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Past Issue in 2023
Volume: 13, Issue: 21
Biological Engineering
Biosynthesis and Genetic Encoding of Non-hydrolyzable Phosphoserine into Recombinant Proteins in Escherichia coli
While site-specific translational encoding of phosphoserine (pSer) into proteins in Escherichia coli via genetic code expansion (GCE) technologies has transformed our ability to study phospho-protein structure and function, recombinant phospho-proteins can be dephosphorylated during expression/purification, and their exposure to cellular-like environments such as cell lysates results in rapid reversion back to the non-phosphorylated form. To help overcome these challenges, we developed an efficient and scalable E. coli GCE expression system enabling site-specific incorporation of a non-hydrolyzable phosphoserine (nhpSer) mimic into proteins of interest. This nhpSer mimic, with the γ-oxygen of phosphoserine replaced by a methylene (CH2) group, is impervious to hydrolysis and recapitulates phosphoserine function even when phosphomimetics aspartate and glutamate do not. Key to this expression system is the co-expression of a Streptomyces biosynthetic pathway that converts the central metabolite phosphoenolpyruvate into non-hydrolyzable phosphoserine (nhpSer) amino acid, which provides a > 40-fold improvement in expression yields compared to media supplementation by increasing bioavailability of nhpSer and enables scalability of expressions. This “PermaPhos” expression system uses the E. coli BL21(DE3) ∆serC strain and three plasmids that express (i) the protein of interest, (ii) the GCE machinery for translational installation of nhpSer at UAG amber stop codons, and (iii) the Streptomyces nhpSer biosynthetic pathway. Successful expression requires efficient transformation of all three plasmids simultaneously into the expression host, and IPTG is used to induce expression of all components. Permanently phosphorylated proteins made in E. coli are particularly useful for discovering phosphorylation-dependent protein–protein interaction networks from cell lysates or transfected cells.Key features• Protocol builds on the nhpSer GCE system by Rogerson et al. (2015), but with a > 40-fold improvement in yields enabled by the nhpSer biosynthetic pathway.• Protein expression uses standard Terrific Broth (TB) media and requires three days to complete.• C-terminal purification tags on target protein are recommended to avoid co-purification of prematurely truncated protein with full-length nhpSer-containing protein.• Phos-tag gel electrophoresis provides a convenient method to confirm accurate nhpSer encoding, as it can distinguish between non-phosphorylated, pSer- and nhpSer-containing variants.Graphical overview
Cancer Biology
Studying Cell Migration (Random and Wound Healing) Parameters with Imaging and MATLAB Analysis
Cell migration is an essential biological process for organisms, in processes including embryonic development, immune response, and cancer metastasis. To elucidate the regulatory machinery of this vital process, methods that mimic in vivo migration, including in vitro wound healing assay and random migration assay, are widely used for cell behavior investigation. However, several concerns are raised with traditional cell migration experiment analysis. First, a manually scratched wound often presents irregular edges, causing the speed analysis difficult. Second, only the migration speed of leading cells is considered in the wound healing assay. Here, we provide a reliable analysis method to trace each cell in the time-lapse images, eliminating the concern about wound shape and creating a more comprehensive understanding of cell migration—not only of collective migration speed but also single-cell directionality and coordination between cells.
Cell Biology
Preparation of Whole-mount Mouse Islets on Vascular Extracellular Matrix for Live Islet Cell Microscopy
Pancreatic islet β cells preferentially secrete insulin toward the plasma membrane, making contact with the capillary extracellular matrix (ECM). Isolated islets separated from the exocrine acinar cells are the best system for cell biology studies of primary β cells, whereas isolated islets lose their capillary network during ex vivo culture. Providing the appropriate extracellular signaling by attaching islets to vascular ECM-coated surfaces can restore the polarized insulin secretion toward the ECM. The guided secretion toward ECM-coated glass coverslips provides a good model for recording insulin secretion in real time to study its regulation. Additionally, β cells attached to the ECM-coated coverslips are suitable for confocal live imaging of subcellular components including adhesion molecules, cytoskeleton, and ion channels. This procedure is also compatible for total internal reflection fluorescence (TIRF) microscopy, which provides optimal signal-to-noise ratio and high spatial precision of structures close to the plasma membrane. In this article, we describe the optimized protocol for vascular ECM-coating of glass coverslips and the process of attachment of isolated mouse islets on the coverslip. This preparation is compatible with any high-resolution microscopy of live primary β cells.Key features• Optimized coating procedure to attach isolated islets, compatible for both confocal and TIRF microscopy.• The ECM-coated glass coverslip functions as the artificial capillary surface to guide secretion toward the coated surface for optimal imaging of secretion events.• Shows the process of islets attachment to the ECM-coated surface in a 6-day ex vivo culture.Graphical overview
Identification of Acetylation Sites of Fatty Acid Synthase (FASN) by Mass Spectrometry and FASN Activity Assay
Lysine acetylation is a conserved post-translational modification and a key regulatory mechanism for various cellular processes, including metabolic control, epigenetic regulation, and cellular signaling transduction. Recent advances in mass spectrometry (MS) enable the extensive identification of acetylated lysine residues of histone and non-histone proteins. However, protein enrichment before MS analysis may be necessary to improve the detection of low-abundant proteins or proteins that exhibit low acetylation levels. Fatty acid synthase (FASN), an essential enzyme catalyzing the de novo synthesis of fatty acids, has been found to be acetylated in various species, from fruit flies to humans. Here, we describe a step-by-step process of antibody-based protein enrichment and sample preparation for acetylation identification of endogenous FASN protein by MS-based proteomics analysis. Meanwhile, we provide a protocol for nicotinamide adenine dinucleotide phosphate (NADPH) absorbance assay for FASN activity measurement, which is one of the primary functional readouts of de novo lipogenesis.Key features• A comprehensive protocol for protein immunoprecipitation and sample preparation for acetylation site identification by mass spectrometry.• Step-by-step procedures for measurement of FASN activity of fruit fly larvae using an absorbance assay.Graphical overview
Drug Discovery
Studying Cellular Focal Adhesion Parameters with Imaging and MATLAB Analysis
Cell signaling is highly integrated for the process of various cell activities. Although previous studies have shown how individual genes contribute to cell migration, it remains unclear how the integration of these signaling pathways is involved in the modulation of cell migration. In our two-hit migration screen, we revealed that serine-threonine kinase 40 (STK40) and mitogen-activated protein kinase (MAPK) worked synergistically, and the suppression of both genes could further lead to suppression in cell migration. Furthermore, based on our analysis of cellular focal adhesion (FA) parameters using MATLAB analysis, we are able to find out the synergistic reduction of STK40 and MAPK that further abolished the increased FA by shSTK40. While FA identification in previous studies includes image analysis using manual selection, our protocol provides a semi-automatic manual selection of FAs using MATLAB. Here, we provide a method that can shorten the amount of time required for manual identification of FAs and increase the precision for discerning individual FAs for various analyses, such as FA numbers, area, and mean signals.
Immunology
Medullary Thymic Epithelial Cell Antigen-presentation Assays
Medullary thymic epithelial cells (mTEC) are bona fide antigen-presenting cells that play a crucial role in the induction of T-cell tolerance. By their unique ability to express a broad range of tissue-restricted self-antigens, mTEC control the clonal deletion (also known as negative selection) of potentially hazardous autoreactive T cells and the generation of Foxp3+ regulatory T cells. Here, we describe a protocol to assess major histocompatibility complex (MHC) class II antigen-presentation capacity of mTEC to CD4+ T cells. We detail the different steps of thymus enzymatic digestion, immunostaining, cell sorting of mTEC and CD4+ T cells, peptide-loading of mTEC, and the co-culture between these two cell types. Finally, we describe the flow cytometry protocol and the subsequent analysis to assess the activation of CD4+ T cells. This rapid co-culture assay enables the evaluation of the ability of mTEC to present antigens to CD4+ T cells in an antigen-specific context.Key features• This protocol builds upon the method used by Lopes et al. (2018 and 2022) and Charaix et al. (2022).• This protocol requires transgenic mice, such as OTIIxRag2-/- mice and the cognate peptide OVA323–339, to assess mTEC antigen presentation to CD4+ T cells.• This requires specific equipment such as a Miltenyi Biotec AutoMACS® Pro Separator, a BD FACSAriaTM III cell sorter, and a BD® LSR II flow cytometer.Graphical overview
Microbiology
Analysis of Plasmodium falciparum Mitochondrial Electron Transport Chain Activity Using Seahorse XFe96 Extracellular Flux Assays
The mitochondrial electron transport chain (ETC) is a multi-component pathway that mediates the transfer of electrons from metabolic reactions that occur in the mitochondrion to molecular oxygen (O2). The ETC contributes to numerous cellular processes, including the generation of cellular ATP through oxidative phosphorylation, serving as an electron sink for metabolic pathways such as de novo pyrimidine biosynthesis and for maintaining mitochondrial membrane potential. Proper functioning of the mitochondrial ETC is necessary for the growth and survival of apicomplexan parasites including Plasmodium falciparum, a causative agent of malaria. The mitochondrial ETC of P. falciparum is an attractive target for antimalarial drugs, due to its essentiality and its differences from the mammalian ETC. To identify novel P. falciparum ETC inhibitors, we have established a real-time assay to assess ETC function, which we describe here. This approach measures the O2 consumption rate (OCR) of permeabilized P. falciparum parasites using a Seahorse XFe96 flux analyzer and can be used to screen compound libraries for the identification of ETC inhibitors and, in part, to determine the targets of those inhibitors.Key features• With this protocol, the effects of candidate inhibitors on mitochondrial O2 consumption in permeabilized asexual P. falciparum parasites can be tested in real time.• Through the sequential injection of inhibitors and substrates into the assay, the molecular targets of candidate inhibitors in the ETC can, in part, be determined.• The assay is applicable for both drug discovery approaches and enquiries into a fundamental aspect of parasite mitochondrial biology.Graphical overviewSeahorse assay experimental workflow. Prior to the assay, coat the cell culture microplate with Cell-Tak to help adhere the parasites to the wells; hydrate the cartridge wells to ensure proper sensor functionality and design the assay template using the Agilent Seahorse Wave Desktop software (Analyze Seahorse data files, Seahorse Wave desktop software|Agilent). On the day of the assay, prepare the inhibitors/substrates that are to be injected into the ports. Then, separate 3 × 108 trophozoite-stage parasites from the uninfected red blood cells (RBCs) and ring-stage parasites using a MACS® magnetic column. Check the purity of the parasites with Giemsa-stained smears. Determine the concentration of infected RBCs in the sample using a hemocytometer and dilute to approximately 5 × 107 parasites per milliliter. Treat infected RBCs with saponin to permeabilize the host cell membrane and seed approximately 5 × 106 parasites (100 μL) per well in mitochondria assay solution (MAS) buffer. Supplement MAS buffer with digitonin to permeabilize the parasite plasma membrane. Load the ports with the prepared inhibitors/substrates and run the assay using a Seahorse XFe96 analyzer. Once the assay is completed, analyze the data using the Wave desktop software. Further data processing can be done using statistical analysis software.
Detailed Protocol to Perform Direct PCR Using Filamentous Fungal Biomass—Tips and Considerations
The precise and rapid detection of fungi is important in various fields, including clinics, industry, and agriculture. While sequencing universal DNA barcodes remains the standard method for species identification and phylogenetic analysis, a significant bottleneck has been the labor-intensive and time-consuming sample preparation for genomic DNA extraction. To address this, we developed a direct PCR method that bypasses the DNA extraction steps, facilitating efficient target DNA amplification. Instead of extracting genomic DNA from fungal mycelium, our method involves adding a small quantity of mycelium directly to the PCR mixture, followed by a heat shock and vortexing. We found these simple adjustments to be sufficient to lyse many filamentous fungal cells, enabling target DNA amplification. This paper presents a comprehensive protocol for executing direct PCR in filamentous fungi. Beyond species identification, this direct PCR approach holds promise for diverse applications, such as diagnostic PCR for genotype screening without fungal DNA extraction. We anticipate that direct PCR will expedite research on filamentous fungi and diagnosis of fungal diseases.Key features• Eliminates the time-consuming genomic DNA extraction step for PCR, enhancing the speed of molecular identification.• Adds a small quantity of mycelium directly into the PCR mix.• Emphasizes the crucial role of heat shock and vortexing in achieving efficient target DNA amplification.• Accelerates the molecular identification of filamentous fungi and rapid diagnosis of fungal diseases.Graphical overview Direct PCR using filamentous fungal biomass
A Guideline for Assessment and Characterization of Bacterial Biofilm Formation in the Presence of Inhibitory Compounds
Campylobacter jejuni, a zoonotic foodborne pathogen, is the worldwide leading cause of acute human bacterial gastroenteritis. Biofilms are a significant reservoir for survival and transmission of this pathogen, contributing to its overall antimicrobial resistance. Natural compounds such as essential oils, phytochemicals, polyphenolic extracts, and D-amino acids have been shown to have the potential to control biofilms formed by bacteria, including Campylobacter spp. This work presents a proposed guideline for assessing and characterizing bacterial biofilm formation in the presence of naturally occurring inhibitory molecules using C. jejuni as a model. The following protocols describe: i) biofilm formation inhibition assay, designed to assess the ability of naturally occurring molecules to inhibit the formation of biofilms; ii) biofilm dispersal assay, to assess the ability of naturally occurring inhibitory molecules to eradicate established biofilms; iii) confocal laser scanning microscopy (CLSM), to evaluate bacterial viability in biofilms after treatment with naturally occurring inhibitory molecules and to study the structured appearance (or architecture) of biofilm before and after treatment.
Spot Assay and Colony Forming Unit (CFU) Analyses–based sensitivity test for Candida albicans and Saccharomyces cerevisiae
Cellular sensitivity is an approach to inhibit the growth of certain cells in response to any non-permissible conditions, as the presence of a cytotoxic agent or due to changes in growth parameters such as temperature, salt, or media components. Sensitivity tests are easy and informative assays to get insight into essential gene functions in various cellular processes. For example, cells having any functionally defective genes involved in DNA replication exhibit sensitivity to non-permissive temperatures and to chemical agents that block DNA replication fork movement. Here, we describe a sensitivity test for multiple strains of Saccharomyces cerevisiae and Candida albicans of diverged genetic backgrounds subjected to several genotoxic chemicals simultaneously. We demonstrate it by testing the sensitivity of DNA polymerase defective yeast mutants by using spot analysis combined with colony forming unit (CFU) efficiency estimation. The method is very simple and inexpensive, does not require any sophisticated equipment, can be completed in 2–3 days, and provides both qualitative and quantitative data. We also recommend the use of this reliable methodology for assaying the sensitivity of these and other fungal species to antifungal drugs and xenobiotic factors.
Molecular Biology
Purification of Long Non-coding RNAs on Replication Forks Using iROND (Isolate RNAs on Nascent DNA)
Fork stability is key to genome DNA duplication and genetic integrity. Long non-coding RNAs (LncRNAs) may play vital roles in fork stabilization and chromatin remodeling. Existing techniques such as NCC-RNA sequencing are useful to identify LncRNAs on nascent chromatin DNA. However, there is still a lack of methods for LncRNAs purification directly from replicative forks, hindering a deep understanding of the functions of LncRNAs in fork regulation. Here, we provide a step-by-step protocol named iROND (isolate RNAs on nascent DNA). iROND was developed and modified from iPOND, a well-known method for purifying fork-associated proteins. iROND relies on click chemistry reaction of 5'-ethynyl-2'-deoxyuridine (EdU)-labeled forks and biotin. After streptavidin pull down, fork-associated LncRNAs and proteins are purified simultaneously. iROND is compatible with downstream RNA sequencing, qPCR confirmation, and immunoblotting. Integrated with functional methods such as RNA fluorescent in situ hybridization (RNA FISH) and DNA fiber assay, it is feasible to screen fork-binding LncRNAs in defined cell lines and explore their functions. In summary, we provide a purification pipeline of fork-associated LncRNAs. iROND is also useful for studying other types of fork-associated non-coding RNAs.Key features• Purify long non-coding RNAs (LncRNAs) directly from replication forks.• Connects to RNA sequencing for screening easily.• Allows testing various genotoxic stress responses.• Provides LncRNA candidate list for downstream functional research.Graphical overviewSchematic overview of isolate RNAs on nascent DNA (iROND) protocol. Cells were pulse-labeled with 5'-ethynyl-2'-deoxyuridine (EdU) for 10 min before paraformaldehyde fixation. EdU-positive forks were ligated with biotin through Click-IT chemistry reaction. Genomic DNA was ultrasonically cracked and crosslinked with streptavidin for pulling down. Both RNA and protein components were purified. RNA components were used for downstream RNA sequencing and qPCR validation. Protein components were used for immunoblotting to evaluate binding dynamics of fork-associated proteins such as helicase, topoisomerase, and DNA polymerases.
Neuroscience
Generation of Human Blood Vessel and Vascularized Cerebral Organoids
Brain organoids have been widely used to study diseases and the development of the nervous system. Many reports have investigated the application of brain organoids, but most of these models lack vascular structures, which play essential roles in brain development and neurological diseases. The brain and blood vessels originate from two different germ layers, making it difficult to induce vascularized brain organoids in vitro. We developed this protocol to generate brain-specific blood vessel and cerebral organoids and then fused them at a specific developmental time point. The fused cerebral organoids exhibited robust vascular network-like structures, which allows simulating the in vivo developmental processes of the brain for further applications in various neurological diseases.Key Features• Culturing vascularized brain organoids using human embryonic stem cells (hESCs).• The new approach generates not only neural cells and vessel-like networks but also brain-resident microglia immune cells in a single organoid.Graphical overviewWorkflow and timeline for vessel organoid and vascularized brain organoid generation. (By Figdraw, ID: RTIURffccf)
A New Behavioral Paradigm for Visual Classical Conditioning in Drosophila
Visual learning in animals is a remarkable cognitive ability that plays a crucial role in their survival and adaptation. Therefore, the ability to learn is highly conserved among animals. Despite lacking a centralized nervous system like vertebrates, invertebrates have demonstrated remarkable learning abilities. Here, we describe a simple behavioral assay that allows the analysis of visual associative learning in individually traceable freely walking adult fruit flies. The setup is based on the simple and widely used behavioral assay to study orientation behavior in flies. A single wing-clipped fly that has been starved for 21 h is placed on a platform where two unreachable opposite visual sets are displayed. This visual learning protocol was initially developed to study the cognitive ability of fruit flies to process numerical information. Through the application of the protocol, flies are able to associate a specific visual set with an appetitive reward. This association is revealed 2 h later during the testing session where we observed a change in their preference upon learning (i.e., change in their spontaneous preference). Moreover, this protocol could potentially be used to associate any other visual object/property to the reward, expanding the opportunities of studying visual learning in freely walking fruit flies at individual level.Graphical overviewGraphical overview of conditional learning protocol. Forty-eight hours before conditioning, the wings of the flies are clipped, and individual flies are left to recover in a fresh food vial. Twenty-one hours before the conditional learning starts, flies are transferred to a starvation vial containing wet paper. The training session consists in placing a drop of sugar next to the place with the lower number of objects (numerosity) and a drop of water next to the larger numerosity. The fly is placed in the arena and left to freely walk for three minutes. Once the session is finished, the fly is placed back in their empty vial for 2 h until the testing session starts.
Measuring Action Potential Propagation Velocity in Murine Cortical Axons
Measuring the action potential (AP) propagation velocity in axons is critical for understanding neuronal computation. This protocol describes the measurement of propagation velocity using a combination of somatic whole cell and axonal loose patch recordings in brain slice preparations. The axons of neurons filled with fluorescent dye via somatic whole-cell pipette can be targeted under direct optical control using the fluorophore-filled pipette. The propagation delays between the soma and 5–7 axonal locations can be obtained by analyzing the ensemble averages of 500–600 sweeps of somatic APs aligned at times of maximal rate-of-rise (dV/dtmax) and axonal action currents from these locations. By plotting the propagation delays against the distance, the location of the AP initiation zone becomes evident as the site exhibiting the greatest delay relative to the soma. Performing linear fitting of the delays obtained from sites both proximal and distal from the trigger zone allows the determination of the velocities of AP backward and forward propagation, respectively.Key features• Ultra-thin axons in cortical slices are targeted under direct optical control using the SBFI-filled pipette.• Dual somatic whole cell and axonal loose patch recordings from 5–7 axonal locations.• Ensemble averaging of 500–600 sweeps of somatic APs and axonal action currents.• Plotting the propagation delays against the distance enables the determination of the trigger zone's position and velocities of AP backward and forward propagation.
Stem Cell
Differentiation of Human Induced Pluripotent Stem Cells (iPSCs)–derived Mesenchymal Progenitors into Chondrocytes
Induced pluripotent stem cells (iPSCs) generated from human sources are valuable tools for studying skeletal development and diseases, as well as for potential use in regenerative medicine for skeletal tissues such as articular cartilage. To successfully differentiate human iPSCs into functional chondrocytes, it is essential to establish efficient and reproducible strategies that closely mimic the physiological chondrogenic differentiation process. Here, we describe a simple and efficient protocol for differentiation of human iPSCs into chondrocytes via generation of an intermediate population of mesenchymal progenitors. These methodologies include step-by-step procedures for mesenchymal derivation, induction of chondrogenic differentiation, and evaluation of the chondrogenic marker gene expression. In this protocol, we describe the detailed procedure for successful derivation of mesenchymal progenitor population from human iPSCs, which are then differentiated into chondrocytes using high-density culture conditions by stimulating with bone morphogenetic protein-2 (BMP-2) or transforming growth factor beta-3 (TGFβ-3). The differentiated iPSCs exhibit temporal expression of cartilage genes and accumulation of a cartilaginous extracellular matrix in vitro, indicating successful chondrogenic differentiation. These detailed methodologies help effective differentiation of human iPSCs into the chondrogenic lineage to obtain functional chondrocytes, which hold great promise for modeling skeletal development and disease, as well as for potential use in regenerative medicine for cell-based therapy for cartilage regeneration.Key features• Differentiation of human iPSCs into chondrocytes using 3D culture methods.• Uses mesenchymal progenitors as an intermediate for differentiation into chondrocytes.
Systems Biology
Workflow for High-throughput Screening of Enzyme Mutant Libraries Using Matrix-assisted Laser Desorption/Ionization Mass Spectrometry Analysis of Escherichia coli Colonies
High-throughput molecular screening of microbial colonies and DNA libraries are critical procedures that enable applications such as directed evolution, functional genomics, microbial identification, and creation of engineered microbial strains to produce high-value molecules. A promising chemical screening approach is the measurement of products directly from microbial colonies via optically guided matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Measuring the compounds from microbial colonies bypasses liquid culture with a screen that takes approximately 5 s per sample. We describe a protocol combining a dedicated informatics pipeline and sample preparation method that can prepare up to 3,000 colonies in under 3 h. The screening protocol starts from colonies grown on Petri dishes and then transferred onto MALDI plates via imprinting. The target plate with the colonies is imaged by a flatbed scanner and the colonies are located via custom software. The target plate is coated with MALDI matrix, MALDI-MS analyzes the colony locations, and data analysis enables the determination of colonies with the desired biochemical properties. This workflow screens thousands of colonies per day without requiring additional automation. The wide chemical coverage and the high sensitivity of MALDI-MS enable diverse screening projects such as modifying enzymes and functional genomics surveys of gene activation/inhibition libraries.Key features• Mass spectrometry analyzes a range of compounds from E. coli colonies as a proxy for liquid culture testing enzyme mutant libraries.• Colonies are transferred to a MALDI target plate by a simple imprinting method.• The screen compares the ratio among several products or searches for the qualitative presence of specific compounds.• The protocol requires a MALDI mass spectrometer.Graphical overviewOverview of the MALDI-MS analysis of microbial colonies for screening mutant libraries. Microbial cells containing a mutant library for enzymes/metabolic pathways are first grown in agar. The colonies are then imprinted onto a MALDI target plate using a filter paper intermediate. An optical image of the MALDI target plate is analyzed by custom software to find the locations of individual colonies and direct subsequent MALDI-MS analyses to the selected colonies. After applying MALDI matrix onto the target plate, MALDI-MS analysis of the colonies is performed. Colonies showing the desired product profiles are found by data analysis via the software, and the colonies are picked for downstream analysis.