for prioritisation of binding clones. In addition, we introduce our suites of mammalian and bacterial vectors, which can be used to functionalise selected nanobodies for various applications such as in imaging and purification.Key features• Standardise the process of building nanobody libraries and finding nanobody binders so that it can be repeated in any lab with reasonable equipment.• Introduce two suites of vectors to functionalise nanobodies for production in either bacterial or mammalian cells.Graphical overview
细胞生物学
Dissecting the Mechanical Control of Mitotic Entry Using a Cell Confinement Setup
利用单细胞限制器解析有丝分裂进入的机械控制
Proliferating cells need to cope with extensive cytoskeletal and nuclear remodeling as they prepare to divide. These events are tightly regulated by the nuclear translocation of the cyclin B1-CDK1 complex, that is partly dependent on nuclear tension. Standard experimental approaches do not allow the manipulation of forces acting on cells in a time-resolved manner. Here, we describe a protocol that enables dynamic mechanical manipulation of single cells with high spatial and temporal resolution and its application in the context of cell division. In addition, we also outline a method for the manipulation of substrate stiffness using polyacrylamide hydrogels. Finally, we describe a static cell confinement setup, which can be used to study the impact of prolonged mechanical stimulation in populations of cells.Key features• Protocol for microfabrication of confinement devices.• Single-cell dynamic confinement coupled with high-resolution microscopy.• Static cell confinement protocol that can be combined with super-resolution STED microscopy.• Analysis of the mechanical control of mitotic entry in a time-resolved manner.Graphical overview
免疫学
Genetic Knock-Ins of Endogenous Fluorescent Tags in RAW 264.7 Murine Macrophages Using CRISPR/Cas9 Genome Editing
利用CRISPR/Cas9基因组编辑技术在RAW 264.7小鼠巨噬细胞中敲入内源性荧光标签
CRISPR/Cas9 genome editing is a widely used tool for creating genetic knock-ins, which allow for endogenous tagging of genes. This is in contrast with random insertion using viral vectors, where expression of the inserted transgene changes the total copy number of a gene in a cell and does not reflect the endogenous chromatin environment or any trans-acting regulation experienced at a locus. There are very few protocols for endogenous fluorescent tagging in macrophages. Here, we describe a protocol to design and test CRISPR guide RNAs and donor plasmids, to transfect them into RAW 264.7 mouse macrophage-like cells using the Neon transfection system and to grow up clonal populations of cells containing the endogenous knock-in at various loci. We have used this protocol to create endogenous fluorescent knock-ins in at least six loci, including both endogenously tagging genes and inserting transgenes in the Rosa26 and Tigre safe harbor loci. This protocol uses circular plasmid DNA as the donor template and delivers the sgRNA and Cas9 as an all-in-one expression plasmid. We designed this protocol for fluorescent protein knock-ins; it is best used when positive clones can be identified by fluorescence. However, it may be possible to adapt the protocol for non-fluorescent knock-ins. This protocol allows for the fairly straightforward creation of clonal populations of macrophages with tags at the endogenous loci of genes. We also describe how to set up imaging experiments in 24-well plates to track fluorescence in the edited cells over time.Key features• CRISPR knock-in of fluorescent proteins in RAW 264.7 mouse macrophages at diverse genomic loci.• This protocol is optimized for the use of the Neon transfection system.• Includes instructions for growing up edited clonal populations from single cells with one single-cell sorting step and efficient growth in conditioned media after cell sorting.• Designed for knocking in fluorescent proteins and screening transfected cells byFACS, but modification for non-fluorescent knock-ins may be possible.Graphical overview
Mesenchymal Stromal Cell (MSC) Functional Analysis—Macrophage Activation and Polarization Assays
间充质干细胞（MSC）功能分析—巨噬细胞激活与极化测定
Stem cell–based therapies have evolved to become a key component of regenerative medicine approaches to human pathologies. Exogenous stem cell transplantation takes advantage of the potential of stem cells to self-renew, differentiate, home to sites of injury, and sufficiently evade the immune system to remain viable for the release of anti-inflammatory cytokines, chemokines, and growth factors. Common to many pathologies is the exacerbation of inflammation at the injury site by proinflammatory macrophages. An increasing body of evidence has demonstrated that mesenchymal stromal cells (MSCs) can influence the immunophenotype and function of myeloid lineage cells to promote therapeutic effects. Understanding the degree to which MSCs can modulate the phenotype of macrophages within an inflammatory environment is of interest when considering strategies for targeted cell therapies. There is a critical need for potency assays to elucidate these intercellular interactions in vitro and provide insight into potential mechanisms of action attributable to the immunomodulatory and polarizing capacities of MSCs, as well as other cells with immunomodulatory potential. However, the complexity of the responses, in terms of cell phenotypes and characteristics, timing of these interactions, and the degree to which cell contact is involved, have made the study of these interactions challenging. To provide a research tool to study the direct interactions between MSCs and macrophages, we developed a potency assay that directly co-cultures MSCs with naïve macrophages under proinflammatory conditions. Using this assay, we demonstrated changes in the macrophage secretome and phenotype, which can be used to evaluate the abilities of the cell samples to influence the cell microenvironment. These results suggest the immunomodulatory effects of MSCs on macrophages while revealing key cytokines and phenotypic changes that may inform their efficacy as potential cellular therapies.Key features• The protocol uses monocytes differentiated into naïve macrophages, which are loosely adherent, have a relatively homogeneous genetic background, and resemble peripheral blood mononuclear cells–derived macrophages.• The protocol requires a plate reader and a flow cytometer with the ability to detect six fluorophores.• The protocol provides a quantitative measurement of co-culture conditions by the addition of a fixed number of freshly thawed or culture-rescued MSCs to macrophages.• This protocol uses assessment of the secretome and cell harvest to independently verify the nature of the interactions between macrophages and MSCs.Graphical overview
微生物学
Efficient Genetic Transformation and Suicide Plasmid-mediated Genome Editing System for Non-model Microorganism Erwinia persicina
非模式微生物桃色欧文氏菌的高效遗传转化及自杀质粒介导的基因组编辑系统
Erwinia persicina is a gram-negative bacterium that causes diseases in plants. Recently, E. persicina BST187 was shown to exhibit broad-spectrum antibacterial activity due to its inhibitory effects on bacterial acetyl-CoA carboxylase, demonstrating promising potential as a biological control agent. However, the lack of suitable genetic manipulation techniques limits its exploitation and industrial application. Here, we developed an efficient transformation system for E. persicina. Using pET28a as the starting vector, the expression cassette of the red fluorescent protein–encoding gene with the strong promoter J23119 was constructed and transformed into BST187 competent cells to verify the overexpression system. Moreover, suicide plasmid–mediated genome editing systems was developed, and lacZ was knocked out of BST187 genome by parental conjugation transfer using the recombinant suicide vector pKNOCK-sacB-km-lacZ. Therefore, both the transformation and suicide plasmid–mediated genome editing system will greatly facilitate genetic manipulations in E. persicina and promote its development and application.Key features• Our studies establish a genetic manipulation system for Erwinia persicina, providing a versatile tool for studying the gene function of non-model microorganisms.• Requires approximately 6–10 days to complete modification of a chromosome locus.Graphical overview
Preparation and Purification of β-1,3-glucan-Linked Candida glabrata Cell Wall Proteases by Ion-Exchange Chromatography, Gel Filtration, and MDPF-Gelatin-Zymography Assay
通过离子交换色谱、凝胶过滤和MDPF-明胶-酶谱分析法准备和纯化β-1,3-葡聚糖连接的光滑念珠菌细胞壁蛋白酶
Candida glabrata is an opportunistic pathogen that may cause serious infections in an immunocompromised host. C. glabrata cell wall proteases directly interact with host cells and affect yeast virulence and host immune responses. This protocol describes methods to purify β-1,3-glucan-bonded cell wall proteases from C. glabrata. These cell wall proteases are detached from the cell wall glucan network by lyticase treatment, which hydrolyzes β-1,3-glucan bonds specifically without rupturing cells. The cell wall supernatant is further fractioned by centrifugal devices with cut-offs of 10 and 50 kDa, ion-exchange filtration(charge), and gel filtration (size exclusion). The enzymatic activity of C. glabrata proteases is verified with MDPF-gelatin zymography and the degradation of gelatin is visualized by loss of gelatin fluorescence. With this procedure, the enzymatic activities of the fractions are kept intact, differing from methods used in previous studies with trypsin digestion of the yeast cell wall. The protein bands may be eventually located from a parallel silver-stained gel and identified with LC–MS/MS spectrometry. The advantage of this methodology is that it allows further host protein degradation assays; the protocol is also suitable for studying other Candida yeast species.Key features• Uses basic materials and laboratory equipment, enabling low-cost studies.• Facilitates the selection and identification of proteases with certain molecular weights.• Enables further functional studies with host proteins, such as structural or immune response–related, or enzymes and candidate protease inhibitors(e.g., from natural substances).• This protocol has been optimized for C. glabrata but may be applied with modifications to other Candida species.Graphical overview
分子生物学
Proximity Labelling to Quantify Kv7.4 and Dynein Protein Interaction in Freshly Isolated Rat Vascular Smooth Muscle Cells
利用近端连接分析在新鲜分离的大鼠血管平滑肌细胞中定量Kv7.4与Dynein蛋白的相互作用
Understanding protein–protein interactions is crucial for unravelling subcellular protein distribution, contributing to our understanding of cellular organisation. Moreover, interaction studies can reveal insights into the mechanisms that cover protein trafficking within cells. Although various techniques such as Förster resonance energy transfer (FRET), co-immunoprecipitation, and fluorescence microscopy are commonly employed to detect protein interactions, their limitations have led to more advanced techniques such as the in situ proximity ligation assay (PLA) for spatial co-localisation analysis. The PLA technique, specifically employed in fixed cells and tissues, utilises species-specific secondary PLA probes linked to DNA oligonucleotides. When proteins are within 40 nm of each other, the DNA oligonucleotides on the probes interact, facilitating circular DNA formation through ligation. Rolling-circle amplification then produces DNA circles linked to the PLA probe. Fluorescently labelled oligonucleotides hybridise to the circles, generating detectable signals for precise co-localisation analysis. We employed PLA to examine the co-localisation of dynein with the Kv7.4 channel protein in isolated vascular smooth muscle cells from rat mesenteric arteries. This method enabled us to investigate whether Kv7.4 channels interact with dynein, thereby providing evidence of their retrograde transport by the microtubule network. Our findings illustrate that PLA is a valuable tool for studying potential novel protein interactions with dynein, and the quantifiable approach offers insights into whether these interactions are changed in disease.