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Past Issue in 2019
Volume: 9, Issue: 17
Biochemistry
![Photoaffinity Labeling of Respiratory Complex I in Bovine Heart Submitochondrial Particles by Photoreactive [125I] amilorides](https://en-cdn.bio-protocol.org/imageup/arcimg/20190902091054730.jpg?t=1758522982)
Photoaffinity Labeling of Respiratory Complex I in Bovine Heart Submitochondrial Particles by Photoreactive [125I] amilorides
The architecture of quinone/inhibitor-access channel in proton-translocating NADH-quinone oxidoreductase (respiratory complex I) was modeled by X-ray crystallography and cryo-EM, however, it remains debatable whether the channel model reflects the physiologically relevant state present throughout the catalytic cycle. Using photoreactive [125I]amilorides, we demonstrated that amiloride-type inhibitors bind to the interfacial region of multiple subunits (49-kDa, ND1, PSST, and 39-kDa subunits), which is difficult to reconcile with the current channel model. This report describes the procedures for photoaffinity labeling of bovine submitochondrial particles by photoreactive [125I]amilorides. The protocol could be widely applicable for the characterization of various biologically active compounds, whose target protein remains to be identified or characterized.
Cancer Biology
A Robust, One-step FRET Assay for Human Heparanase
Heparanase, an endo-β-D-glucuronidase, cleaves cell surface and extracellular matrix heparan sulfate (HS) chains at distinct sites and plays important biological roles including modulation of cell growth and metastasis. Although a number of different types of heparanase assays have been reported to date, most are labor intensive, complex and/or expensive to carry out. We reasoned that a simpler heparanase assay could be developed using heparin labeled with Dabcyl and EDANS as donor and acceptor fluorophores so as to generate a FRET signal. Our results show that a more robust heparanase assay could be developed based on the principle studied herein and more homogeneous preparation of heparin. Yet, the assay in its current form could be used for routine screening of potential inhibitors in a high-throughput manner as well as for studying heparanase activity expressed in tumors as well as biological fluids like plasma.
Cell Biology
In vitro Intestinal Epithelial Wound-healing Assays Using Electric Cell-Substrate Impedance Sensing Instrument
Here, we describe an in vitro epithelial wound-healing assay using Electric Cell-Substrate Impedance Sensing (ECIS) technology. The ECIS technology is a real time cell growth assay based on a small (250 μm diameter) active gold electrode which resistance is measured continuously. When intestinal epithelial cells reach confluency on the gold electrode, resistances reach a plateau. For the wound-healing assays, confluent intestinal epithelial monolayers are subjected to a current of 40 kHz frequency, 1,400 μA amplitude, and 30-second duration. This kills the cells around the small active gold electrode, causing detachment and generating a wound that is healed by surrounding cells that have not been submitted to the current pulse. Wound healing is then assessed by continuous resistance measurements for approximately 30 h after wound. Both cell wounding and measurements of the subsequent healing process are carried out under computer control that takes online measurements each 30 s and stores the data. ECIS technology can be used to study the underlying causes for impaired mucosal healing and to test the efficacy of drugs in mucosal healing.
Developmental Biology
Estrous Cycle Monitoring in Mice with Rapid Data Visualization and Analysis
The estrous cycle provides a readout of reproductive health in female laboratory rodents, and estrous cycle stage can be an important physiological variable. Accurate assessment of estrous cycle stage is also important in producing timed pregnancies for developmental studies. Here, we provide a protocol for evaluation of estrous cycle stage through a minimally invasive procedure of acquiring cells lining the vaginal cavity and immediate microscopic visual assessment of these cells without drying or staining. When performed over several consecutive days, the pattern of progression through the four main stages of the estrous cycle, and disruptions to this pattern, can be determined. We also present software that enables more efficient cycle stage data analysis and pattern visualization. These protocols and tools will thus facilitate the incorporation of female animals in laboratory experiments and enhance the assessment of relationships between the reproductive cycle and overall physiology and behavior.
A Widely Applicable Urea-based Fluorescent/Colorimetric mRNA in situ Hybridization Protocol
In situ hybridization methods are routinely employed to detect nucleic acid sequences, allowing to localize gene expression or to study chromosomal organization in their native context. These methods rely on the pairwise binding of a labeled probe to the target endogenous nucleic acid sequence–the hybridization step, followed by detection of annealed sequences by means of fluorescent or colorimetric reactions. Successful hybridization requires permeabilization of tissues, followed by denaturation of nucleic acids strands, which is usually carried out in a formamide-based buffer and at high temperatures. Such reaction conditions, besides posing a health hazard (both concerning manipulation and waste disposal), can be excessively harsh for the delicate tissues of some species or developmental stages. We detail here an alternative method for in situ hybridization, where the toxic formamide is replaced with a urea solution. This substitution improved both tissues preservation and signal-to-noise detection, in several animal species. The protocol described here, originally developed for the hydrozoan jellyfish Clytia hemisphaerica, provides guidelines for adapting formamide-based traditional protocols to the urea variant. Urea-based protocols have already been successfully applied to diverse invertebrate and vertebrate species, showing the ease of such a modification, and providing the scientific community with a promising, safer and versatile tool.
Immunology
A Refined Protocol for Identifying Citrulline-specific Monoclonal Antibodies from Single Human B Cells from Rheumatoid Arthritis Patient Material
We describe here a detailed, refined protocol for the generation of citrulline-specific monoclonal antibodies from single human B cells from rheumatoid arthritis (RA) patients. This protocol provides a detailed guide of the procedure starting from single B cells of your choice and followed by amplification of the variable region of immunoglobulin genes by RT-PCR, subsequent immunoglobulin gene cloning, recombinant IgG1 monoclonal antibody (mAb) production and quality controls. The produced mAbs can be used for further studies including reactivity towards candidate antigens and functionality both in vitro and in vivo. This protocol can be used to generate antigen-specific mAbs from B cells derived from different tissues and compartments, including peripheral blood, synovial fluid, digested biopsies, bone marrow aspirations, and bronchoalveolar lavage fluid. Notably, although examples are given on how to identify citrulline-specific autoantibodies the general methods can also be applied to other reactivities.
Isolation of Neutrophil Nuclei for Use in NETosis Assays
Neutrophils are critical immune cells that protect our body against invading pathogens. They generate antibacterial DNA structures called neutrophil extracellular traps (NET). Recently we identified a new mechanism that enables NET formation. We observed that following recognition of lipopolysaccharides, inflammatory caspases cleave Gasdermin D and enable NET generation (Chen et al., 2018). This protocol describes how we purify neutrophil nuclei to visualize NET formation by live microscopy. After neutrophil purification from murine bone marrow, neutrophils are lysed in a hypotonic buffer using a nitrogen cavitation device to prevent lysis of neutrophil granules and subsequent contamination by granules proteases. Lysed neutrophils are then centrifuged, and nuclei are counted. The protocol described here is straightforward and enables the study of early changes happening in the nuclei of neutrophils undergoing NETosis with limited contamination by granule proteases.
Microbiology
Yeast Single-cell RNA-seq, Cell by Cell and Step by Step
Single-cell RNA-seq (scRNA-seq) has become an established method for uncovering the intrinsic complexity within populations. Even within seemingly homogenous populations of isogenic yeast cells, there is a high degree of heterogeneity that originates from a compact and pervasively transcribed genome. Research with microorganisms such as yeast represents a major challenge for single-cell transcriptomics, due to their small size, rigid cell wall, and low RNA content per cell. Because of these technical challenges, yeast-specific scRNA-seq methodologies have recently started to appear, each one of them relying on different cell-isolation and library-preparation methods. Consequently, each approach harbors unique strengths and weaknesses that need to be considered. We have recently developed a yeast single-cell RNA-seq protocol (yscRNA-seq), which is inexpensive, high-throughput and easy-to-implement, tailored to the unique needs of yeast. yscRNA-seq provides a unique platform that combines single-cell phenotyping via index sorting with the incorporation of unique molecule identifiers on transcripts that allows to digitally count the number of molecules in a strand- and isoform-specific manner. Here, we provide a detailed, step-by-step description of the experimental and computational steps of yscRNA-seq protocol. This protocol will ease the implementation of yscRNA-seq in other laboratories and provide guidelines for the development of novel technologies.
High Resolution Respirometry in Candida albicans
Many Candida species, such as the opportunistic human pathogen Candida albicans, are Crabtree-Negative yeasts and are therefore highly dependent on the energy generated through oxidative phosphorylation. Respiration control is linked to a range of aspects of C. albicans cell physiology that appear to be important for virulence, most notably its ability to switch from yeast to hyphal forms and the maintenance of the cell wall. The following protocol allows for the measurement and characterization of respiration in C. albicans using high resolution respirometry. We outline how addition of respiration inhibitors can be used to assay the “mode” of respiration, mitochondrial health and the level of electron transport that is coupled to ATP synthase activity in living cell cultures. These data provide useful insight into the effects of external factors, such as exposure to anti-fungal compounds, or internal changes such as genetic alterations on respiratory performance.
Development of an Efficient Transformation System for Halotolerant Yeast Debaryomyces hansenii CBS767
Debaryomyces hansenii is one of the most osmotolerant and halotolerant yeasts. Further, its association with traditional cheese and meat products imparting special flavors to these products project this yeast with enormous biotechnological potential in the agrofood sector. However, lack of an efficient transformation system in D. hansenii still direct the complementation based assay in S. cerevisiae mutants for functional analysis of D. hansenii genes. Here, we have described the development of an efficient transformation system for D. hansenii that is based on a histidine auxotrophic recipient strain, DBH9 (generated by UV induced random mutagenesis), and the DhHIS4 gene as the selectable marker (Minhas et al., 2009). Moreover, the same method has also been employed for gene disruption in D. hansenii by homologous recombination.
Neuroscience
Co-culture of Human Stem Cell Derived Neurons and Oligodendrocyte Progenitor Cells
Crosstalk between neurons and oligodendrocytes is important for proper brain functioning. Multiple co-culture methods have been developed to study oligodendrocyte maturation, myelination or the effect of oligodendrocytes on neurons. However, most of these methods contain cells derived from animal models. In the current protocol, we co-culture human neurons with human oligodendrocytes. Neurons and oligodendrocyte precursor cells (OPCs) were differentiated separately from pluripotent stem cells according to previously published protocols. To study neuron-glia cross-talk, neurons and OPCs were plated in co-culture mode in optimized conditions for additional 28 days, and prepared for OPC maturation and neuronal morphology analysis. To our knowledge, this is one of the first neuron-OPC protocols containing all human cells. Specific neuronal abnormalities not observed in mono-cultures of Tuberous Sclerosis Complex (TSC) neurons, became apparent when TSC neurons were co-cultured with TSC OPCs. These results show that this co-culture system can be used to study human neuron-OPC interactive mechanisms involved in health and disease.
A Novel Dual Lentiviral CRISPR-based Transcriptional Activation System for Gene Expression Regulation in Neurons
Robust and efficient gene expression control enables the study of a gene’s function in the central nervous system. Advances in CRISPR-based technology provide new avenues not only for gene editing, but for complex transcriptional control. Here, we describe a protocol to generate high-titer lentiviruses with neuron-optimized CRISPR-activation constructs (dual lentiviruses consisting of a gene-specific single guide RNA and the CRISPR-activator) for use in primary neurons in vitro or in the adult brain in vivo. This protocol enables modular, scalable, and multiplexable gene regulation in the nervous system and does not require a transgenic model organism.
A High-throughput and Pathophysiologically Relevant Astrocyte-motor Neuron Co-culture Assay for Amyotrophic Lateral Sclerosis Therapeutic Discovery
Amyotrophic lateral sclerosis (ALS) is an adult onset neurological disorder characterized by loss of motor neurons leading to progressive muscle wasting and eventually death. Astrocytes play a key role in disease pathogenesis. However, the ability to study astrocytic support towards motor neurons in ALS has been limited by a lack of sustainable high-throughput human cell models. Moreover, the ability to assess how astrocytic support of motor neurons is influenced by drug treatment or nutritional supplementation has been hampered by the lack of robust methodology. We have developed a high-throughput astrocyte motor neuron co-culture assay, which, by using Hb9-GFP+ motor neurons enables researchers to assess how ALS affects the ability of astrocytes to support motor neurons in 384-well plates. Moreover, astrocyte function can be manipulated by nutritional supplementation or drug treatment to identify possible therapeutic targets.
Plant Science
PEG-mediated, Stable, Nuclear and Chloroplast Transformation of Cyanidioschizon merolae
The ability to achieve nuclear or chloroplast transformation in plants has been a long standing goal, especially in microalgae research. Over past years there has been only little success, but transient and stable nuclear transformation has been achieved in multiple species. Our newly developed method allows for relatively simple transformation of Cyanidioschizon merolae in both nuclear and chloroplast genome by means of homologous recombination between the genome and a transformation vector. The use of chloramphenicol resistance gene as the selectable marker allows for plate-based efficient selection of mutant colonies. Overall, the method allows the generation of mutant strains within 6 months.