往期刊物2021
卷册: 11, 期号: 9
生物化学
Click Chemistry for Imaging in-situ Protein Palmitoylation during the Asexual Stages of Plasmodium falciparum
恶性疟原虫无性期原位蛋白质棕榈酰化的点击化学成像
Palmitoylation refers to the modification of the cysteine thiols in proteins by fatty acids, most commonly palmitic acid, through ‘thioester bond’ formation. In vivo, palmitoylation of proteins is catalyzed by palmitoyl acyltransferases (PATs or DHHC-PATs). Palmitoylation has recently emerged as a crucial post-translational modification in malarial parasites. The expression and activity of palmitoyl transferases vary across different developmental stages of the malarial parasite’s life cycle. The abundance of palmitoylated proteins at a given stage is a measure of overall PAT activity. The PAT activity can also change in response to external signals or inhibitors. Here, we describe a protocol to ‘image’ palmitoyl-transferase activity during the asexual stages using Click Chemistry and fluorescence microscopy. This method is based on metabolic labeling of a clickable analog of palmitic acid by parasitic cells, followed by CuAAC (Copper-catalyzed Alkyne-Azide Cycloaddition reaction) Click Chemistry to render palmitoylated proteins fluorescent. Fluorescence allows the quantitation of intracellular palmitoylation in parasite cells across various development stages. Using this method, we observed that intracellular palmitoylation increases as the parasite transitions from ring to schizont stages and appears to be most abundant during the schizont stages in Plasmodium falciparum.
Proteomics Profiling of S-sulfurated Proteins in Acinetobacter baumannii
鲍曼不动杆菌硫酸化蛋白的蛋白质组学分析
Hydrogen sulfide (H2S) is emerging as an important modulator in bacterial cytoprotection against the host immune response in infected animals, which may well be attributed to downstream highly oxidized sulfur species, termed reactive sulfur species (RSS), derived from H2S. One mechanism by which H2S/RSS may signal in the cell is through proteome S-sulfuration (persulfidation), which is the conversion of protein thiols (-SH) to protein persulfides (-SSH). While several analytical methods have been developed to profile sites of protein persulfidation, few have been applied to bacterial cells. The analytical workflow presented here was recently utilized to profile proteome persulfidation in the major human pathogen Acinetobacter baumannii treated with an exogenous sulfide source, Na2S. The data obtained using this protocol allow quantitation of the change in persulfidation status of each cysteine in the proteome normalized to the change in protein abundance, thus identifying sites of persulfidation that may constitute regulatory modifications. These can be validated using follow-up biochemical studies.
Chromatographic Assays for the Enzymatic Degradation of Chitin
几丁质酶解的色谱分析
Chitin is an insoluble linear polymer of β(1→4)-linked N-acetylglucosamine. Enzymatic cleavage of chitin chains can be achieved using hydrolytic enzymes, called chitinases, and/or oxidative enzymes, called lytic polysaccharide monooxygenases (LPMOs). These two groups of enzymes have different modes of action and yield different product types that require different analytical methods for detection and quantitation. While soluble chromogenic substrates are readily available for chitinases, proper insight into the activity of these enzymes can only be obtained by measuring activity toward their polymeric, insoluble substrate, chitin. For LPMOs, only assays using insoluble chitin are possible and relevant. Working with insoluble substrates complicates enzyme assays from substrate preparation to product analysis. Here, we describe typical set-ups for chitin degradation reactions and the chromatographic methods used for product analysis.Graphical abstract:Overview of chromatographic methods for assessing the enzymatic degradation of chitin
Characterising Plant Deubiquitinases with in vitro Activity-based Labelling and Ubiquitin Chain Disassembly Assays
植物去泛素酶的体外活性标记和泛素链分解分析
Post-translational modification of proteins by ubiquitin is an essential cellular signaling mechanism in all eukaryotes. Ubiquitin is removed from target proteins by a wide range of deubiquitinase (DUB) enzymes with different activities and substrate specificities. Understanding how DUBs function in vitro is a vital first step to uncovering their cellular roles. Here, we provide protocols for the rapid analysis of DUB activity in vitro by activity-based labelling with the suicide probe, HA-ubiquitin vinyl sulfone (HA-UbVS), and ubiquitin chain disassembly assays. We have previously used these methods to analyse the activity of the Arabidopsis thaliana DUB, UBP6, but in principle, these protocols are applicable to any DUB of interest.
生物工程
Inkjet 3D Printing of Polymers Resistant to Fungal Attachment
抗真菌附着聚合物的喷墨3D打印
Inkjet 3D printing is an additive manufacturing method that allows the user to produce a small batch of customized devices for comparative study versus commercial products. Here, we describe the use of a commercial 2D ink development system (Dimatix material printing) to manufacture small batches of 3D medical or other devices using a recently characterized fungal anti-attachment material. Such printed devices may resist problems that beset commercial medical products due to colonization by the fungal pathogen Candida albicans. By sequentially introducing the cross-section bitmaps of the product’s CAD model and elevating the print head height using the auto-clicking script, we were able to create complex self-support geometries with the 2D ink development system. The use of this protocol allows researchers to produce a small batch of specimens for characterization from only a few grams of raw material. Additionally, we describe the testing of manufactured specimens for fungal anti-attachment. In comparison with most commercial AM systems, which require at least a few hundred grams of ink for printing trials, our protocol is well suited for smaller-scale production in material studies.
生物物理学
Spin Labeling of RNA Using “Click” Chemistry for Coarse-grained Structure Determination via Pulsed Electron-electron Double Resonance Spectroscopy
利用脉冲电子-电子双共振光谱测定粗粒结构的“点击”化学的RNA自旋标记
Understanding the function of oligonucleotides on a molecular level requires methods for studying their structure, conformational changes, and internal dynamics. Various biophysical methods exist to achieve this, including the whole toolbox of Electron Paramagnetic Resonance (EPR or ESR) spectroscopy. An EPR method widely used in this regard is Pulsed Electron-Electron Double Resonance (PELDOR or DEER), which provides distances in the nanometer range between electron spins in biomolecules with Angstrom precision, without restriction to the size of the biomolecule, and in solution. Since oligonucleotides inherently do not contain unpaired electrons, these have to be introduced in the form of so-called spin labels. Firstly, this protocol describes how nitroxide spin labels can be site-specifically attached to oligonucleotides using “Click” chemistry. The reaction provides little byproducts, high yields, and is conveniently performed in aqueous solution. Secondly, the protocol details how to run the PELDOR experiment, analyze the data, and derive a coarse-grained structure. Here, emphasis is placed on the pitfalls, requirements for a good dataset, and limits of interpretation; thus, the protocol gives the user a guideline for the whole experiment i.e., from spin labeling, via the PELDOR measurement and data analysis, to the final coarse-grained structure.Graphical abstract:Schematic overview of the workflow described in this protocol: First, the spin-labeling of RNA is described, which is performed as a "Click"-reaction between the alkyne-functionalized RNA strand and the azide group of the spin label. Next, step-by-step instructions are given for setting up PELDOR/DEER distance measurements on the labeled RNA, and for data analysis. Finally, guidelines are provided for building a structural model from the previously analyzed data.
癌症生物学
ATAC-Seq-based Identification of Extrachromosomal Circular DNA in Mammalian Cells and Its Validation Using Inverse PCR and FISH
基于ATAC-Seq的哺乳动物细胞染色体外环状DNA鉴定及其反向PCR和FISH验证
Recent studies from multiple labs including ours have demonstrated the importance of extrachromosomal circular DNA (eccDNA) from yeast to humans (Shibata et al., 2012; Dillon et al., 2015; Møller et al., 2016; Kumar et al., 2017; Turner et al., 2017; Kim et al., 2020). More recently, it has been found that cancer cells obtain a selective advantage by amplifying oncogenes on eccDNA, which drives genomic instability (Wu et al., 2019; Kim et al., 2020). Previously, we have purified circular DNA and enriched the population using rolling circle amplification followed by high-throughput sequencing for the identification of eccDNA based on the unique junctional sequence. However, eccDNA identification by rolling circle amplification is biased toward small circles. Here, we report a rolling circle-independent method to detect eccDNA in human cancer cells. We demonstrate a sensitive and robust step-by-step workflow for finding novel eccDNAs using ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) combined with a Circle_finder bioinformatics algorithm to predict the eccDNAs, followed by its validation using two independent methods, inverse PCR and metaphase FISH (Fluorescence in situ Hybridization).
免疫学
Analysis of Monocyte Cell Fate by Adoptive Transfer in a Murine Model of TLR7-induced Systemic Inflammation
TLR7诱导的系统性炎症小鼠模型中通过过继转移单核细胞命运分析
Myeloid plasticity is a hallmark of the innate immune response to Toll-like receptor (TLR) activation. Here, we provide a protocol for monocyte cell fate tracking by adoptive transfer in the context of systemic inflammation induced by TLR7 activation, the principal innate immune receptor sensing viral RNA in mice. Defined monocyte subsets are isolated from the bone marrow of donor mice by cell sorting and adoptively transferred into the systemic circulation of congenic hosts, with or without concurrent activation of TLR7 via the topical application of the small molecule agonist, imiquimod, in a cream formulation that induces a systemic inflammatory response. Advantages are the precise definition of donor cell populations and resulting cell fate without the need for host conditioning in a model that recapitulates key aspects of the systemic inflammatory response to TLR7 stimulation.
微生物学
COVID-19 Sample Pooling: From RNA Extraction to Quantitative Real-time RT-PCR
COVID-19样本汇集:从RNA提取到实时定量RT-PCR
The COVID-19 pandemic requires mass screening to identify those infected for isolation and quarantine. Individually screening large populations for the novel pathogen, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is costly and requires a lot of resources. Sample pooling methods improve the efficiency of mass screening and consume less reagents by increasing the capacity of testing and reducing the number of experiments performed, and are therefore especially suitable for under-developed countries with limited resources. Here, we propose a simple, reliable pooling strategy for COVID-19 testing using clinical nasopharyngeal (NP) and/or oropharyngeal (OP) swabs. The strategy includes the pooling of 10 NP/OP swabs for extraction and subsequent testing via quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), and may also be applied to the screening of other pathogens.
High-throughput Method for Detecting Siderophore Production by Rhizosphere Bacteria
根际细菌产生铁载体的高通量检测方法
Siderophores, a key substance that microorganisms produce to obtain iron under iron-limited conditions, play an important role in regulating interactions between beneficial bacteria and pathogenic bacteria. A large number of bacteria were isolated from the rhizosphere, and we used the method presented here to assay the siderophore production by these rhizosphere bacteria. This method is a modified version of the universal chrome azurol S (CAS) assay that uses a 96-channel manual pipetting workstation. By combining the liquid CAS assay with the multi-channel pipette workstation, high-throughput and rapid detection of siderophore production can be achieved. In summary, this method can be used to gain a general understanding of siderophore production by rhizosphere bacteria.
Isolation of GFP-expressing Malarial Hypnozoites by Flow Cytometry Cell Sorting
流式细胞术分离表达GFP的疟原虫催眠素
Hypnozoites are dormant liver-stage parasites unique to relapsing malarial species, including the important human pathogen Plasmodium vivax, and pose a barrier to the elimination of malaria. Little is known regarding the biology of these stages, largely due to their inaccessible location. Hypnozoites can be cultured in vitro but these cultures always consist of a mixture of hepatocytes, developing forms, and hypnozoites. Here, using a GFP-expressing line of the hypnozoite model parasite Plasmodium cynomolgi, we describe a protocol for the FACS-based isolation of malarial hypnozoites. The purified hypnozoites can be used for a range of ‘-omics’ studies to dissect the biology of this cryptic stage of the malarial life cycle.
Assessing Swarming of Aerobic Bacteria from Human Fecal Matter
人类粪便中需氧细菌群集的评估
Swarming – swift movement across a surface via flagella propulsion – is a unique property of many bacteria. The role of swarming, particularly among bacterial populations of the human gut microbiome, is not yet fully understood; although, it is becoming an area of increased scientific and clinical inquiry. To further characterize bacterial swarming in human health, an effective assay for swarming that utilizes complex material, such as fecal matter, is necessary. Until now, the vast majority of swarming assays have only been able to accommodate bacteria grown in culture, most often Pseudomonas. These assays tend to use a standard lysogenic broth (LB) agar medium; however, the reagents involved have not been tailored to the inoculation of complex material. In this paper, we offer a specialized protocol for eliciting the swarming of bacteria from frozen human fecal samples. We describe the simple, yet reproducible steps required to perform the assay, identifying an ideal volume of 7.5 μl for inoculation of material, as well as an ideal agar concentration of 0.4%. This protocol typically allows researchers to identify swarming within 24 h after incubation in a standard incubator.
分子生物学
Identification of R-loop-forming Sequences in Drosophila melanogaster Embryos and Tissue Culture Cells Using DRIP-seq
DRIP-seq法鉴定黑腹果蝇胚胎和组织培养细胞R-loop形成序列
R-loops are non-canonical nucleic structures composed of an RNA–DNA hybrid and a displaced ssDNA. Originally identified as a source of genomic instability, R-loops have been shown over the last decade to be involved in the targeting of proteins and to be associated with different histone modifications, suggesting a regulatory function. In addition, R-loops have been demonstrated to form differentially during the development of different tissues in plants and to be associated with diseases in mammals. Here, we provide a single-strand DRIP-seq protocol to identify R-loop-forming sequences in Drosophila melanogaster embryos and tissue culture cells. This protocol differs from earlier DRIP protocols in the fragmentation step. Sonication, unlike restriction enzymes, generates a homogeneous and highly reproducible nucleic acid fragment pool. In addition, it allows the use of this protocol in any organism with minimal optimization. This protocol integrates several steps from published protocols to identify R-loop-forming sequences with high stringency, suitable for de novo characterization.Graphic abstract:Figure 1. Overview of the strand-specific DRIP-seq protocol
神经科学
Activity-based Anorexia for Modeling Vulnerability and Resilience in Mice
基于活动的厌食症小鼠脆弱性和恢复力模型的建立
Activity-based anorexia (ABA) is a widely used rodent model of anorexia nervosa. It involves combining limited access to food with unlimited access to a running wheel, leading to a paradoxical decrease in food intake, hyperactivity, and life-threatening weight loss. Although initially characterized in rats, ABA has been tested in mice with results that vary based on strain, sex, age, the amount of time food is available, and the number of days of food restriction. Here, we present our ABA protocol for modeling both vulnerability and resilience to diet and exercise in C57BL/6 female mice. While vulnerable mice exhibit the expected increase in running, reduction in food intake, and excessive weight loss, resilient mice exhibit an adaptive increase in food intake, decrease in total wheel running, and weight stabilization. In contrast to previous ABA studies in which resilience is defined by the relative rate of weight loss, our protocol leads to a resilient phenotype that more closely resembles the maintenance of a stable bodyweight exhibited by most humans who diet and exercise without developing anorexia nervosa. This protocol will be useful for future studies aimed at identifying the physiological and neural adaptations underlying both resilience and vulnerability to this eating disorder.
植物科学
Histological Methods to Detect Early-stage Plant Defense Responses during Artificial Inoculation of Lolium perenne with Epichloë festucae
羊茅人工接种多年生黑麦草过程中植物早期防御反应的组织学检测
Epichloë species form agriculturally important symbioses with many cool season grasses. To study these symbioses, such as the interaction of Epichloë festucae with perennial ryegrass (Lolium perenne), host plants can be infected by artificial inoculation of etiolated seedlings. This inoculation is performed by placing mycelium into an incision in the meristem, as previously described by Latch and Christensen (1985). In recent years, this method has been broadly used to study this interaction at the molecular level using different Epichloë festucae mutants that can cause incompatible interactions. We have developed and adapted methods to study four of the most important host plant responses to infection, including cell death, callose deposition, lignin production, and hydrogen peroxide (H2O2) production, which are useful in defining the host response to infection at a very early time point.
Age, Wound Size and Position of Injury – Dependent Vascular Regeneration Assay in Growing Leaves
年龄、伤口大小和损伤部位 – 生长叶片中依赖于损伤的维管再生试验
Recurring damage to the aerial organs of plants necessitates their prompt repair, particularly their vasculature. While vascular regeneration assays for aerial plant parts such as the stem and inflorescence stalk are well established, those for leaf vasculature remain unexplored. Recently, we established a new vascular regeneration assay in growing leaves and discovered the underlying molecular mechanism. Here, we describe the detailed stepwise method for the incision and regeneration assay used to study leaf vascular regeneration. By using a combination of micro-surgical perturbations, brightfield microscopy, and other experimental approaches, we further show that the age of the leaf as well as the position and size of the injury determine the overall success rate of regeneration. This easy-to-master vascular regeneration assay is an efficient and rapid method to study the mechanism of vascular regeneration in growing leaves. The assay can be readily combined with cellular and molecular biology techniques.