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Past Issue in 2018
Volume: 8, Issue: 24
Biochemistry
Preparation of Silk Fibroin Nanoparticles and Enzyme-Entrapped Silk Fibroin Nanoparticles
After silk fiber is degummed in boiling 0.2% Na2CO3 solution, the degummed fibroin fiber could be dissolved in highly concentrated neutral salts such as CaCl2. The partially degraded polypeptides of silk fibroin, commonly called as regenerated liquid silk, could be obtained via water dialysis. The silk fibroin nanoparticles (SFNs) or enzyme-entrapped SFNs are prepared rapidly from the liquid silk by using acetone. The globular particles with a range of 35-125 nm in diameter, are water-insoluble but well dispersed and stable in aqueous solution. The nanoparticles are potentially useful in biomaterials such as cosmetics, anti-UV skincare products, and surface improving materials, especially in enzyme/drug delivery system as vehicle. Here, a detailed protocol for SFNs and enzyme-entrapped SFNs preparation is described.
Cell Biology
Quantitative Analysis of Cargo Density in Single-extracellular Vesicles by Imaging
Function of extracellular vesicles such as exosomes and microvesicles is determined by their wide ranges of cargoes inside them. Even in the pure exosomes or microvesicles the cargo contents are very heterogeneous. To understand this heterogeneous nature of extracellular vesicles, we need information of the vesicles, which will give us some parameters including vesicle size, number and cargo content of each vesicle. Here, we describe a new method to quantify cargo density in single-extracellular vesicles. Staining of extracellular vesicles in a membrane lipid content-proportionate manner and immobilization of extracellular vesicles onto glass substrate allow us to obtain cargo density information of single-extracellular vesicles. This protocol will be useful to analyze the effects of various drugs or genetic manipulation on vesicle generation and maturation including cargo sorting into heterogeneous extracellular vesicles.
Microirradiation for Precise, Double-strand Break Induction in vivo in Caenorhabditis elegans
DNA double-strand breaks (DSBs) are toxic lesions that every cell must accurately repair in order to survive. The repair of DSBs is an integral part of a cell life cycle and can lead to lethality if repaired incorrectly. Laser microirradiation is an established technique which has been used in yeast, mammalian cell culture, and Drosophila cell culture to study the regulation of DSB repair. Up to our studies, this method has not been adapted for use in a whole, live, multicellular organism to study this repair in vivo. We have recently shown that this system can be used for study of the recruitment of vital repair proteins to microirradiation-induced breaks in the transparent nematode Caenorhabditis elegans. With the integration of microirradiation and imaging technology, we can precisely induce DSBs in target nuclei and study the recruitment of fluorescently tagged repair proteins from the time of damage induction. Whole, live worms are plated and immobilized for targeting of nuclei, and immediately following induction the targeted region can be imaged for up to an hour and a half post-microirradiation. This method is the first that allows for study of DNA repair protein kinetics in vivo in an intact organism, which can be adapted in numerous ways to allow for study of repair kinetics in various aspects of the repair process.
Immunology
Method for Studying the Effect of Gene Silencing on Bacterial Infection-induced ERK1/2 Signaling in Bone-marrow Derived Macrophages
Macrophages are highly phagocytic cells that utilize various pathogen recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). These PAMPs can be present within the microbe, such as bacterial CpG DNA, and are recognized by Toll-like receptor 9 (TLR9), a PRR present on the endosomal membrane of macrophages. PAMPs can also be present on the surface of microbes, such as Lipopolysaccharide (LPS), which decorates the outer membrane of gram-negative bacteria like Salmonella typhimurium and Escherichia coli. LPS is recognized by TLR4 present on the plasma membrane of macrophages, and LPS-TLR4 association leads to activation of signaling cascades including MAPK phosphorylation, which in turn promotes macrophage activation and microbial killing. This protocol describes the method for studying the role of a gene of interest in Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) signaling, induced by bacterial infection in primary bone-marrow derived macrophages (BMDMs).
Monitoring Natural Killer Cell Function in Human Ovarian Cancer Cells of Ascitic Fluid
Natural killer (NK) cells are the major effectors of the innate immune system when activated resulting in modulation of immune response of the host defense through target cell lysis and secretion of cytokines. Precise functions of NK cells are essential for the treatment outcome of different virus infections and malignant diseases. NK cells impart cytotoxic effect to the target cells lacking MHC class I molecules and thus the final readout of the activity is death of target cells. The NK cell function is evaluated by the 51Cr-release and/or flow cytometry-based assays. In the present protocol, we have determined the activation of NK cells by the liberation of IL-10 and IFNγ, and subsequently its function by enumerating the number of dead tumor cells originally isolated from the ascitic fluid of ovarian cancer patients. The entire assay is based on cells of the healthy donors and patients. Besides determining function, this method is able to demarcate between NK-cell sensitive and insensitive tumor cells. This technique enables researchers to study NK cell functions in healthy donors or in patients to reveal their impact on different malignancies and to further discover new therapeutic strategies.
Quantitation of Regulatory Activity for the Complement Alternative Pathway Using an Adaptation of the AP50 in vitro Assay
Complement pathways function to identify and remove pathogens and infected cells. There are three complement pathways: the classical, lectin and alternative pathway (AP). While all pathways are activated following pathogen stimuli, the AP is constitutively active and tightly controlled by activators (e.g., Factor B, Factor D) and negative regulators (e.g., Factor H). Complement activity can be measured by well-established methods that are often used in a diagnostic setting to determine the CH50 (50% complement hemolytic activity) or AP50, specifically to measure AP activity. The protocol here has adapted the traditional AP50 method designed to measure AP activity in human sera, to measure the positive or negative AP regulatory activity within a given test sample. The assay relies on the ability of AP components in human serum to lyse rabbit erythrocytes under in vitro conditions specific for the AP with subsequent release of hemoglobin that is quantitated by measurement of optical density. Our method has added test substances, such as cell culture media with defined changes in individual complement components and determined the ability to either promote or inhibit AP activity in vitro. Thus, this protocol reflects the overall functional ability of a sample to effect AP activity and can be used in the research laboratory to determine AP regulatory activity in a complex biological sample, or to test the ability of drugs or novel biomolecules to regulate AP activity.
Eimeria vermiformis Infection Model of Murine Small Intestine
Eimeria vermiformis is a tissue specific, intracellular protozoan that infects the murine small intestinal epithelia, which has been widely used as a coccidian model to study mucosal immunology. This mouse infection model is valuable to investigate the mechanisms of host protection against primary and secondary infection in the small intestine. Here, we describe the generation of an E. vermiformis stock solution, preparation of sporulated E. vermiformis to infect mice and determination of oocysts burden. This protocol should help to establish a highly reproducible natural infection challenge model to study immunity in the small intestine. The information obtained from using this mouse model can reveal fundamental mechanisms of interaction between the pathogen and the immune response, e.g., provided by intraepithelial lymphocytes (IEL) at the basolateral site of epithelial cells but also a variety of other immune cell populations present in the gut.
Microbiology
Production, Titration and Imaging of Zika Virus in Mammalian Cells
Since the outbreak of Zika virus (ZIKV) in Latin America and the US in 2016, this flavivirus has emerged as a major threat for public health. Indeed, it is now clear that ZIKV is vertically transmitted from the infected mother to the fetus and this may lead to severe neurological development defects including (but not restricted to) neonate microcephaly. Although ZIKV has been identified in the late 1940s, very little was known about its epidemiology, symptoms and molecular biology before its reemergence 60 years later. Recently, tremendous efforts have been made to develop molecular clones and tools as well as cell culture and animal models to better understand ZIKV fundamental biology and pathogenesis and to develop so-far-unavailable antiviral drugs and vaccines. This bio-protocol describes basic experimental procedures to produce ZIKV stocks and to quantify their concentration in infectious virus particles as well as to image and study this pathogen within infected cells using confocal microscopy-based imaging.
Systematic Quantification of GFP-tagged Protein Foci in Schizosaccharomyces pombe Nuclei
DNA damage repair proteins form foci in response to DNA damaging agents. The efficiency and integrity of the DNA repair pathway of a particular eukaryotic (mutant) strain is usually determined by the number of foci formed compared with their wild-type counterpart. Conventionally, focus number is determined visually, and this low accuracy may obscure the identification of a weaker phenotype, particularly when the output is low. Here, using the homologous recombination protein Rhp54 as an example, we present a protocol that can increase the consistency of foci identification among samples and can significantly improve the efficiency of foci quantification for large sample sizes. A similar method can be applied to other foci-forming proteins.
Analysis of DNA Exchange Using Thymidine Analogs (ADExTA) in Trypanosoma cruzi
Trypanosoma cruzi is a protozoan parasite belonging to the Trypanosomatidae family. Although the trypanosomatids multiply predominantly by clonal generation, the presence of DNA exchange in some of them has been puzzling researchers over the years, mainly because it may represent a novel form that these organisms use to gain variability. Analysis of DNA Exchange using Thymidine Analogs (ADExTA) is a method that allows the in vitro detection and measurement of rates of DNA exchange, particularly in trypanosomatid cells, in a rapid and simple manner by indirect immunofluorescence assay (IFA). The method can be used to detect DNA exchange within one trypanosomatid lineage or among different lineages by paired analysis. The principle of this assay is based on the incorporation of two distinguishable halogenated thymidine analogs called 5′-chloro-2′-deoxyuridine (CldU) and 5′-iodo-2′-deoxyuridine (IdU) during DNA replication. After mixing the two cell cultures that had been previously incorporated with CldU and IdU separately, the presence of these unusual deoxynucleosides in the genome can be detected by specific antibodies. For this, a DNA denaturation step is required to expose the sites of thymidine analogs incorporated. Subsequently, a secondary reaction using fluorochrome-labeled antibodies will generate distinct signals under fluorescence analysis. By using this method, DNA exchange verification (i.e., the presence of both CldU and IdU in the same cell) is possible using a standard fluorescence microscope. It typically takes 2-3 days from the thymidine analogs incorporation to results. Of note, ADExTA is relatively cheap and does not require transfections or harsh genetic manipulation. These features represent an advantage when compared to other time-consuming protocols that demand DNA manipulation to introduce distinct drug-resistance markers in different cells for posterior selection.
Molecular Biology
Variable Dose Analysis: A Novel High-throughput RNAi Screening Method for Drosophila Cells
Genetic screens are a powerful approach to identify previously uncharacterized genes involved in specific biological processes. Several technologies have been developed for high-throughput screens using reagents such as RNAi or CRISPR, and each approach is associated with specific advantages and disadvantages. Variable Dose Analysis (VDA), is an RNAi-based method developed in Drosophila cells that improves signal-to-noise ratio compared to previous methods. VDA assays are performed by co-transfecting cells with a plasmid expressing shRNA, (a type of RNAi that can be easily expressed from a DNA plasmid) against a gene of interest and a second plasmid expressing a fluorescent reporter protein. Fluorescent protein expression, can be used as an indirect readout of shRNA expression and therefore target gene knockdown efficiency. Using this approach, we can measure phenotypes over a range of knockdown efficiencies in a single sample. When applied to genetic interaction screens, VDA results in improved consistency between screens and reliable detection of known interactions. Furthermore, because phenotypes are analyzed over a range of target gene knockdown efficiencies, VDA allows the detection of phenotypes and genetic interactions involving essential genes at sub-lethal knockdown efficiency. This therefore represents a powerful approach to high-throughput screening applicable to a wide range of biological questions.
Neuroscience
Three-chamber Social Approach Task with Optogenetic Stimulation (Mice)
The formation of social relationships via social interactions and memory are essential for one’s physical and mental health. To date, rodent studies have used the three-chamber social approach test to measure social approach, social novelty, and social memory. In recent years, techniques including optogenetics have been developed to acutely control the activity of genetically defined populations of neurons. Recent studies have even combined optogenetics with advanced temporal gene expression control systems to label certain populations of neurons during learning and subsequently reactivated for memory testing. We combined optogenetic targeting with the three-chamber social approach test to examine particular neural circuits of interest during social memory encoding or retrieval. First, we stereotaxically infected specific brain areas with viral-encoding opsins that acutely activate or inhibit the firing of the neurons. Next, we subjected the mice to the three-chamber behavioral paradigm while delivering light during social memory encoding or retrieval. Lastly, the mice were tested with the delivery of light in a counter-balanced manner which allows each subject to be its own internal control. Thus, the optogenetic stimulation coupled with the three-chamber social approach test is a well-validated paradigm to explore the contribution of diverse brain circuits in various social cognition processes.
Partial Transection of Adult Rat Optic Nerve as a Model of Secondary Degeneration in the Central Nervous System
Injury to the central nervous system is characterized by damage that spreads from the initial point of impact into the surrounding adjacent tissue, in a phenomenon referred to as secondary degeneration. The optic nerve can be used to effectively model injury and secondary degeneration to white matter tracts. Partial transection of the dorsal aspect of the nerve leaves the ventral aspect initially undamaged but vulnerable to secondary degeneration, allowing study of tissue exclusively vulnerable to secondary degeneration. Thus the partial optic nerve transection model of secondary degeneration is a valuable tool to study the pathology of spreading damage following neurotrauma and can be used to assess potential efficacy of therapeutic strategies.
Plant Science
Visualization and Quantification of Cell-to-cell Movement of Proteins in Nicotiana benthamiana
Cell-to-cell movement of proteins through plasmodesmata is a widely-established mechanism for intercellular signaling in plants. Current techniques to study intercellular protein translocation rely on single-cell transformation using particle bombardment or transgenic lines expressing photo-inducible fluorophores. The method presented here allows visualization and objective quantification of (effector) protein movement between N. benthamiana leaf cells. Agroinfiltration is performed using a single binary vector encoding a GFP-tagged protein of interest that is either mobile or non-mobile (MP; non-MP), together with an ER-anchored mCherry. Upon creation of mosaic-like transformation patterns, cell-to-cell movement of the MP can be followed by monitoring translocation of the GFP signal from mCherry labeled transformed cells into neighboring non-transformed cells. This process can be visualized using confocal microscopy and quantified following protoplast isolation and flow cytometric cell analysis. This method overcomes the limitations of existing methods as it allows rapid and objective quantification of protein translocation without the need of creating transgenic plants.