Methods and Protocols — Open Access Journal

Cell-free protein synthesis (CFPS) is a platform technology that provides new opportunities for protein expression, metabolic engineering, therapeutic development, education, and more. The advantages of CFPS over in vivo protein expression include its open system, the elimination of reliance on living cells, and the ability to focus all system energy on production of the protein of interest. Over the last 60 years, the CFPS platform has grown and diversified greatly, and it continues to evolve today. Both new applications and new types of extracts based on a variety of organisms are current areas of development. However, new users interested in CFPS may find it challenging to implement a cell-free platform in their laboratory due to the technical and functional considerations involved in choosing and executing a platform that best suits their needs. Here we hope to reduce this barrier to implementing CFPS by clarifying the similarities and differences amongst cell-free platforms, highlighting the various applications that have been accomplished in each of them, and detailing the main methodological and instrumental requirement for their preparation. Additionally, this review will help to contextualize the landscape of work that has been done using CFPS and showcase the diversity of applications that it enables. Full article

Introduction: The prevalence and incidence of sport-related concussion have continued to increase over the past decade, and researchers from various backgrounds strive for evidenced-based clinical assessment and management. When diagnosing and managing a concussion, a battery of tests from several domains (e.g., symptom reporting, neurocognitive, physiology) must be used. In this study, we propose and develop an objective, evidence-based protocol to assess the pathophysiology of the brain by using non-invasive methods. Methods: Contact sport athletes (n = 300) will be assessed at the beginning of the season in a healthy state to establish baseline values, and then prospectively followed if a mild traumatic brain injury (mTBI) occurs on approximately days 1–2, 3–5, 7–10, 21, 30, and subsequently thereafter, depending on the severity of injury. The protocol includes spontaneous measurements at rest, during head postural change, controlled breathing maneuvers for cerebrovascular reactivity, a neurovascular coupling stimuli, and a baroreflex/autoregulation maneuver. Physiological data collection will include cerebral blood flow velocity, cerebral oxygenation, respiratory gases for end-tidal oxygen and carbon dioxide, finger photoplethysmography for blood pressure, seismocardiography for cardiac mechanics, and electrocardiography. Conclusion, Limitations, and Ethics: The protocol will provide an objective, physiological evidence-based approach in an attempt to better diagnose concussion to aid in return-to-play or -learn. Ethics approval has been granted by the University Research Ethics Board. Full article

Reverse genetic systems for RNA viruses are the platforms to introduce mutations into the RNA genomes and thus have helped understand their life cycle and harness them for human purposes to develop vaccines and delivery systems. These systems are based on the complementary DNA (cDNA) of the RNA viruses, whose transcripts derived from bacterial RNA polymerases act not only as the primary mRNA for phage protein synthesis, but also as the template for phage RNA replicases (aka. RNA-dependent RNA polymerases). Here, we present a protocol optimized for the small RNA phages of Leviviridae (i.e., leviphages) infecting Pseudomonas aeruginosa. This protocol includes three fundamental steps: (i) Creation of a promoter-fused cDNA, (ii) generation of a clone into mini-Tn7-based vector, and (iii) introduction of the clone into non-susceptible hosts. As the representative example, we describe the reverse genetic system for PP7, which infects a set of P. aeruginosa strains such as PAO1. The cDNA was fused to the T7 promoter, which was cloned in mini-Tn7-Gm. This construct was introduced into P. aeruginosa PAK and E. coli HB101. Functional assembly of PP7 phages from the culture supernatants were assessed by plaque formation on PAO1 and the phage particles were observed under transmission microscope. We found that the host cells should be cultured at 30 °C for the maximal phage production (~10¹² pfu/mL). The reverse genetic systems will provide a new insight into the life cycle of the RNA phages and help develop engineered variants with new traits for phage applications regarding selective diagnosis and efficient therapy. Full article

Uganda has persistently had high adolescent pregnancy prevalence; 25% for the last 10 years. This protocol presents the design of a Cluster Randomized Controlled Trial (CRCT) to investigate the effectiveness of School-Based Sexual and Reproductive Health (SBSRH) interventions on prevention of pregnancy among school girls aged 15–19 years in the Hoima District, Uganda. 18 secondary schools (clusters) will be selected using cluster sampling and allocated 1:1 into control or intervention group stratified by geographical location. 1080 (60 each cluster) participants/girls aged 15–19 years will be selected using simple random sampling. The intervention group will receive tailored SRH information, in-school medical care and referral over 12 months. The control group will receive no intervention from the research team; however, they can access alternative services elsewhere if they wish. Data will be obtained at baseline, 6 months and 12 months. The outcomes are reduction in occurrence of pregnancy, utilization of SRH services and sexual behavioral change. To our knowledge, this is the first CRCT providing combined SRH interventions for prevention of adolescent pregnancy in Uganda. If effective, it could have great potential in preventing adolescent pregnancy. Trial Registration: Pan African Clinical Trial Registry (PACTR201810882140200) Registered on 16 October 2018. Full article

A major drawback of nanocomposite scaffolds in bone tissue engineering is dimensional shrinkage after the fabrication process. Shrinkage yields gaps between the scaffold and host bone in the defect site and eventually causes failure in osteointegration by micromovement. The present study was conducted using titanium (Ti) mesh and Gelfoam^® to prevent radial and axial micromovement, respectively. A critical-sized defect (CSD) was created in the center of the calvarium of Sprague Dawley rats to implant porous polydopamine-laced hydroxyapatite collagen calcium silicate (HCCS-PDA), a novel nanocomposite scaffold. Gelfoam^® was applied around the edge of the defect, and then the HCCS-PDA scaffold was inserted in the defect area. Ti mesh was placed between the periosteum and skin right, above the inserted scaffold site. There were two test groups, with a fixture (Gelfoam^® and Ti mesh) and without a fixture, each group contained five animals. The rats were sacrificed after three months post-operation. The explanted calvaria underwent micro-CT scanning and a push-out test to quantify osteointegration and mechanical strength between the scaffold and host bone. Histological analysis of undecalcified bone was performed by grinding resin infiltrated calvaria blocks to prepare 10 μm slices. Osteointegration was higher in the group with fixation than without fixation. Movement of the HCCS-PDA scaffold in the gap resulted in diminished osteointegration. With fixation, the movement was inhibited and osteointegration became prominent. Here we present a successful method of preventing axial and radial movement of scaffolds using Gelfoam^® and Ti mesh. Applying this fixture, we expect that an HCCS-PDA scaffold can repair CSD more effectively. Full article

Peripheral nerve stimulation has emerged as a platform therapy to treat a wide range of disorders. Continued development and translation of these strategies requires that researchers have access to reliable, customizable electrodes for nerve stimulation. Here, we detail procedures to build three different configurations of cuff electrodes with varying numbers and orientations of contacts for nerve stimulation in rats. These designs are built with simple, widely available materials, using platinum–iridium electrodes assembled into polyurethane tubing. Moreover, the designs can easily be customized to increase versatility and individualize for specific stimulation applications. This protocol provides a resource to facilitate the construction and customization of stimulation cuffs to support preclinical nerve stimulation research. Full article

Lytic Campylobacter phages, which can be used to combat this pathogen in animals and on food products, have been studied for more than 30 years. Though, due to some peculiarities of the phages, which hampered their isolation and particularly their molecular analysis for a long time, progress in this research field was rather slow. Meanwhile, the situation has changed and much more is known about the biology and genetics of those phages. In this article, we address specific issues that should be considered when Campylobacter phages are studied, starting with the isolation and propagation of the phages and ending with a thorough characterization including whole-genome sequencing. The basis for advice and recommendations given here is a careful review of the scientific literature and experiences that we have had ourselves with Campylobacter phages. Full article

Phage display is a powerful technique for drug discovery in biomedical research in particular for antibody libraries. But, several technical challenges are associated with the selection process. For instance, during the panning step, the successful elution of the phages bound to the antigen is critical in order to avoid losing the most promising binders. Here, we present an efficient protocol to establish, screen and select synthetic libraries of domain antibodies using phage display. We do not only present suitable solutions to the above-mentioned challenges to improve elution by 50-fold, but we also present a step by step in-depth protocol with miniaturized volumes and optimized procedures to save material, costs and time for a successful phage display with domain antibodies. Hence, this protocol improves the selection process for an efficient handling process. The here presented library is based on the variable domain (vNAR) of the naturally occurring novel antibody receptor (IgNAR) from cartilage fishes. Diversity was introduced in the Complementarity-Determining Region 3 (CDR3) of the antigen-binding site with different composition and length. Full article

Unnatural proteins are crucial biomacromolecules and have been widely applied in fundamental science, novel biopolymer materials, enzymes, and therapeutics. Cell-free protein synthesis (CFPS) system can serve as a robust platform to synthesize unnatural proteins by highly effective site-specific incorporation of unnatural amino acids (UNAAs), without the limitations of cell membrane permeability and the toxicity of unnatural components. Here, we describe a quick and simple method to synthesize unnatural proteins in CFPS system based on Escherichia coli crude extract, with unnatural orthogonal aminoacyl-tRNA synthetase and suppressor tRNA evolved from Methanocaldococcus jannaschii. The superfolder green fluorescent protein (sfGFP) and p-propargyloxyphenylalanine (pPaF) were used as the model protein and UNAA. The synthesis of unnatural sfGFPs was characterized by microplate spectrophotometer, affinity chromatography, and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). This protocol provides a detailed procedure guiding how to use the powerful CFPS system to synthesize unnatural proteins on demand. Full article

The application of high-throughput nucleic acid and protein sequencing technologies is transforming our understanding of plant microbiomes and their interactions with their hosts in health and disease. However, progress in studying host-microbiome interactions in above-ground compartments of the tree (the phyllosphere) has been hampered due to high concentrations of phenolic compounds, lignin, and other compounds in tree bark that severely limit the success of DNA, RNA, and protein extraction. Here we present modified sample-preparation and kit-based protocols for the extraction of host and microbiome DNA and RNA from oak (Quercus robus and Quercus petraea) bark tissue for subsequent high-throughput sequencing. In addition, reducing the quantity of bark tissue used for an established protein extraction protocol yielded high quality protein for parallel analysis of the oak-microbiota metaproteome. These procedures demonstrate the successful extraction of nucleic acids and proteins from oak tissue using as little as 50 mg of sample input, producing sufficient quantities for nucleic acid sequencing and protein mass spectrometry of tree stem tissues and their associated microbiota. Full article

Air puff systems are at once trivially straightforward and dauntingly complex. On the one hand, they are little but a pressure source, valve, and tube connected together. On the other, the air passing through them is a compressible medium, expanding approximately adiabatically while travelling at high velocity through a compliant tube, and exiting as a turbulent jet with velocity peak and profile varying non-linearly in its near-field. This complexity puts precise mathematical prediction of puff properties out of reach of most labs. There are, however, a number of phenomena fundamental to air puff system design that are worth understanding to a first order of approximation, or at least qualitatively. Using a simplified, “electronic–hydraulic analogy” model, this paper discusses these phenomena in just enough depth for the reader to confidently specify parts for an air puff delivery system, to measure its key parameters, and/or to describe a given system unambiguously in publications, thus maximizing reproducibility. Full article

Hydrogen sulfide (H₂S) is the most recently established gaseous vasodilator, enzymatically produced from cysteine metabolism, involved in a number of pathophysiological processes. However, its accurate detection in vivo is critical due to its volatility and tendency to form sulfane sulfur derivatives, thus limiting the data interpretation of its biological roles. We developed new applications of the simple and rapid method to measure H₂S release in cell culture systems, based on the lead acetate strip test. This test, previously prevalently used in microbiology, was compared with the agar trap method, applied, in parallel, on both cell cultures and cell-free samples. Sulfane sulfur represents the major species derived from intracellular H₂S. Various fluorescent probes are available for quantitation of H₂S derivatives intracellularly. We present here an alternative to the classic imaging method for sulfane sulfur evaluation, running on a flow cytometer, based on SSP4 probe labeling. Flow cytometry turned out to be more direct, fully quantitative and less time-consuming compared to microscopy and more precise with respect to the fluorescence multi-plate reader assay. The new application methods for H₂S determination appear to be fully suitable for the analysis of H₂S release and sulfane sulfur content in biological samples. Full article

Single-particle tracking (SPT) has been used and developed over the last 25 years as a method to investigate molecular dynamics, structure, interactions, and function in the cellular context. SPT is able to show how fast and how far individual molecules move, identify different dynamic populations, measure the duration and strength of intermolecular interactions, and map out structures on the nanoscale in cells. In combination with other techniques such as macromolecular crystallography and molecular dynamics simulation, it allows us to build models of complex structures, and develop and test hypotheses of how these complexes perform their biological roles in health as well as in disease states. Here, we use the example of the epidermal growth factor receptor (EGFR), which has been studied extensively by SPT, demonstrating how the method has been used to increase our understanding of the receptor’s organization and function, including its interaction with the plasma membrane, its activation, clustering, and oligomerization, and the role of other receptors and endocytosis. The examples shown demonstrate how SPT might be employed in the investigation of other biomolecules and systems. Full article

Recent improvements in optical tools that can perturb brain activity and simultaneously reveal the elicited alterations in the associated regions offer an exceptional means to understand and map the connectivity of the brain. In this work, we exploit a combination of recently developed optical tools to monitor neural population at the meso-scale level and to mould the cortical patterns of targeted neuronal population. Our goal was to investigate the propagation of neuronal activity over the mouse cortex that is triggered by optogenetic stimulation in the contralateral hemisphere. Towards this aim, we developed a wide-field fluorescence microscope that is characterized by a double illumination path allowing for the optogenetic stimulation of the transfected area in the left hemisphere and the simultaneous recording of cortical activity in the right hemisphere. The microscope was further implemented with a custom shutter in order to split the LED illumination path, resulting in a half-obscured field of view. By avoiding the spectral crosstalk between GCaMP6f and channelrhodopsin 2 (ChR2), this system offered the possibility of simultaneous “pumping and probing” of inter-hemispheric functional connectivity on Thy1-GCaMP6f mice. Full article

Preparing pioloform/formvar support films on transmission electron microscopy (TEM) grids is a routine laboratory procedure in practically all electron microscopy units. In current practice, these grids are manually placed on the support film one by one using special tweezers, a process requiring a steady hand. The work is often ergonomically awkward to continue for a longer period of time. In this article, we describe a low-cost, computer vision-guided robot arm that automatically places the grids on the film. The success rate of the prototype robot is 90%, which is comparable to an experienced laboratory technician. Full article

The identification of nearly all proteins in a biological system using data-dependent acquisition (DDA) tandem mass spectrometry has become routine for organisms with relatively small genomes such as bacteria and yeast. Still, the quantification of the identified proteins may be a complex process and often requires multiple different software packages. In this protocol, I describe a flexible strategy for the identification and label-free quantification of proteins from bottom-up proteomics experiments. This method can be used to quantify all the detectable proteins in any DDA dataset collected with high-resolution precursor scans and may be used to quantify proteome remodeling in response to drug treatment or a gene knockout. Notably, the method is statistically rigorous, uses the latest and fastest freely-available software, and the entire protocol can be completed in a few hours with a small number of data files from the analysis of yeast. Full article

Optogenetics is an emerging method that uses light to manipulate electrical activity in excitable cells exploiting the interaction between light and light-sensitive depolarizing ion channels, such as channelrhodopsin-2 (ChR2). Initially used in the neuroscience, it has been adopted in cardiac research where the expression of ChR2 in cardiac preparations allows optical pacing, resynchronization and defibrillation. Recently, optogenetics has been leveraged to manipulate cardiac electrical activity in the intact heart in real-time. This new approach was applied to simulate a re-entrant circuit across the ventricle. In this technical note, we describe the development and the implementation of a new software package for real-time optogenetic intervention. The package consists of a single LabVIEW program that simultaneously captures images at very high frame rates and delivers precisely timed optogenetic stimuli based on the content of the images. The software implementation guarantees closed-loop optical manipulation at high temporal resolution by processing the raw data in workstation memory. We demonstrate that this strategy allows the simulation of a ventricular tachycardia with high stability and with a negligible loss of data with a temporal resolution of up to 1 ms. Full article

Ligand binding to receptors is one of the most important regulatory elements in biology as it is the initiating step in signaling pathways and cascades. Thus, precisely localizing binding sites and measuring interaction forces between cognate receptor–ligand pairs leads to new insights into the molecular recognition involved in these processes. Here we present a detailed protocol about applying a technique, which combines atomic force microscopy (AFM)-based recognition imaging and force spectroscopy for studying the interaction between (membrane) receptors and ligands on the single molecule level. This method allows for the selection of a single receptor molecule reconstituted into a supported lipid membrane at low density, with the subsequent quantification of the receptor–ligand unbinding force. Based on AFM tapping mode, a cantilever tip carrying a ligand molecule is oscillated across a membrane. Topography and recognition images of reconstituted receptors are recorded simultaneously by analyzing the downward and upward parts of the oscillation, respectively. Functional receptor molecules are selected from the recognition image with nanometer resolution before the AFM is switched to the force spectroscopy mode, using positional feedback control. The combined mode allows for dynamic force probing on different pre-selected molecules. This strategy results in higher throughput when compared with force mapping. Applied to two different receptor–ligand pairs, we validated the presented new mode. Full article