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Past Issue in 2023
Volume: 13, Issue: 12
Biochemistry
Purification of Recombinant Human Amphiphysin 1 and its N-BAR Domain
Bin/Amphiphysin/Rvs (BAR) proteins are known as classical membrane curvature generators during endocytosis. Amphiphysin, a member of the N-BAR sub-family of proteins that contain a characteristic amphipathic sequence at the N-terminus of the BAR domain, is involved in clathrin-mediated endocytosis. Full-length amphiphysin contains a ~ 400 amino acid long disordered linker connecting the N-BAR domain and a C-terminal Src homology 3 (SH3) domain. We express and purify recombinant amphiphysin and its N-BAR domain along with an N-terminal glutathione-S-transferase (GST) tag. The GST tag allows extraction of the protein of interest using affinity chromatography and is removed in the subsequent protease treatment and ion-exchange chromatography steps. In the case of the N-BAR domain, cleavage of the GST tag was found to cause precipitation. This issue can be minimized by adding glycerol to the protein purification buffers. In the final step, size exclusion chromatography removes any potential oligomeric species. This protocol has also been successfully used to purify other N-BAR proteins, such as endophilin, Bin1, and their corresponding BAR domains.Graphical overview
Cell Biology
Ten-fold Robust Expansion Microscopy
Expansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y, and z. By systematic exploration of the ExM recipe space, we developed a novel ExM method termed Ten-fold Robust Expansion Microscopy (TREx) that, as the original ExM method, requires no specialized equipment or procedures. TREx enables ten-fold expansion of both thick mouse brain tissue sections and cultured human cells, can be handled easily, and enables high-resolution subcellular imaging with a single expansion step. Furthermore, TREx can provide ultrastructural context to subcellular protein localization by combining antibody-stained samples with off-the-shelf small molecule stains for both total protein and membranes.
Synthesis of Bacteria-mimetic Gold Nanoparticles for Phagocytosis by Immune Cells
Cell-based carrier exhibits inherent advantages as the next generation of drug delivery system, namely high biocompatibility and physiological function. Current cell-based carriers are constructed via direct payload internalization or conjugation between cell and payload. However, the cells involved in these strategies must be firstly extracted from the body and the cell-based carrier must be prepared in vitro. Herein, we synthesize bacteria-mimetic gold nanoparticles (GNPs) for the construction of cell-based carrier in mice. Both β-cyclodextrin (β-CD)-modified GNPs and adamantane (ADA)-modified GNPs are coated by E. coli outer membrane vesicles (OMVs). The E. coli OMVs induce the phagocytosis of GNPs by circulating immune cells, leading to intracellular degradation of OMVs and subsequent supramolecular self-assembly of GNPs driven by β-CD-ADA host–guest interactions. In vivo construction of cell-based carrier based on bacteria-mimetic GNPs avoids the immunogenicity induced by allogeneic cells and restriction by the number of separated cells. Due to the inflammatory tropism, endogenous immune cells carry the intracellular GNP aggregates to the tumor tissues in vivo. Graphical overviewCollect the outer membrane vesicles (OMVs) of E. coli by gradient centrifugation (a) and coat on gold nanoparticles (GNP) surface (b) to prepare OMV-coated cyclodextrin (CD)-GNPs and OMV-coated adamantane (ADA)-GNPs (c) via ultrasonic method
Environmental science
Isolation and Quantification of Mandelonitrile from Arabidopsis thaliana Using Gas Chromatography/Mass Spectrometry
Mandelonitrile is a nitrogen-containing compound, considered an essential secondary metabolite. Chemically, it is a cyanohydrin derivative of benzaldehyde, with relevant functions in different physiological processes including defense against phytophagous arthropods. So far, procedures for detecting mandelonitrile have been effectively applied in cyanogenic plant species such as Prunus spp. Nevertheless, its presence in Arabidopsis thaliana, considered a non-cyanogenic species, has never been determined. Here, we report the development of an accurate protocol for mandelonitrile quantification in A. thaliana within the context of A. thaliana–spider mite interaction. First, mandelonitrile was isolated from Arabidopsis rosettes using methanol; then, it was derivatized by silylation to enhance detection and, finally, it was quantified using gas chromatography–mass spectrometry. The selectivity and sensitivity of this method make it possible to detect low levels of mandelonitrile (LOD 3 ppm) in a plant species considered non-cyanogenic that, therefore, will have little to no cyanogenic compounds, using a small quantity of starting material (≥100 mg).
Immunology
A New Methodology for the Quantification of Neutrophil Extracellular Traps in Patient Plasma
Neutrophil extracellular traps (NETs) are web-like structures made up of decondensed chromatin fibers along with neutrophil granular proteins that are extruded by neutrophils after activation or in response to foreign microorganisms. NETs have been associated with autoimmune and inflammatory diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, coronavirus disease 2019 (COVID-19), and others. There are reliable methods available to quantitate NETs from neutrophils, but their accurate quantification in patient plasma or serum remains a challenge. We developed a highly sensitive ELISA to detect NETs in serum/plasma and designed a novel smear immunofluorescence assay to detect NETs in as little as 1 μL of serum/plasma. We further validated our technology on plasma samples from SLE patients and healthy donors that carry interferon regulatory factor 5 genetic risk. The multiplex ELISA combines the use of three antibodies against myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and DNA to detect the NET complexes with higher specificities. The immunofluorescence smear assay can visually detect intact structures of NETs in 1 μL of serum/plasma and provide similar results that correlate with findings from the multiplex ELISA. Furthermore, the smear assay is a relatively simple, inexpensive, and quantifiable method of NET detection for small volumes.Graphical overview
Selection of Molecules with Immunological Potential from Excretory and Secretory Products from the Nematode Haemonchus placei by Cell Proliferation and Gene Expression Assays
The nematode Haemonchus placei is a pathogenic parasite, the most seriously affecting ruminant’s health and being responsible for enormous economic losses all over the world. The present protocol describes different in vitro techniques to select potential candidate antigens with immune-protective activity from excretory and secretory products (ESP) from H. placei transitory infective larvae (xL3). ESP from xL3 were obtained from the in vitro infective larvae (L3) maintained in Hank’s medium at 37 °C with 5% CO2 for 48 h. Then, the presence of ESP proteins was confirmed by SDS-PAGE to be used in an in vitro proliferation assay with bovine peripheral blood mononuclear cells (PBMCs). The ESP were exposed to the PBMCs during two different periods (24 and 48 h). Genes associated with immune response against the nematode were analyzed using relative gene expression and bioinformatic tools. These are simple, economic, and helpful tools to identify potential immune-protective molecules under in vitro conditions for confirming the efficacy of future in vivo assays.Graphical overview
Microbiology
β-lactamase (Bla) Reporter-based System to Study Flagellar Type 3 Secretion in Salmonella
Export of type 3 secretion (T3S) substrates is traditionally evaluated using trichloroacetic acid (TCA) precipitation of cultured cell supernatants followed by western blot analysis of the secreted substrates. In our lab, we have developed β-lactamase (Bla), lacking its Sec secretion signal, as a reporter for the export of flagellar proteins into the periplasm via the flagellar T3S system. Bla is normally exported into the periplasm through the SecYEG translocon. Bla must be secreted into the periplasm in order to fold into an active conformation, where it acts to cleave β-lactams (such as ampicillin) to confer ampicillin resistance (ApR) to the cell. The use of Bla as a reporter for flagellar T3S allows the relative comparison of translocation efficiency of a particular fusion protein in different genetic backgrounds. In addition, it can also be used as a positive selection for secretion. Graphical overviewUtilization of β-lactamase (Bla) lacking its Sec secretion signal and fused to flagellar proteins to assay the secretion of exported flagellar substrates, into the periplasm, through the flagellar T3S system. A. Bla is normally transported into the periplasm space through the Sec secretion pathway, where it folds into an active conformation and allows resistance to ampicillin (ApR). B. Bla, lacking its Sec secretion signal, is fused to flagellar proteins to assay the secretion of exported flagellar proteins into the periplasm through the flagellar T3S system.
Neuroscience
Alginate Gel Immobilization of Caenorhabditis elegans for Optical Calcium Imaging of Neurons
A fascinating question in neuroscience is how sensory stimuli evoke calcium dynamics in neurons. Caenorhabditis elegans is one of the most suitable models for optically recording high-throughput calcium spikes at single-cell resolution. However, calcium imaging in C. elegans is challenging due to the difficulties associated with immobilizing the organism. Currently, methods for immobilizing worms include entrapment in a microfluidic channel, anesthesia, or adhesion to a glass slide. We have developed a new method to immobilize worms by trapping them in sodium alginate gel. The sodium alginate solution (5%), polymerized with divalent ions, effectively immobilizes worms in the gel. This technique is especially useful for imaging neuronal calcium dynamics during olfactory stimulation. The highly porous and transparent nature of alginate gel allows the optical recording of cellular calcium oscillations in neurons when briefly exposed to odor stimulation.
A Standardized Protocol for Early-life Stress-induced Social Defeat in Mice
Neuropsychiatric diseases, like depression, have a considerable and persistent impact on human health; however, little is known about their underlying pathogenesis. Social defeat is a model for stress-induced psychopathologies that could present with behaviors resembling those observed in humans with depression. However, previous animal models of social defeat mainly focus on adults. Here, we re-design the protocol of early-life stress-induced social defeat paradigm, which is based on a classic resident–intruder model. Briefly, each two-week-old experimental mouse of C57BL/6 strain is introduced into the home cage of an unfamiliar CD1 aggressor mouse for 30 min per day for 10 consecutive days. Later, all experimental mice are raised individually for another month. Finally, the mice are identified as defeated through social interaction and open field tests. This model has been shown to be etiological and predictive and provide high validity and could be a powerful tool to investigate the underlying pathogenesis of early onset depression. Graphical overview
Triplet-primed PCR and Melting Curve Analysis for Rapid Molecular Screening of Spinocerebellar Ataxia Types 1, 2, and 3
There are more than 40 types of spinocerebellar ataxia (SCA), most of which are caused by abnormal expansion of short tandem repeats at various gene loci. These phenotypically similar disorders require molecular testing at multiple loci by fluorescent PCR and capillary electrophoresis to identify the causative repeat expansion. We describe a simple strategy to screen for the more common SCA1, SCA2, and SCA3 by rapidly detecting the abnormal CAG repeat expansion at the ATXN1, ATXN2, and ATXN3 loci using melting curve analysis of triplet-primed PCR products. Each of the three separate assays employs a plasmid DNA carrying a known repeat size to generate a threshold melt peak temperature, which effectively distinguishes expansion-positive samples from those without a repeat expansion. Samples that are screened positive based on their melt peak profiles are subjected to capillary electrophoresis for repeat sizing and genotype confirmation. These screening assays are robust and provide accurate detection of the repeat expansion while eliminating the need for fluorescent PCR and capillary electrophoresis for every sample.