Methods Protoc., Volume 6, Issue 2 (April 2023) – 23 articles

Traditional Western blotting is one of the most used analytical techniques in biological research. However, it can be time-consuming and suffer from a lack of reproducibility. Consequently, devices with different degrees of automation have been developed. These include semi-automated techniques and fully automated devices that replicate all stages downstream of the sample preparation, including sample size separation, immunoblotting, imaging, and analysis. We directly compared traditional Western blotting with two different automated systems, iBind™ Flex, which is a semi-automated system designed to perform the immunoblotting, and JESS Simple Western™, a fully automated and capillary-based system performing all steps downstream of sample preparation and loading, including imaging and image analysis. We found that a fully automated system can save time and importantly offer valuable sensitivity. This is particularly beneficial for limited sample amounts. The downside of automation is the cost of devices and reagents. Nevertheless, automation can be a good option to increase output and facilitate sensitive protein analyses. Full article

Outer membrane vesicles (OMVs) are lipid structures containing various biomolecules in their native environment and are spontaneously shed by gram-negative bacteria. OMVs perform several biological functions critical to both bacterial physiology and pathogenicity. Scientific research on OMV function and biogenesis requires a standardized and robust method of isolating these vesicles from bacterial cultures that reliably provide high-purity OMVs. Herein, we describe an optimized protocol to isolate OMVs from overnight cultures of three different strains of nontypeable Haemophilus influenzae (NTHi) for use in different downstream applications. Involving mainly differential centrifugation of the culture supernatant, the procedure described is relatively simple, efficient, and generates high-quality OMV preparations from each strain tested with sufficient yields, while preserving the native outer membrane composition. Full article

While the general reliability of the Y balance test has been previously found to be excellent, earlier reviews highlighted a need for a more consistent methodology between studies. The purpose of this test–retest intrarater reliability study was to assess the intrarater reliability of the YBT using different methodologies regarding normalisation for leg length, number of repetitions, and score calculation. Sixteen healthy adult novice recreational runners aged 18–55 years, both women and men, were reviewed in a laboratory environment. Mean calculated scores, intraclass correlation coefficient, standard error of measurement, and minimal detectable change were calculated and analysed between different leg length normalisation and score calculation methods. The number of repetitions needed to reach a plateauing of results was analysed from the mean proportion of maximal reach per successful repetition. The intrarater reliability of the YBT was found to be good to excellent, and it was not affected by the method of score calculation or leg length measurement. The test results plateaued after the sixth successful repetition. Based on this study, it is suggested to use anterior superior iliac spine–medial malleolus length for leg length normalisation because this method was proposed in the original YBT protocol. At least seven successful repetitions should be performed to reach a result plateau. The average of the best three repetitions should be used to mitigate possible outliers and account for the learning effects seen in this study. Full article

Medicinal and herbal plants are abundant sources of phytochemicals, which are biologically active compounds with potential health benefits. The characterisation of phytochemicals has been the subject of many studies, but there is a lack of comprehensive assays to accurately assess the main phytochemical categories and their antioxidant properties. To address this, the present study has developed a multiparametric protocol comprising eight biochemical assays, which quantify the major categories of phytochemicals, including polyphenols, tannins and flavonoids, as well as their antioxidant and scavenging potential. The presented protocol offers several advantages over other methods, including higher sensitivity and significantly lower cost, making it a simpler and more affordable approach compared to commercial kits. The protocol was tested on two datasets with seventeen distinct herbal and medicinal plants, and the results demonstrated its effectiveness in accurately characterising the phytochemical composition of plant samples. The modular design of the protocol allows its adaptation to any spectrophotometric instrumentation, while all assays are simple to follow and require a minimum number of analytical steps. Full article

The application of the CRISPR/Cas9-based genome editing technique to the yeast Saccharomyces cerevisiae has made it possible to simultaneously modify several sites, particularly to integrate several expression cassettes. The existing methods provide high efficiency for such modifications; however, common protocols include several preparatory steps, namely, the construction of an intermediate Cas9-expressing strain, the assembly of a plasmid bearing several single guide RNA (sgRNA) expression cassettes, and the surrounding integrated DNA fragments with long flanks for recombination with target loci. Since these preparatory steps are time consuming and may not be desirable in some types of experiments, we explored the possibility of multiple integration without these steps. We have demonstrated that it is possible to skip them simultaneously and integrate up to three expression cassettes into separate sites by transforming the recipient strain with the Cas9 expression plasmid, three differently marked sgRNA plasmids, and three donor DNAs flanked with short (70 bp) arms for recombination. This finding increases the flexibility of choosing the optimal experimental design for multiple editing of the genome of S. cerevisiae and can significantly accelerate such experiments. Full article

A histological examination is an important tool in embryology, developmental biology, and correlated areas. Despite the amount of information available about tissue embedding and different media, there is a lack of information regarding best practices for embryonic tissues. Embryonic tissues are considered fragile structures, usually small in size, and frequently challenging to position correctly in media for the subsequent histological steps. Here, we discuss the embedding media and procedures that provided us with appropriate preservation of tissue and easier orientation of embryos at early development. Fertilized Gallus gallus eggs were incubated for 72 h, collected, fixed, processed, and embedded with paraplast, polyethylene glycol (PEG), or historesin. These resins were compared by the precision of tissue orientation, the preview of the embryos in the blocks, microtomy, contrast in staining, preservation, average time, and cost. Paraplast and PEG did not allow correct embryo orientation, even with agar–gelatin pre-embedded samples. Additionally, structural maintenance was hindered and did not allow detailed morphological assessment, presenting tissue shrinkage and disruption. Historesin provided precise tissue orientation and excellent preservation of structures. Assessing the performance of the embedding media contributes significantly to future developmental research, optimizing the processing of embryo specimens and improving results. Full article

Malaria is a parasitic infection caused by a protozoon of the genus Plasmodium, transmitted to humans by female biting mosquitoes of the genus Anopheles. Chloroquine and its derivates have caused the parasite to develop drug resistance in endemic areas. For this reason, new anti-malarial drugs as treatments are crucial. This work aimed to evaluate the humoral response. with hyper-immune sera, of mice immunized with six derivatives of tetrahydro-(2H)-1,3,5-thiadiazine-2-thione (bis-THTT) by indirect ELISA test. The cross-reactivity between the compounds as antigens and their microbial activity on Gram-positive and Gram-negative bacteria was evaluated. The results of the humoral evaluation by indirect ELISA show that three bis-THTTs react with almost all of the above. Besides, three compounds used as antigens stimulate the BALB/c mice’s immune system. The best combination of two antigens as a combined therapy displays similar absorbances between the antigens in the mixture, showing similar recognition by antibodies and their compounds. In addition, our results showed that different bis-THTT presented antimicrobial activity on Gram-positive bacteria, mainly on Staphylococcus aureus strains, and no inhibitory activity was observed on the Gram-negative bacteria tested. Full article

Cell-free protein synthesis (CFPS) is a method utilized for producing proteins without the limits of cell viability. The plug-and-play utility of CFPS is a key advantage over traditional plasmid-based expression systems and is foundational to the potential of this biotechnology. A key limitation of CFPS is the varying stability of DNA types, limiting the effectiveness of cell-free protein synthesis reactions. Researchers generally rely on plasmid DNA for its ability to support robust protein expression in vitro. However, the overhead required to clone, propagate, and purify plasmids reduces the potential of CFPS for rapid prototyping. While linear templates overcome the limits of plasmid DNA preparation, linear expression templates (LETs) were under-utilized due to their rapid degradation in extract based CFPS systems, limiting protein synthesis. To reach the potential of CFPS using LETs, researchers have made notable progress toward protection and stabilization of linear templates throughout the reaction. The current advancements range from modular solutions, such as supplementing nuclease inhibitors and genome engineering to produce strains lacking nuclease activity. Effective application of LET protection techniques improves expression yields of target proteins to match that of plasmid-based expression. The outcome of LET utilization in CFPS is rapid design–build–test–learn cycles to support synthetic biology applications. This review describes the various protection mechanisms for linear expression templates, methodological insights for implementation, and proposals for continued efforts that may further advance the field. Full article

Increasing evidence strongly supports the key role of the tumour microenvironment in response to systemic therapy, particularly immune checkpoint inhibitors (ICIs). The tumour microenvironment is a complex tapestry of immune cells, some of which can suppress T-cell immunity to negatively impact ICI therapy. The immune component of the tumour microenvironment, although poorly understood, has the potential to reveal novel insights that can impact the efficacy and safety of ICI therapy. Successful identification and validation of these factors using cutting-edge spatial and single-cell technologies may enable the development of broad acting adjunct therapies as well as personalised cancer immunotherapies in the near future. In this paper we describe a protocol built upon Visium (10x Genomics) spatial transcriptomics to map and characterise the tumour-infiltrating immune microenvironment in malignant pleural mesothelioma. Using ImSig tumour-specific immune cell gene signatures and BayesSpace Bayesian statistical methodology, we were able to significantly improve immune cell identification and spatial resolution, respectively, improving our ability to analyse immune cell interactions within the tumour microenvironment. Full article

The human milk microbiota (HMM) of healthy women can vary substantially, as demonstrated by recent advances in DNA sequencing technology. However, the method used to extract genomic DNA (gDNA) from these samples may impact the observed variations and potentially bias the microbiological reconstruction. Therefore, it is important to use a DNA extraction method that is able to effectively isolate gDNA from a diverse range of microorganisms. In this study, we improved and compared a DNA extraction method for gDNA isolation from human milk (HM) samples to commercial and standard protocols. We evaluated the extracted gDNA using spectrophotometric measurements, gel electrophoresis, and PCR amplifications to assess its quantity, quality, and amplifiability. Additionally, we tested the improved method’s ability to isolate amplifiable gDNA from fungi, Gram-positive and Gram-negative bacteria to validate its potential for reconstructing microbiological profiles. The improved DNA extraction method resulted in a higher quality and quantity of the extracted gDNA compared to the commercial and standard protocols and allowed for polymerase chain reaction (PCR) amplification of the V3–V4 regions of the 16S ribosomal gene in all the samples and the ITS-1 region of the fungal 18S ribosomal gene in 95% of the samples. These results suggest that the improved DNA extraction method demonstrates better performance for gDNA extraction from complex samples such as HM. Full article

Insulin is a hormone produced by β-cells of the pancreas and controls the amount of sugar in the blood. Since its discovery over 100 years ago, insulin has been used as a life-saving treatment for people with diabetes. Historically, the biological activity or bioidentity of insulin products has been assessed using an in vivo model. However, reduction in animal experiments is a goal for many worldwide, and there is a need to develop in vitro bioassays to reliably test the biological activity of insulin products. This article describes an in vitro cell-based method to assess the biological activity of insulin glargine, insulin aspart, and insulin lispro in a step-by-step manner. Full article

Mitochondrial dysfunction and cytosolic oxidative stress are pathological biomarkers interlinked in several chronic diseases and cellular toxicity promoted by high-energy radiation or xenobiotics. Thus, assessing the activities of the mitochondrial redox chain complexes and the cytosolic antioxidant enzymes in the same cell culture system is a valuable approach to addressing the challenge of chronic diseases or unveiling the molecular mechanisms underlying the toxicity of physical and chemical stress agents. The present article gathers the experimental procedures to obtain, from isolated cells, a mitochondria-free cytosolic fraction and a mitochondria-rich fraction. Furthermore, we describe the methodologies to evaluate the activity of the main antioxidant enzymes in the mitochondria-free cytosolic fraction (superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase), and the activity of the individual mitochondrial complexes I, II and IV, as well as the conjugated activity of complexes I–III and complexes II–III in the mitochondria-rich fraction. The protocol to test the citrate synthase activity was also considered and used to normalize complexes. The procedures were optimized within an experimental setup to allow that each condition to be tested only requires sampling of one T-25 flask of cells 2D cultured, as the typical results presented and discussed here. Full article

Long-read sequencing methods allow a comprehensive analysis of transcriptomes in identifying full-length transcripts. This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies^® is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes. Full article

Despite the decline in under-five mortality by over 60% in the last three decades, majority of child mortality is still attributable to communicable and infectious diseases that are not only preventable, but they are also treatable. We evaluated the potential impact of a participatory community engagement and innovative community incentivization (C3I) strategy for improving the coverage of child health interventions in a rural setting in Pakistan. We first undertook formative research to assess community knowledge and the likelihood of collective community strategy and conditional incentives for improving existing preventive and care-seeking practices for childhood diarrhea and pneumonia. We developed options for community incentivization and improving group practices, taking local norms and customs into account in the design of the community mobilization strategies and messages. These interventions were then formally evaluated prospectively in a three-arm cluster randomized controlled trial. Clusters were randomly assigned by a computer algorithm using restricted randomization by an external statistician (1:1:1) into three groups: community mobilization and incentivization (CMI); community mobilization only using an enhanced communication package (CM); and control group. The C3I was an innovative strategy as it involved serial incremental targets of collective improvement in community behavior related to improvement in the coverage of a composite indicator of fully immunized children (FIC), oral rehydration salt (ORS), and the sanitation index (SI). The evaluation was done by an independent data collection and analysis team at baseline and end line (after 24 months). Full article

The importance of physical activity (PA) for the health and wellbeing of older adults is well documented, yet many older adults are insufficiently active. This issue is more salient in regional and rural areas, where evidence of the most critical components of interventions that explain PA participation and maintenance in older populations is sparse. This realist review will (1) systematically identify and synthesise literature on PA interventions in community-dwelling older adults in regional and rural areas, and (2) explore how and why those interventions increase PA in that population. Using a realist synthesis framework and the behaviour change wheel (BCW), context–mechanism–outcome (C-M-O) patterns of PA interventions for older adults in regional and rural areas will be synthesised. Thematic analysis will be employed to compare, contrast, and refine emerging C-M-O patterns to understand how contextual factors trigger mechanisms that influence regional and rural community-dwelling older adults’ participation in PA interventions. This realist review will be the first to adopt a BCW analysis and a realist synthesis framework to explore PA interventions in community-dwelling older adults in regional and rural areas. This review will provide recommendations for evidence-based interventions to improve PA participation and adherence by revealing the important mechanisms apparent in this context. Systematic review registration: (PROSPERO CRD42023402499). Full article

Background: Evidence-based recommendations for the treatment of knee and hip osteoarthritis are similar internationally. Nevertheless, clinical practice varies across countries. Instruments for measuring quality have been developed to improve health care through targeted interventions. Studies on health service quality must consider the structural and cultural characteristics of countries, because each of their strengths and weaknesses differ. However, such instruments for health-related patient-reported outcomes for osteoarthritis have not yet been validated in German and Italian languages. Objectives: In order to be able to set targeted measures for the improvement of prevention and non-surgical treatment of osteoarthritis in South Tyrol, Italy, the quality of care must be recorded. Therefore, the aim of the project is to update, translate, and validate the OsteoArthritis Quality Indicator (OA-QI) questionnaire version 2, an established and validated questionnaire in Norwegian and English, for Germany and Italy. The second aim is to determine the quality of care for osteoarthritis of the hip and knee in a sample of patients who consult general practice in South Tyrol, and for comparison with patients who are admitted to rehabilitative spa-treatments for osteoarthritis in the state of Salzburg, Austria. Discussion: The results of this study will enable the identification and closure of gaps in osteoarthritis care. Although it is expected that body weight and exercise will play special roles, other areas of nonsurgical care might also be involved. Full article

Prior to the federal law passed in December 2019, many states passed an increased age of sale law prohibiting youth under age 21 (or Tobacco 21) from purchasing tobacco products, including e-cigarettes. Although previous research has documented tobacco retail sale violations, fewer studies have examined age verification and illegal tobacco sales in the context of Tobacco 21 or repeated purchase attempts in various settings. In this study conducted between 2019 and 2022, buyers aged 18 to 20 years made repeated unsupervised purchase attempts of cigarettes, cigars, e-cigarettes, tobacco-free nicotine pouches, and smokeless tobacco at over 180 tobacco or e-cigarette retailers in New Jersey, New York City, and Pitt County (North Carolina). Buyers documented whether they were asked for identification and whether they were able to successfully purchase a tobacco or nicotine product at each visit. The primary outcome will be the percent of retailers that checked the buyers’ identification at store visits and the percent of visits that resulted in a successful underage tobacco product purchase. We will compare the results across time periods, study sites, products, and buyer characteristics (i.e., gender, minoritized racial/ethnic identity) while controlling for repeated visits. These results will inform improvements to current compliance check inspection programs as well as interventions that reduce youth access to tobacco. Full article

Background: The isokinetic dynamometry is considered a gold standard in muscle strength testing. The reliability of lower limb isokinetic strength measurements has not been thoroughly evaluated. Objective: To examine the test−retest reliability of isokinetic ankle plantar and dorsiflexion, ankle inversion and eversion, knee extension and flexion and hip abduction and adduction strength in physically active adults using Biodex System 4 Pro. Methods: Peak torques (PTs) and average peak torques (APTs) of the dominant and nondominant lower limbs were tested twice in 19 physically active adults 7 to 14 days apart. Results: The intraclass correlation coefficients (ICC) values varied from excellent to moderate and coefficient of variation of typical error (CV_TE) values were 6.6–19.5%. Change in the mean expressed as a percent varied from −3.1% to 9.6%. There was no difference in the reliability between PT and APT values. Dominant lower limb was more reliable in every case if there was difference between limbs. Conclusion: Test−retest reliability of isokinetic ankle, knee and hip strength in physically active adults using Biodex System 4 is mostly good or excellent. However, the observed range of the random variation has to be noted when using it in scientific follow-up studies or evaluation of patient progress in clinical settings. Full article

We describe a workflow for efficient, environmentally attentive, and sustainable practices related to routine agarose gel electrophoresis. The methods reduce plastic waste and improve efficiency, especially for the exhaustive screening of difficult-to-obtain plasmids. Sustainability is increased when agarose is used ten times over by virtue of a thorough recycling regimen. The workflow optimizes workspaces and standardizes lab practices for handling potentially hazardous waste, minimizing environmental harm. Safety, efficiency, and sustainability improve laboratory productivity, help minimize environmental contamination, and increase cost-effectiveness. Full article

Tn5 transposase use in biotechnology has substantially advanced the sequencing applications of genome-wide analysis of cells. This is mainly due to the ability of Tn5 transposase to efficiently transpose DNA essentially randomly into any target DNA without the aid of other factors. This concise review is focused on the advances in Tn5 applications in multi-omics technologies, genome-wide profiling, and Tn5 hybrid molecule creation. The possibilities of other transposase uses are also discussed. Full article

Understanding the mechanisms and kinetics of membrane damage is of interest to researchers in several overlapping fields of biology. In this study, we describe the development and validation of a simple ${}^{32}$PO${}_4^{3-}$ release radioassay used to track nanometer-scale damage to the bacterial cell membrane. Nanoscale membrane damage will result in the release of small cytoplasmic molecules, such as amino acids, sugars, and osmolytes. Our radioassay tracks the release of these molecules using the release of cytoplasmic ${}^{32}$PO${}_4^{3-}$ as a proxy. Our assay can both detect ${}^{32}$PO${}_4^{3-}$ release and track release kinetics in the order of minutes. We demonstrate the use of our radioassay using A. baumannii treated with colistin and $\Omega 76$: two agents known to cause membrane damage. Our assay tracks greater membrane damage in A. baumannii treated with both these agents, compared to an untreated control. Our assay fills a niche that is not covered by traditional ${}^{51}$Cr release radioassays and fluorescent staining techniques. Furthermore, our assay can potentially be used to track membrane damage in other membrane systems such as lipid vesicles, animal cells, and organelles. Full article

Trauma triggers critical molecular and cellular signaling cascades that drive biological outcomes and recovery. Variations in the gene expression of common endogenous reference housekeeping genes (HKGs) used in data normalization differ between tissue types and pathological states. Systematically, we investigated the gene stability of nine HKGs (Actb, B2m, Gapdh, Hprt1, Pgk1, Rplp0, Rplp2, Tbp, and Tfrc) from tissues prone to remote organ dysfunction (lung, liver, kidney, and muscle) following extremity trauma. Computational algorithms (geNorm, Normfinder, ΔCt, BestKeeper, RefFinder) were applied to estimate the expression stability of each HKG or combinations of them, within and between tissues, under both steady-state and systemic inflammatory conditions. Rplp2 was ranked as the most suitable in the healthy and injured lung, kidney, and skeletal muscle, whereas Rplp2 and either Hprt1 or Pgk1 were the most suitable in the healthy and injured liver, respectively. However, the geometric mean of the three most stable genes was deemed the most stable internal reference control. Actb and Tbp were the least stable in normal tissues, whereas Gapdh and Tbp were the least stable across all tissues post-trauma. Ct values correlated poorly with the translation from mRNA to protein. Our results provide a valuable resource for the accurate normalization of gene expression in trauma-related experiments. Full article

A common laboratory method involves gel electrophoresis followed by photographic documentation of the results, a procedure which is performed worldwide by students and experienced scientists alike. Proprietary Gel Documentation Systems are convenient and useful for documentation of electrophoresis results, but the systems can be prohibitively expensive to purchase and repair, they contain features that are not necessary for everyday documentation, and some users may not find the systems intuitive to operate. We describe our gel documentation setup that meets the everyday needs for documentation in our lab. The setup is inexpensive, modular, user friendly, and increases sustainability through extending the working life of obsolete cell phones, iPads, or other electronic devices containing a camera. More importantly, the setup completely shields users from potentially damaging ultraviolet radiation. Full article