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Past Issue in 2020
Volume: 10, Issue: 3
Biophysics
Evaluating Whole Blood Clotting in vitro on Biomaterial Surfaces
Biomaterial-associated thrombosis is still a major concern for blood-contacting implants. After the medical device is implanted and comes in contact with blood, several complex reactions occur, which may lead to thrombus formation and failure of the device. Therefore, it is essential to evaluate the biomaterial interaction with the whole blood. Several studies have been reported in the literature that evaluate different steps in the coagulation cascade, such as protein adsorption, plasma activation, and platelet adhesion in vitro, however, evaluation of whole blood clotting on biomaterial surfaces is not widely reported. Here, a protocol to evaluate whole blood clotting in vitro on 2D biomaterials surfaces via a simple and fast hemolysis assay is presented. Whole human blood is placed onto the biomaterial surfaces and is allowed to clot for different time periods. After the specific time intervals, the surfaces are transferred into deionized (DI) water to release the free hemoglobin and the absorbance of this solution is measured. The absorbance value is proportional to the free hemoglobin concentration in the DI water due to lysis of red blood cells and gives an indirect correlation to the extent of blood clotting on the biomaterial surfaces. This protocol provides a fast, facile and effective method to measure the anti-thrombogenic properties of biomaterials.
Cancer Biology
Identification of Target Protein for Bio-active Small Molecule Using Photo-cross Linked Beads and MALDI-TOF Mass Spectrometry
Development of methods for protein identification is one of the important aspects of proteomics. Here, we report a protocol for the preparation of compound conjugated beads by photo-crosslinking, affinity purification, gel electrophoresis, and highly sensitive mass spectrometric assay for drug-target identification. Although there are several other methods used for drug-target identification, such as biochemical fractionation or radioactive ligand binding assay, affinity purification is widely used for its straight-forward and easy approach. To identify the target protein of an inhibitor of cancer cell-accelerated fibroblast migration, we prepared the inhibitor-conjugated beads by photo-crosslinking. Proteins were pulled down from cell lysates by the compound beads and separated by SDS-PAGE, and a specifically pulled down protein was cut out, trypsin-digested, analyzed using matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF-MS) and identified by peptide mass fingerprinting (PMF) method. Since the photo-crosslinking enables the immobilization of ligands on an affinity matrix in a functional group-independent manner, we do not have to determine the functional group of the compound to conjugate the matrix. In addition, as compared to other MS techniques such as electrospray ionization, MALDI offers a less complex sample preparation procedure and higher sensitivity, and thus is better suited for the rapid identification of proteins isolated by gel electrophoresis.
Measurement of ATPase Activity of Valosin-containing Protein/p97
Valosin-containing protein (VCP; also known as p97) is a type II ATPase regulating several cellular processes. Using proteomic techniques, we identified a chemical compound that binds to the D1 ATPase domain of VCP. The protocol described here was to study the effect of the compound on ATPase activity in vitro of purified VCP protein. ATPases are enzymes that hydrolyze ATP in a reaction resulting the release of an inorganic phosphate. This reaction can be measured using several methods, such as colorimetric, fluorescence, and radiometric assays, in addition to the bioluminescence assay mentioned here. Since the remaining ATP level after the reaction was detected using a luciferase assay, the luminescent signal indicates the ATPase activity inversely. This protocol is sensitive, rapid, and can be used for high-throughput screening assays to study the effect of compounds on ATPase function.
Detection of Individual RNA in Fixed Cells and Tissues by Chromogenic ISH
Visualization of RNA molecules in situ helps to better understand the functions of expressed genes. Currently, most conventional in situ hybridization methods for visualization of individual RNAs are based on fluorescence detection. Herein we present a chromogenic in situ hybridization protocol for visualization of single RNA molecules in fixed cells and tissues. The protocol is based on padlock probing and rolling circle amplification to generate detectable chromogenic signal from single RNA molecules. Chromogenic signal can avoid background autofluorescence and can be preserved for a longer period than fluorescence signal.
Approaching RNA-seq for Cell Line Identification
Cancer cell lines serve as invaluable model systems for cancer biology research and help in evaluating the efficacy of new therapeutic agents. However, cell line contamination and misidentification have become one of the most pressing problems affecting biomedical research. Available methods of cell line authentication suffer from limited access, time-consuming and often costly for many researchers, hence a new and cost-effective approach for cell line authentication is needed. In this regard, we developed a new method called CeL-ID for cell line authentication using genomic variants as a byproduct derived from RNA-seq data. CeL-ID was trained and tested on publicly available more than 900 RNA-seq dataset derived from the Cancer Cell Line Encyclopedia (CCLE) project; including most frequently used adult and pediatric cancer cell lines. We generated cell line specific variant profiles from RNA-seq data using our in-house pipeline followed by pair-wise variant profile comparison between cell lines using allele frequencies and depth of coverage values of the entire variant set. Comparative analysis of variant profiles revealed that they differ significantly from cell line to cell line whereas identical, synonymous and derivative cell lines share high variant identity and their allelic fractions are highly correlated, which is the basis of this cell line authentication protocol. Additionally, CeL-ID also includes a method to estimate the possible cross-contamination using a linear mixture model with any possible CCLE cells in case no perfect match was detected.
Cell Biology
Live Mitochondrial or Cytosolic Calcium Imaging Using Genetically-encoded Cameleon Indicator in Mammalian Cells
Calcium (Ca2+) imaging aims at investigating the dynamic changes in live cells of its concentration ([Ca2+]) in different pathophysiological conditions. Ca2+ is an ubiquitous and versatile intracellular signal that modulates a large variety of cellular functions thanks to a cell type-specific toolkit and a complex subcellular compartmentalization.Many Ca2+ sensors are presently available (chemical and genetically encoded) that can be specifically targeted to different cellular compartments. Using these probes, it is now possible to monitor Ca2+ dynamics of living cells not only in the cytosol but also within specific organelles. The choice of a specific sensor depends on the experimental design and the spatial and temporal resolution required.Here we describe the use of novel Förster resonance energy transfer (FRET)-based fluorescent Ca2+ probes to dynamically and quantitatively monitor the changes in cytosolic and mitochondrial [Ca2+] in a variety of cell types and experimental conditions. FRET-based sensors have the enormous advantage of being ratiometric, a feature that makes them particularly suitable for quantitative and in vivo applications.
Rapid Nickel-based Isolation of Extracellular Vesicles from Different Biological Fluids
Extracellular vesicles (EVs) are membranous structures that cells massively release in extracellular fluids. EVs are cargo of cellular components such as lipids, proteins, and nucleic acids that can work as a formidable source in liquid biopsy studies searching for disease biomarkers. We recently demonstrated that nickel-based isolation (NBI) is a valuable method for fast, efficient, and easy recovery of heterogeneous EVs from biological fluids. NBI exploits nickel cations to capture negatively charged vesicles. Then, a mix of balanced chelating agents elutes EVs while preserving their integrity and stability in solution. Here, we describe steps and quality controls to functionalize a matrix of agarose beads, obtain an efficient elution of EVs, and extract nucleic acids carried by them. We demonstrate the versatility of NBI method in isolating EVs from media of primary mouse astrocytes, from human blood, urine, and saliva processed in parallel, as well as outer membrane vesicles (OMVs) from cultured Gram-negative bacteria.
Developmental Biology
Mouse Embryonic Tooth Germ Dissection and Ex vivo Culture Protocol
A tooth germ ex vivo organ culture allows visualization of its development in different stages, thus enabling investigation of the molecular mechanisms of regulatory factors. Tooth germs can be rapidly dissected from E13 mouse embryos and placed on cell culture inserts for observation of subsequent tooth germ development in a three-dimensional situation in real time. This method is also suitable for other organs that develop by epithelial-mesenchymal interactions, including salivary gland, hair, lung, and kidney. In addition, siRNAs or growth factors can be easily added to ex vivo tooth germ cultures to investigate the detailed molecular function of specific genes. The present protocol provides an efficient and practical method for isolation and ex vivo culture of embryonic tooth germs.
Microbiology
Expression and Purification of Adeno-associated Virus Virus-like Particles in a Baculovirus System and AAVR Ectodomain Constructs in E. coli
Adeno-associated virus (AAV) is a promising gene therapy vector and the biophysical characterization of its interactions with host proteins is a critical foundation for engineering tissue targeting and immune escape. Presented here are protocols for the production of: (a) the outer protein shells (virus-like particles or VLPs) for serotype 2 (AAV-2) and (b) two fragments from the binding ectodomain of AAV’s cellular receptor, AAVR. His6PKD1-2 comprises the first two polycystic kidney disease (PKD) domains, the minimal required for efficient binding of AAV, expressed with an N-terminal histidine tag. MBP-PKD1-5 is a fusion of the maltose binding protein with all five of the PKD domains of the AAVR receptor. Presented are the expression and purification of milligram quantities, ample for in vitro analyses. For AAV-2, the protocol offers an alternative to the use of (infectious) wild-type virus or transducing vectors. One of the methods for producing transducing vector is in Sf9 cells, and the production of VLPs is based on this. For AAVR, the protocols enable biochemical and biophysical characterization of virus-binding. The minimal two-domain construct allows more saturated binding to symmetry-equivalent sites on the virus, while the larger construct might be better expected to reflect the native receptor.
Rapid Detection of Proliferative Bacteria by Electrical Stimulation
Detecting live bacteria is an important task for antimicrobial susceptibility testing (AST) in the medical sector and for quality-monitoring in biological industries. Current methods for live-bacteria detection suffer limitations in speed or sensitivity. In a recent paper, we reported that electrical response dynamics in membrane potential enable single-cell rapid detection of live bacteria. The electrical response can be observed within a minute after electrical stimulation. Thus, it has potential in accelerating AST and the monitoring of biological samples. This method also enables experiments for biophysical and microbiological investigations into bacterial electrophysiology. With the hope that more researchers, scientists and engineers will use electrical stimulation for their assays, here we detail each step of the electrical stimulation experiment.
Transcervical Mouse Infections with Chlamydia trachomatis and Determination of Bacterial Burden
Chlamydia trachomatis is an obligate human pathogen. It infects the genital tract of humans ascending into the fallopian tube, exacerbated by chronic pelvic pain, pelvic inflammatory disease, and fallopian tube scaring resulting in infertility and other malignancies. The major hurdle in controlling chlamydial spread is that the infection remains asymptomatic, thus leading to chronic, recurrent and persistent infections, with no vaccines developed so far. Being a human pathogen, we do not have an in vivo model of C. trachomatis infection. C. trachomatis do not cause ascending infections and fallopian tube pathology in the mouse urogenital tract when infected vaginally. To overcome this hurdle trans cervical method of infection must be adapted. In this protocol the method of establishing trans-cervical chlamydial infection with the procedure to determine the bacterial load is detailed. This method will facilitate to deliver the bacteria past the cervix establishing an ascending infection into the uterine horns reciprocating human fallopian tube infections.
Competition Assays to Quantify the Effect of Biocontrol Yeasts against Plant Pathogenic Fungi on Fruits
Yeasts such as Aureobasidium pullulans are unicellular fungi that occur in all environments and play important roles in biotechnology, medicine, food and beverage production, research, and agriculture. In the latter, yeasts are explored as biocontrol agents for the control of plant pathogenic fungi (e.g., Botrytis cinerea, Fusarium sp.); mainly on flowers and fruits. Eventually, such yeasts must be evaluated under field conditions, but such trials require a lot of time and resources and are often difficult to control. Experimental systems of intermediate complexity, between in vitro Petri dish assays and field trials, are thus required. For pre- and post-harvest applications, competition assays on fruits are reproducible, economical and thus widely used. Here, we present a general protocol for competition assays with fruits that can be adapted depending on the biocontrol yeast, plant pathogen, type of assay or fruit to be studied.
Molecular Biology
Structural Alignment and Covariation Analysis of RNA Sequences
RNA molecules adopt defined structural conformations that are essential to exert their function. During the course of evolution, the structure of a given RNA can be maintained via compensatory base-pair changes that occur among covarying nucleotides in paired regions. Therefore, for comparative, structural, and evolutionary studies of RNA molecules, numerous computational tools have been developed to incorporate structural information into sequence alignments and a number of tools have been developed to study covariation. The bioinformatic protocol presented here explains how to use some of these tools to generate a secondary-structure-aware multiple alignment of RNA sequences and to annotate the alignment to examine the conservation and covariation of structural elements among the sequences.
Neuroscience
Implanting and Recycling Neuropixels Probes for Recordings in Freely Moving Mice
Recording neural activity in unrestricted animals is necessary to unravel the neural basis of ethological behaviors. Recently, Neuropixels probes have made important strides in improving yield and lowering noise, but have limited use cases in freely moving animals. Although there are a number of studies demonstrating the use of these probes in headfixed mice, there are not established protocols for the use and reuse of them in a freely moving mouse. We therefore designed a novel device (the AMIE) that maximizes the potential value of these powerful probes. Here, we provide the technical drawings for the AMIE and detail its preparation, implantation, and explantation. With our approach, researchers can record hundreds of neurons during freely moving behavior across weeks of experiments, and then recycle valuable probes for future use.
Stem Cell
In vitro Self-organized Mouse Small Intestinal Epithelial Monolayer Protocol
Developing protocols to obtain intestinal epithelial monolayers that recapitulate in vivo physiology to overcome the limitations of the organoids’ closed geometry has become of great interest during the last few years. Most of the developed culture models showed physiological-relevant cell composition but did not prove self-renewing capacities. Here, we show a simple method to obtain mouse small intestine-derived epithelial monolayers organized into proliferative crypt-like domains, containing stem cells, and differentiated villus-like regions, closely resembling the in vivo cell composition and distribution. In addition, we adapted our model to a tissue culture format compatible with functional studies and prove close to physiological barrier properties of our in vitro epithelial monolayers. Thus, we have set-up a protocol to generate physiologically relevant intestinal epithelial monolayers to be employed in assays where independent access to both luminal and basolateral compartments is needed, such as drug absorption, intracellular trafficking and microbiome-epithelium interaction assays.