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Biology Methods and Protocols
Number of Followers: 0  Open Access journalISSN (Online) 2396-8923 Published by Oxford University Press  [425 journals]
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- Assessing RNA integrity by digital RT-PCR: Influence of extraction,
storage, and matrices
First page: bpae053
Abstract: AbstractThe development of high-throughput sequencing has greatly improved our knowledge of microbial diversity in aquatic environments and its evolution in highly diverse ecosystems. Relevant microbial diversity description based on high-throughput sequencing relies on the good quality of the nucleic acid recovered. Indeed, long genetic fragments are more informative for identifying mutation combinations that characterize variants or species in complex samples. This study describes a new analytical method based on digital Polymerase Chain Reaction (PCR) partitioning technology for assessing the fragmentation of nucleic acid and more specifically viral RNA. This method allows us to overcome limits associated with hydrolysis probe-based assay by focusing on the distance between different amplicons, and not, as usual, on the size of amplicons. RNA integrity can thus be determined as a new fragmentation index, the so-called Fragment size 50. The application of this method has provided information on issues that are inherent in environmental analyses, such as the storage impact of raw samples or extracted RNA, extraction methods, and the nature of the sample on the integrity of viral RNA. Finally, the estimation of fragment size by digital PCR (dPCR) showed a very strong similarity with the fragment size sequenced using Oxford Nanopore Technology. In addition to enabling objective improvements in analytical methods, this approach could become a systematic quality control prior to any long-read sequencing, avoiding insufficiently productive sequencing runs or biases in the representativeness of sequenced fragments.
PubDate: Sat, 27 Jul 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae053
Issue No: Vol. 9, No. 1 (2024)
- Deep learning pipeline reveals key moments in human embryonic development
predictive of live birth after in vitro fertilization
First page: bpae052
Abstract: AbstractDemand for in vitro fertilization (IVF) treatment is growing; however, success rates remain low partly due to difficulty in selecting the best embryo to be transferred. Current manual assessments are subjective and may not take advantage of the most informative moments in embryo development. Here, we apply convolutional neural networks (CNNs) to identify key windows in pre-implantation human development that can be linked to embryo viability and are therefore suitable for the early grading of IVF embryos. We show how machine learning models trained at these developmental time points can be used to refine overall embryo viability assessment. Exploiting the well-known capabilities of transfer learning, we illustrate the performance of CNN models for very limited datasets, paving the way for the use on a clinic-by-clinic basis, catering for local data heterogeneity.
PubDate: Fri, 19 Jul 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae052
Issue No: Vol. 9, No. 1 (2024)
- Molecular identification of polymorphic transposable elements in
populations of the invasive ant Cardiocondyla obscurior
First page: bpae050
Abstract: AbstractTransposable elements (TEs) are found in virtually every eukaryotic genome and are important for generating de novo genetic variation. However, outside of costly and time-consuming whole-genome sequencing approaches, the set of available methods to study TE polymorphisms in non-model species is very limited. The Transposon Display (TD) is a simple yet effective technique to characterize polymorphisms across samples by identifying amplified fragment length polymorphisms using primers targeting specific TE families. So far, this technique has almost exclusively been used in plants. Here, we present an optimized TD protocol for insect species with small genomes such as ants (ca. 200–600 Mb). We characterized TE polymorphisms between two distinct genetic lineages of the invasive ant Cardiocondyla obscurior, as well as between neighboring populations of the New World lineage. We found active LTR/Ty3 retrotransposons, that contributed to the genetic diversification of populations in this species.
PubDate: Sat, 13 Jul 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae050
Issue No: Vol. 9, No. 1 (2024)
- The use of droplet-based microfluidic technologies for accelerated
selection of Yarrowia lipolytica and Phaffia rhodozyma yeast mutants
First page: bpae049
Abstract: AbstractMicroorganisms are widely used for the industrial production of various valuable products, such as pharmaceuticals, food and beverages, biofuels, enzymes, amino acids, vaccines, etc. Research is constantly carried out to improve their properties, mainly to increase their productivity and efficiency and reduce the cost of the processes. The selection of microorganisms with improved qualities takes a lot of time and resources (both human and material); therefore, this process itself needs optimization. In the last two decades, microfluidics technology appeared in bioengineering, which allows for manipulating small particles (from tens of microns to nanometre scale) in the flow of liquid in microchannels. The technology is based on small-volume objects (microdroplets from nano to femtolitres), which are manipulated using a microchip. The chip is made of an optically transparent inert to liquid medium material and contains a series of channels of small size (<1 mm) of certain geometry. Based on the physical and chemical properties of microparticles (like size, weight, optical density, dielectric constant, etc.), they are separated using microsensors. The idea of accelerated selection of microorganisms is the application of microfluidic technologies to separate mutants with improved qualities after mutagenesis. This article discusses the possible application and practical implementation of microfluidic separation of mutants, including yeasts like Yarrowia lipolytica and Phaffia rhodozyma after chemical mutagenesis will be discussed.
PubDate: Wed, 10 Jul 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae049
Issue No: Vol. 9, No. 1 (2024)
- Machine learning of cellular metabolic rewiring
First page: bpae048
Abstract: AbstractMetabolic rewiring allows cells to adapt their metabolism in response to evolving environmental conditions. Traditional metabolomics techniques, whether targeted or untargeted, often struggle to interpret these adaptive shifts. Here, we introduce MetaboLiteLearner, a lightweight machine learning framework that harnesses the detailed fragmentation patterns from electron ionization (EI) collected in scan mode during gas chromatography/mass spectrometry to predict changes in the metabolite composition of metabolically adapted cells. When tested on breast cancer cells with different preferences to metastasize to specific organs, MetaboLiteLearner predicted the impact of metabolic rewiring on metabolites withheld from the training dataset using only the EI spectra, without metabolite identification or pre-existing knowledge of metabolic networks. Despite its simplicity, the model learned captured shared and unique metabolomic shifts between brain- and lung-homing metastatic lineages, suggesting cellular adaptations associated with metastasis to specific organs. Integrating machine learning and metabolomics paves the way for new insights into complex cellular adaptations.
PubDate: Tue, 02 Jul 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae048
Issue No: Vol. 9, No. 1 (2024)
- The landscape of RNA 3D structure modeling with transformer networks
First page: bpae047
Abstract: AbstractTransformers are a powerful subclass of neural networks catalyzing the development of a growing number of computational methods for RNA structure modeling. Here, we conduct an objective and empirical study of the predictive modeling accuracy of the emerging transformer-based methods for RNA structure prediction. Our study reveals multi-faceted complementarity between the methods and underscores some key aspects that affect the prediction accuracy.
PubDate: Tue, 02 Jul 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae047
Issue No: Vol. 9, No. 1 (2024)
- High-throughput evaluation of hemolytic activity through precise
measurement of colony and hemolytic zone sizes of engineered Bacillus
subtilis on blood agar
First page: bpae044
Abstract: AbstractBiosurfactants have remarkable characteristics, such as environmental friendliness, high safety, and excellent biodegradability. Surfactin is one of the best-known biosurfactants produced by Bacillus subtilis. Because the biosynthetic pathways of biosurfactants, such as surfactin, are complex, mutagenesis is a useful alternative to typical metabolic engineering approaches for developing high-yield strains. Therefore, there is a need for high-throughput and accurate screening methods for high-yield strains derived from mutant libraries. The blood agar lysis method, which takes advantage of the hemolytic activity of biosurfactants, is one way of determining their concentration. This method includes inoculating microbial cells onto blood-containing agar plates, and biosurfactant production is assessed based on the size of the hemolytic zone formed around each colony. Challenges with the blood agar lysis method include low experimental reproducibility and a lack of established protocols for high-throughput screening. Therefore, in this study, we investigated the effects of the inoculation procedure and media composition on the formation of hemolytic zones. We also developed a workflow to evaluate the number of colonies using robotics. The results revealed that by arranging colonies at appropriate intervals and measuring the areas of colonies and hemolytic rings using image analysis software, it was possible to accurately compare the hemolytic activity among several colonies. Although the use of the blood agar lysis method for screening is limited to surfactants exhibiting hemolytic activity, it is believed that by considering the insights gained from this study, it can contribute to the accurate screening of strains with high productivity.
PubDate: Thu, 27 Jun 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae044
Issue No: Vol. 9, No. 1 (2024)
- KCL TEST: an open-source inspired asymptomatic SARS-CoV-2 surveillance
programme in an academic institution
First page: bpae046
Abstract: AbstractRapid and accessible testing was paramount in the management of the COVID-19 pandemic. Our university established KCL TEST: a SARS-CoV-2 asymptomatic testing programme that enabled sensitive and accessible PCR testing of SARS-CoV-2 RNA in saliva. Here, we describe our learnings and provide our blueprint for launching diagnostic laboratories, particularly in low-resource settings. Between December 2020 and July 2022, we performed 158277 PCRs for our staff, students, and their household contacts, free of charge. Our average turnaround time was 16 h and 37 min from user registration to result delivery. KCL TEST combined open-source automation and in-house non-commercial reagents, which allows for rapid implementation and repurposing. Importantly, our data parallel those of the UK Office for National Statistics, though we detected a lower positive rate and virtually no delta wave. Our observations strongly support regular asymptomatic community testing as an important measure for decreasing outbreaks and providing safe working spaces. Universities can therefore provide agile, resilient, and accurate testing that reflects the infection rate and trend of the general population. Our findings call for the early integration of academic institutions in pandemic preparedness, with capabilities to rapidly deploy highly skilled staff, as well as develop, test, and accommodate efficient low-cost pipelines.
PubDate: Thu, 20 Jun 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae046
Issue No: Vol. 9, No. 1 (2024)
- Early detection and diagnosis of cancer with interpretable machine
learning to uncover cancer-specific DNA methylation patterns
First page: bpae028
Abstract: AbstractCancer, a collection of more than two hundred different diseases, remains a leading cause of morbidity and mortality worldwide. Usually detected at the advanced stages of disease, metastatic cancer accounts for 90% of cancer-associated deaths. Therefore, the early detection of cancer, combined with current therapies, would have a significant impact on survival and treatment of various cancer types. Epigenetic changes such as DNA methylation are some of the early events underlying carcinogenesis. Here, we report on an interpretable machine learning model that can classify 13 cancer types as well as non-cancer tissue samples using only DNA methylome data, with 98.2% accuracy. We utilize the features identified by this model to develop EMethylNET, a robust model consisting of an XGBoost model that provides information to a deep neural network that can generalize to independent data sets. We also demonstrate that the methylation-associated genomic loci detected by the classifier are associated with genes, pathways and networks involved in cancer, providing insights into the epigenomic regulation of carcinogenesis.
PubDate: Thu, 20 Jun 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae028
Issue No: Vol. 9, No. 1 (2024)
- A protocol to isolate, identify, and verify glucose- or
carbohydrate-binding receptors
First page: bpae045
Abstract: AbstractSensing, transport, and utilization of glucose is pivotal to the maintenance of energy homeostasis in animals. Although transporters involved in mobilizing glucose across different cellular compartments are fairly well known, the receptors that bind glucose to mediate its effects independently of glucose metabolism remain largely unrecognized. Establishing precise and reproducible methods to identify glucose receptors in the brain or other peripheral organs will pave the way for comprehending the role of glucose signaling pathways in maintaining, regulating, and reprogramming cellular metabolic needs. Identification of such potential glucose receptors will also likely lead to development of effective therapeutics for treatment of diabetes and related metabolic disorders. Commercially available biotin or radiolabeled glucose conjugates have low molecular weight; therefore, they do not provide enough sensitivity and density to isolate glucose receptors. Here, we describe a protocol to isolate, identify, and verify glucose-binding receptor/s using high molecular weight glucose (or other carbohydrate) conjugates. We have produced 30 kDa glucose– (or other carbohydrate–) biotin–polyacrylamide (PAA) conjugates with mole fractions of 80:5:15% respectively. These conjugates are used with biotin-streptavidin biochemistry, In-cell ELISA, and surface plasmon resonance (SPR) methods to isolate, identify, and verify glucose- or carbohydrate-binding receptors. We first demonstrate how streptavidin-coated magnetic beads are employed to immobilize glucose–biotin–PAA conjugates. Then, these beads are used to enrich and isolate glucose-binding proteins from tissue homogenates or from single-cell suspensions. The enriched or isolated proteins are subjected to mass spectrometry/proteomics to reveal the identity of top candidate proteins as potential glucose receptors. We then describe how the In-cell ELISA method is used to verify the interaction of glucose with its potential receptor through stable expression of the receptor in-vitro. We further demonstrate how a highly sensitive SPR method can be used to measure the binding kinetics of glucose with its receptor. In summary, we describe a protocol to isolate, identify, and verify glucose- or carbohydrate-binding receptors using magnetic beads, In-cell ELISA, and SPR. This protocol will form the future basis of studying glucose or carbohydrate receptor signaling pathways in health and in disease.
PubDate: Wed, 19 Jun 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae045
Issue No: Vol. 9, No. 1 (2024)
- Multimodal pretraining for unsupervised protein representation learning
First page: bpae043
Abstract: AbstractProteins are complex biomolecules essential for numerous biological processes, making them crucial targets for advancements in molecular biology, medical research, and drug design. Understanding their intricate, hierarchical structures, and functions is vital for progress in these fields. To capture this complexity, we introduce Multimodal Protein Representation Learning (MPRL), a novel framework for symmetry-preserving multimodal pretraining that learns unified, unsupervised protein representations by integrating primary and tertiary structures. MPRL employs Evolutionary Scale Modeling (ESM-2) for sequence analysis, Variational Graph Auto-Encoders (VGAE) for residue-level graphs, and PointNet Autoencoder (PAE) for 3D point clouds of atoms, each designed to capture the spatial and evolutionary intricacies of proteins while preserving critical symmetries. By leveraging Auto-Fusion to synthesize joint representations from these pretrained models, MPRL ensures robust and comprehensive protein representations. Our extensive evaluation demonstrates that MPRL significantly enhances performance in various tasks such as protein–ligand binding affinity prediction, protein fold classification, enzyme activity identification, and mutation stability prediction. This framework advances the understanding of protein dynamics and facilitates future research in the field. Our source code is publicly available at https://github.com/HySonLab/Protein_Pretrain.
PubDate: Tue, 18 Jun 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae043
Issue No: Vol. 9, No. 1 (2024)
- PROMER technology: A new real-time PCR tool enabling multiplex detection
of point mutation with high specificity and sensitivity
First page: bpae041
Abstract: AbstractReal-time polymerase chain reaction (real-time PCR) is a powerful tool for the precise quantification of nucleic acids in various applications. In cancer management, the monitoring of circulating tumor DNA (ctDNA) from liquid biopsies can provide valuable information for precision care, including treatment selection and monitoring, prognosis, and early detection. However, the rare and heterogeneous nature of ctDNA has made its precise detection and quantification challenging, particularly for ctDNA containing hotspot mutations. We have developed a new real-time PCR tool, PROMER technology, which enables the precise and sensitive detection of ctDNA containing cancer-driven single-point mutations. The PROMER functions as both a PRObe and priMER, providing enhanced detection specificity. We validated PROMER technology using synthetic templates with known KRAS point mutations and demonstrated its sensitivity and linearity of quantification. Using genomic DNA from human cancer cells with mutant and wild-type KRAS, we confirmed that PROMER PCR can detect mutant DNA. Furthermore, we demonstrated the ability of PROMER technology to efficiently detect mutation-carrying ctDNA from the plasma of mice with human cancers. Our results suggest that PROMER technology represents a promising new tool for the precise detection and quantification of DNA containing point mutations in the presence of a large excess of wild-type counterpart.
PubDate: Tue, 04 Jun 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae041
Issue No: Vol. 9, No. 1 (2024)
- Mapping adipocyte interactome networks by HaloTag-enrichment-mass
spectrometry
First page: bpae039
Abstract: AbstractMapping protein interaction complexes in their natural state in vivo is arguably the Holy Grail of protein network analysis. Detection of protein interaction stoichiometry has been an important technical challenge, as few studies have focused on this. This may, however, be solved by artificial intelligence (AI) and proteomics. Here, we describe the development of HaloTag-based affinity purification mass spectrometry (HaloMS), a high-throughput HaloMS assay for protein interaction discovery. The approach enables the rapid capture of newly expressed proteins, eliminating tedious conventional one-by-one assays. As a proof-of-principle, we used HaloMS to evaluate the protein complex interactions of 17 regulatory proteins in human adipocytes. The adipocyte interactome network was validated using an in vitro pull-down assay and AI-based prediction tools. Applying HaloMS to probe adipocyte differentiation facilitated the identification of previously unknown transcription factor (TF)–protein complexes, revealing proteome-wide human adipocyte TF networks and shedding light on how different pathways are integrated.
PubDate: Wed, 29 May 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae039
Issue No: Vol. 9, No. 1 (2024)
- IntelliGenes: Interactive and user-friendly multimodal AI/ML application
for biomarker discovery and predictive medicine
First page: bpae040
Abstract: AbstractArtificial intelligence (AI) and machine learning (ML) have advanced in several areas and fields of life; however, its progress in the field of multi-omics is not matching the levels others have attained. Challenges include but are not limited to the handling and analysis of high volumes of complex multi-omics data, and the expertise needed to implement and execute AI/ML approaches. In this article, we present IntelliGenes, an interactive, customizable, cross-platform, and user-friendly AI/ML application for multi-omics data exploration to discover novel biomarkers and predict rare, common, and complex diseases. The implemented methodology is based on a nexus of conventional statistical techniques and cutting-edge ML algorithms, which outperforms single algorithms and result in enhanced accuracy. The interactive and cross-platform graphical user interface of IntelliGenes is divided into three main sections: (i) Data Manager, (ii) AI/ML Analysis, and (iii) Visualization. Data Manager supports the user in loading and customizing the input data and list of existing biomarkers. AI/ML Analysis allows the user to apply default combinations of statistical and ML algorithms, as well as customize and create new AI/ML pipelines. Visualization provides options to interpret a diverse set of produced results, including performance metrics, disease predictions, and various charts. The performance of IntelliGenes has been successfully tested at variable in-house and peer-reviewed studies, and was able to correctly classify individuals as patients and predict disease with high accuracy. It stands apart primarily in its simplicity in use for nontechnical users and its emphasis on generating interpretable visualizations. We have designed and implemented IntelliGenes in a way that a user with or without computational background can apply AI/ML approaches to discover novel biomarkers and predict diseases.
PubDate: Wed, 29 May 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae040
Issue No: Vol. 9, No. 1 (2024)
- An optimized ligation-mediated PCR method for chromosome walking and
fusion gene chromosomal breakpoints identification
First page: bpae037
Abstract: AbstractMolecular techniques that recover unknown sequences next to a known sequence region have been widely applied in various molecular studies, such as chromosome walking, identification of the insertion site of transposon mutagenesis, fusion gene partner, and chromosomal breakpoints, as well as targeted sequencing library preparation. Although various techniques have been introduced for efficiency enhancement, searching for relevant single molecular event present in a large-sized genome remains challenging. Here, the optimized ligation-mediated polymerase chain reaction (PCR) method was developed and successfully identified chromosomal breakpoints far away from the exon of the new exon junction without the need for nested PCR. In addition to recovering unknown sequences next to a known sequence region, the high efficiency of the method could also improve the performance of targeted next-generation sequencing (NGS).
PubDate: Sat, 25 May 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae037
Issue No: Vol. 9, No. 1 (2024)
- Two methods of isolation of rat aortic smooth muscle cells with high yield
First page: bpae038
Abstract: AbstractVascular smooth muscle cells (VSMCs) are an integral part of blood vessels and are the focus of intensive research in vascular biology, translational research, and cardiovascular diseases. Though immortalized vascular smooth muscle cell lines are available, their use is limited, underscoring the need for primary VSMCs. There are several methods for isolating primary cells from mice. However, the isolation method from rat blood vessels requires optimization, given the differences in the aorta of mice and rats. Here we compare two methods for VSMCs isolation from rats: enzymatic digestion and the “block” method. We observed a significantly higher yield of VSMCs using the enzymatic digestion method. We further confirmed that VSMCs expressed well-established VSMC-specific markers (calponin) with both methods and observed the persistence of this marker up to 9 passages, suggesting a continuation of the secretory phenotype of VSMCs. Overall, this work compares two methods and demonstrates a practical and effective method for isolating VSMCs from rat aorta, providing vascular biologists with a valuable and reliable experimental tool.
PubDate: Wed, 22 May 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae038
Issue No: Vol. 9, No. 1 (2024)
- A one-step low-cost molecular test for SARS-CoV-2 detection suitable for
community testing using minimally processed saliva
First page: bpae035
Abstract: AbstractThe gold standard for coronavirus disease 2019 diagnostic testing relies on RNA extraction from naso/oropharyngeal swab followed by amplification through reverse transcription-polymerase chain reaction (RT-PCR) with fluorogenic probes. While the test is extremely sensitive and specific, its high cost and the potential discomfort associated with specimen collection made it suboptimal for public health screening purposes. In this study, we developed an equally reliable, but cheaper and less invasive alternative test based on a one-step RT-PCR with the DNA-intercalating dye SYBR Green, which enables the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly from saliva samples or RNA isolated from nasopharyngeal (NP) swabs. Importantly, we found that this type of testing can be fine-tuned to discriminate SARS-CoV-2 variants of concern. The saliva RT-PCR SYBR Green test was successfully used in a mass-screening initiative targeting nearly 4500 asymptomatic children under the age of 12. Testing was performed at a reasonable cost, and in some cases, the saliva test outperformed NP rapid antigen tests in identifying infected children. Whole genome sequencing revealed that the antigen testing failure could not be attributed to a specific lineage of SARS-CoV-2. Overall, this work strongly supports the view that RT-PCR saliva tests based on DNA-intercalating dyes represent a powerful strategy for community screening of SARS-CoV-2. The tests can be easily applied to other infectious agents and, therefore, constitute a powerful resource for an effective response to future pandemics.
PubDate: Wed, 22 May 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae035
Issue No: Vol. 9, No. 1 (2024)
- Point-of-care potentials of lateral flow-based field screening for
Mycoplasma bovis infections: a literature review
First page: bpae034
Abstract: AbstractPoint-of-care (POC) field screening for tools for Mycoplasma bovis (M. bovis) is still lacking due to the requirement for a simple, robust field-applicable test that does not entail specialized laboratory equipment. In accordance with the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) guidelines, this review identifies the methodologies that were retrieved based on our search strategy that have been reported for the diagnosis of m. bovis infection between 2014 and diagnostics. A search criterion was generated to curate 103 articles, which were reduced in number (to 46), following the screening guidelines of PRISMA. The 43 articles included in the study present 25 different assay methods. The assay methods were grouped as microbiological culture, serological assay, PCR-based assay, LAMP-based assay, NGS-based assay, or lateral flow assay. We, however, focus our discussion on the three lateral flow-based assays relative to others, highlighting the advantages they present above the other techniques and their potential applicability as a POC diagnostic test for M. bovis infections. We therefore call for further research on developing a lateral flow-based screening tool that could revolutionize the diagnosis of M. bovis infection.
PubDate: Wed, 22 May 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae034
Issue No: Vol. 9, No. 1 (2024)
- High-throughput surface epitope immunoaffinity isolation of extracellular
vesicles and downstream analysis
First page: bpae032
Abstract: AbstractExtracellular vesicles (EVs), including exosomes, have significant potential for diagnostic and therapeutic applications. The lack of standardized methods for efficient and high-throughput isolation and analysis of EVs, however, has limited their widespread use in clinical practice. Surface epitope immunoaffinity (SEI) isolation utilizes affinity ligands, including antibodies, aptamers, or lectins, that target specific surface proteins present on EVs. Paramagnetic bead-SEI isolation represents a fit-for-purpose solution for the reproducible, high-throughput isolation of EVs from biofluids and downstream analysis of RNA, protein, and lipid biomarkers that is compatible with clinical laboratory workflows. This study evaluates a new SEI isolation method for enriching subpopulations of EVs. EVs were isolated from human plasma using a bead-based SEI method designed for on-bead and downstream analysis of EV-associated RNA and protein biomarkers. Western blot analysis confirmed the presence of EV markers in the captured nanoparticles. Mass spectrometry analysis of the SEI lysate identified over 1500 proteins, with the top 100 including known EV-associated proteins. microRNA (miRNA) sequencing followed by RT-qPCR analysis identified EV-associated miRNA transcripts. Using SEI, EVs were isolated using automated high-throughput particle moving instruments, demonstrating equal or higher protein and miRNA yield and recovery compared to manual processing. SEI is a rapid, efficient, and high-throughput method for isolating enriched populations of EVs; effectively reducing contamination and enabling the isolation of a specific subpopulation of EVs. In this study, high-throughput EV isolation and RNA extraction have been successfully implemented. This technology holds great promise for advancing the field of EV research and facilitating their application for biomarker discovery and clinical research.
PubDate: Fri, 17 May 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae032
Issue No: Vol. 9, No. 1 (2024)
- Isolation of short RNAs with homogeneous 3′-ends using quaternary-amine
anion exchange chromatography
First page: bpae033
Abstract: AbstractVisualizing RNA–protein interactions through structural approaches requires the use of RNA molecules purified to homogeneity. We describe here a simple and effective method, free of acrylamide contamination and without using UV radiation, to separate in vitro synthesized, heterogeneous RNA transcripts (up to ∼15 nucleotides) at single-nucleotide resolution by quaternary-amine anion exchange chromatography. The quality of short RNAs isolated through this method is validated by gel electrophoresis, mass spectrometry, and crystallization with a protein-binding partner.
PubDate: Fri, 17 May 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae033
Issue No: Vol. 9, No. 1 (2024)
- Meaning and prediction of ‘excess mortality’: a comparison of Covid-19
and pre-Covid-19 mortality data in 31 Eurostat countries from 1965 to 2021
First page: bpae031
Abstract: AbstractDetermining ‘excess mortality’ makes it possible to compare the burden of disasters between countries and over time, and thus also to evaluate the success of mitigation measures. However, the debate on coronavirus disease 2019 (Covid-19) has exposed that calculations of excess mortalities vary considerably depending on the method and its specification. Moreover, it is often unclear what exactly is meant by ‘excess mortality’. We define excess mortality as the excess over the number of deaths that would have been expected counter-factually, that is without the catastrophic event in question. Based on this definition, we use a very parsimonious calculation method, namely the linear extrapolation of death figures from previous years to determine the excess mortality during the Covid-19 pandemic. But unlike most other literature on this topic, we first evaluated and optimized the specification of our method using a larger historical data set in order to identify and minimize estimation errors and biases. The result shows that excess mortality rates in the literature are often inflated. Moreover, they would have exhibited considerable excess mortalities in the period before Covid-19, if this value had already been of public interest at that time. Three conclusions can be drawn from this study and its findings: (i) All calculation methods for current figures should first be evaluated against past figures. (ii) To avoid alarm fatigue, thresholds should be introduced which would differentiate between ‘usual fluctuations’ and ‘remarkable excess’. (iii) Statistical offices could provide more realistic estimates.
PubDate: Fri, 17 May 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae031
Issue No: Vol. 9, No. 1 (2024)
- Comparing skin swabs, buccal swabs, and toe clips for amphibian genetic
sampling, a case study with a small anuran (Acris blanchardi)
First page: bpae030
Abstract: AbstractMultiple methods for collecting genetic samples from amphibians exist, each with their own implications for study design, animal welfare, and costs. Toe clipping is one common method, but there is ongoing debate regarding its potential detriment. Less invasive methods should be implemented, if efficacious, as amphibians are a particularly vulnerable vertebrate group. Skin and buccal swabbing are less invasive methods for genetic sampling, but the potential for contamination and a lower yield of DNA may exist. To compare these methods, we gathered skin swabs, buccal swabs, and toe clips from the same individuals of a relatively small anuran species, Blanchard’s Cricket Frog (Acris blanchardi). We then compared DNA yield, DNA purity, amplification success rate, and genotypic data quality among sample types. We found toe clips and buccal swabs generated similar DNA yield and purity, with skin swabs yielding significantly less DNA of significantly lower purity than the other sample types. Amplification success rate was significantly higher using toe clips compared to the other sample types, though buccal swab samples amplified more readily than skin swabs. Genotypic data from toe clips and buccal swabs did not differ significantly in quality, but skin swab data quality was significantly lowest among sample types. Thus, skin swabbing could produce erroneous data in some situations, but buccal swabbing is likely an effective substitute to toe clipping, even for small species. Our results can help future researchers select which genetic sampling method might best suit their research needs.
PubDate: Thu, 16 May 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae030
Issue No: Vol. 9, No. 1 (2024)
- A highly sensitive stem-loop RT-qPCR method to study siRNA intracellular
pharmacokinetics and pharmacodynamics
First page: bpae029
Abstract: AbstractSmall interfering RNA (siRNA) is a powerful tool for sequence-specific silencing of disease-related genes. In this study, we established and validated a stem-loop reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method applicable for both chemically unmodified and modified siRNA, aiming to elucidate mechanistic intracellular pharmacokinetic and pharmacodynamic (PK/PD) properties of siRNA. We conducted a comprehensive evaluation of factors affecting intracellular siRNA quantification. Our study revealed that immobilization-based siRNA extraction introduced high variation, making it unsuitable for absolute quantification. Conversely, direct cell lysis followed by stem-loop RT-qPCR demonstrated excellent reproducibility, with a quantification range from 0.0002 to 20 femtomole (fmole) for unmodified siRNA and 0.02 to 20 fmole for modified siRNA. The design of a 6-bp overlapping RT primer facilitated the distinction of full-length antisense from its 3′-metabolites, and pre-annealing of antisense to RT primer enhanced sensitivity and reproducibility. Differences in siRNA loss during storage and sample processing were noted among microcentrifuge tubes from various manufacturers. Endogenous miR-16 served as a reference for normalizing cytoplasmic siRNA, while protein concentration post-immunoprecipitation lysis was used to normalize RNA-induced silencing complex (RISC)-loaded siRNA levels. This method successfully enabled a detailed characterization of the time profiles of cytoplasmic and RISC-loaded siRNA, advancing the in vitro–in vivo translation of siRNA therapeutics.
PubDate: Mon, 06 May 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae029
Issue No: Vol. 9, No. 1 (2024)
- Macro-based collagen quantification and segmentation in picrosirius
red-stained heart sections using light microscopy
First page: bpae027
Abstract: AbstractPicrosirius red staining constitutes an important and broadly used tool to visualize collagen and fibrosis in various tissues. Although multiple qualitative and quantitative analysis methods to evaluate fibrosis are available, many require specialized devices and software or lack objectivity and scalability. Here, we aimed to develop a versatile and powerful “QuantSeg” macro in the FIJI image processing software capable of automated, robust, and quick collagen quantification in cardiac tissue from light micrographs. To examine different patterns of fibrosis, an optional segmentation algorithm was implemented. To ensure the method’s validity, we quantified the collagen content in a set of wild-type versus plakoglobin-knockout murine hearts exhibiting extensive fibrosis using both the macro and an established, fluorescence microscopy-based method, and compared results. To demonstrate the capabilities of the segmentation feature, rat hearts were examined post-myocardial infarction. We found the QuantSeg macro to robustly detect the differences in fibrosis between knockout and control hearts. In sections with low collagen content, the macro yielded more consistent results than using the fluorescence microscopy-based technique. With its wide range of output parameters, ease of use, cost effectiveness, and objectivity, the QuantSeg macro has the potential to become an established method for analysis of PSR-stained tissue. The novel segmentation feature allows for automated evaluation of different patterns of cardiac fibrosis for the first time.
PubDate: Sat, 27 Apr 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae027
Issue No: Vol. 9, No. 1 (2024)
- Multiparameter flow cytometric detection and analysis of rare cells in in
vivo models of cancer metastasis
First page: bpae026
Abstract: AbstractRapid and reliable circulating tumor cell (CTC) and disseminated tumor cell (DTC) detection are critical for rigorous evaluation of in vivo metastasis models. Clinical data show that each step of the metastatic cascade presents increasing barriers to success, limiting the number of successful metastatic cells to fewer than 1 in 1,500,000,000. As such, it is critical for scientists to employ approaches that allow for the evaluation of metastatic competency at each step of the cascade. Here, we present a flow cytometry-based method that enables swift and simultaneous comparison of both CTCs and DTCs from single animals, enabling evaluation of multiple metastatic steps within a single model system. We present the necessary gating strategy and optimized sample preparation conditions necessary to capture CTCs and DTCs using this approach. We also provide proof-of-concept experiments emphasizing the appropriate limits of detection of these conditions. Most importantly, we successfully recover CTCs and DTCs from murine blood and bone marrow. In Supplemental materials, we expand the applicability of our method to lung tissue and exemplify a potential multi-plexing strategy to further characterize recovered CTCs and DTCs. This approach to multiparameter flow cytometric detection and analysis of rare cells in in vivo models of metastasis is reproducible, high throughput, broadly applicable, and highly adaptable to a wide range of scientific inquiries. Most notably, it simplifies the recovery and analysis of CTCs and DTCs from the same animal, allowing for a rapid first look at the comparative metastatic competency of various model systems throughout multiple steps of the metastatic cascade.
PubDate: Sat, 27 Apr 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae026
Issue No: Vol. 9, No. 1 (2024)
- Detection of SARS-CoV-2 B.1.1.529 (Omicron) variant by SYBR Green-based
RT-qPCR
First page: bpae020
Abstract: AbstractThe coronavirus disease 2019 (COVID-19) pandemic is unceasingly spreading across the globe, and recently a highly transmissible Omicron SARS-CoV-2 variant (B.1.1.529) has been discovered in South Africa and Botswana. Rapid identification of this variant is essential for pandemic assessment and containment. However, variant identification is mainly being performed using expensive and time-consuming genomic sequencing. In this study, we propose an alternative RT-qPCR approach for the detection of the Omicron BA.1 variant using a low-cost and rapid SYBR Green method. We have designed specific primers to confirm the deletion mutations in the spike (S Δ143-145) and the nucleocapsid (N Δ31-33) which are characteristics of this variant. For the evaluation, we used 120 clinical samples from patients with PCR-confirmed SARS-CoV-2 infections, and displaying an S-gene target failure (SGTF) when using TaqPath COVID-19 kit (Thermo Fisher Scientific, Waltham, USA) that included the ORF1ab, S, and N gene targets. Our results showed that all the 120 samples harbored S Δ143-145 and N Δ31-33, which was further confirmed by whole-genome sequencing of 10 samples, thereby validating our SYBR Green-based protocol. This protocol can be easily implemented to rapidly confirm the diagnosis of the Omicron BA.1 variant in COVID-19 patients and prevent its spread among populations, especially in countries with high prevalence of SGTF profile.
PubDate: Sat, 27 Apr 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae020
Issue No: Vol. 9, No. 1 (2024)
- Correction to: An improved method for measuring catalase activity in
biological samples
First page: bpae025
Abstract: This is a correction to: Mahmoud Hussein Hadwan, Marwah Jaber Hussein, Rawa M Mohammed, Asad M Hadwan, Hawraa Saad Al-Kawaz, Saba S M Al-Obaidy, Zainab Abbas Al Talebi, An improved method for measuring catalase activity in biological samples, Biology Methods and Protocols, Volume 9, Issue 1, 2024, bpae015, https://doi.org/10.1093/biomethods/bpae015
PubDate: Fri, 26 Apr 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae025
Issue No: Vol. 9, No. 1 (2024)
- An extended multiplicative error model of allometry: Incorporating
systematic components, non-normal distributions, and piecewise
heteroscedasticity
First page: bpae024
Abstract: AbstractAllometry refers to the relationship between the size of a trait and that of the whole body of an organism. Pioneering observations by Otto Snell and further elucidation by D’Arcy Thompson set the stage for its integration into Huxley’s explanation of constant relative growth that epitomizes through the formula of simple allometry. The traditional method to identify such a model conforms to a regression protocol fitted in the direct scales of data. It involves Huxley’s formula-systematic part and a lognormally distributed multiplicative error term. In many instances of allometric examination, the predictive strength of this paradigm is unsuitable. Established approaches to improve fit enhance the complexity of the systematic relationship while keeping the go-along normality-borne error. These extensions followed Huxley’s idea that considering a biphasic allometric pattern could be necessary. However, for present data composing 10 410 pairs of measurements of individual eelgrass leaf dry weight and area, a fit relying on a biphasic systematic term and multiplicative lognormal errors barely improved correspondence measure values while maintaining a heavy tails problem. Moreover, the biphasic form and multiplicative-lognormal-mixture errors did not provide complete fit dependability either. However, updating the outline of such an error term to allow heteroscedasticity to occur in a piecewise-like mode finally produced overall fit consistency. Our results demonstrate that when attempting to achieve fit quality improvement in a Huxley’s model-based multiplicative error scheme, allowing for a complex allometry form for the systematic part, a non-normal distribution-driven error term and a composite of uneven patterns to describe the heteroscedastic outline could be essential.
PubDate: Thu, 18 Apr 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae024
Issue No: Vol. 9, No. 1 (2024)
- Teaching scientific evidence and critical thinking for policy making
First page: bpae023
Abstract: AbstractWhile there is worldwide tendency to promote the use of scientific evidence to inform policy making, little has been done to train scientists and policy makers for this interaction. If we want to bridge the gap between academia, scientific knowledge, and policy, we must begin by providing formal training and skill building for actors and stakeholders. Scientists are not trained to communicate and inform policy, and policy makers are not trained to understand scientific process and assess evidence. Building an environment where this collaboration can flourish depends on teaching competencies and abilities specific for decision-making processes. As professors of policy with a background in science, we have started teaching preliminary courses on the use of scientific evidence in policy making. Feedback from students and institutions has been positive, paving the way for similar courses in other schools and institutions and maybe even new career paths. This article is intended to share our experience in designing and teaching courses aimed at training policy makers. Moving forward we plan to include training for science majors, thus encompassing the two main sides of this dialogue and opening new career opportunities for scientists and policy makers.
PubDate: Thu, 11 Apr 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae023
Issue No: Vol. 9, No. 1 (2024)
- Intra-arterial delivery of neurospheres into isolated perfused porcine
colons: a proof of concept
First page: bpae022
Abstract: AbstractCell replacement in aganglionic intestines is a promising, yet merely experimental tool for the therapy of congenital dysganglionosis of the enteric nervous system like Hirschsprung disease. While the injection of single cells or neurospheres to a defined and very restricted location is trivial, the translation to the clinical application, where large aganglionic or hypoganglionic areas need to be colonized (hundreds of square centimetres), afford a homogeneous distribution of multiple neurospheres all over the affected tissue areas. Reaching the entire aganglionic area in vivo is critical for the restoration of peristaltic function. The latter mainly depends on an intact nervous system that extends throughout the organ. Intra-arterial injection is a common method in cell therapy and may be the key to delivering cells or neurospheres into the capillary bed of the colon with area-wide distribution. We describe an experimental method for monitoring the distribution of a defined number of neurospheres into porcine recta ex vivo, immediately after intra-arterial injection. We designed this method to localize grafting sites of single neurospheres in precise biopsies which can further be examined in explant cultures. The isolated perfused porcine rectum allowed us to continuously monitor the perfusion pressure. A blockage of too many capillaries would lead to an ischaemic situation and an increase of perfusion pressure. Since we could demonstrate that the area-wide delivery of neurospheres did not alter the overall vascular resistance, we showed that the delivery does not significantly impair the local circulation.
PubDate: Tue, 02 Apr 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae022
Issue No: Vol. 9, No. 1 (2024)
- Pyrimethamine reduced tumour growth in pre-clinical cancer models: a
systematic review to identify potential pre-clinical studies for
subsequent human clinical trials
First page: bpae021
Abstract: AbstractPyrimethamine (PYR), a STAT3 inhibitor, has been shown to reduce tumour burden in mouse cancer models. It is unclear how much of a reduction occurred or whether the PYR dosages and route of administration used in mice were consistent with the FDA's recommendations for drug repurposing. Search engines such as ScienceDirect, PubMed/MEDLINE, and other databases, including Google Scholar, were thoroughly searched, as was the reference list. The systematic review includes fourteen (14) articles. The risk of bias (RoB) was assessed using SYRCLE's guidelines. Due to the heterogeneity of the data, no meta-analysis was performed. According to the RoB assessment, 13/14 studies fall into the moderate RoB category, with one study classified as high RoB. None adhered to the ARRIVE guideline for transparent research reporting. Oral (FDA-recommended) and non-oral routes of PYR administration were used in mice, with several studies reporting very high PYR dosages that could lead to myelosuppression, while oral PYR dosages of 30 mg/kg or less are considered safe. Direct human equivalent dose translation is probably not the best strategy for comparing whether the used PYR dosages in mice are in line with FDA-approved strength because pharmacokinetic profiles, particularly PYR's half-life (t1/2), between humans (t1/2 = 96 h) and mice (t1/2 = 6 h), must also be considered. Based on the presence of appropriate control and treatment groups, as well as the presence of appropriate clinically proven chemotherapy drug(s) for comparison purposes, only one study (1/14) involving liver cancer can be directed into a clinical trial. Furthermore, oesophageal cancer too can be directed into clinical trials, where the indirect effect of PYR on the NRF2 gene may suppress oesophageal cancer in patients, but this must be done with caution because PYR is an investigational drug for oesophageal cancer, and combining it with proven chemotherapy drug(s) is recommended.
PubDate: Fri, 29 Mar 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae021
Issue No: Vol. 9, No. 1 (2024)
- An optimized protocol for generating appendage-bearing skin organoids from
human-induced pluripotent stem cells
First page: bpae019
Abstract: AbstractOrganoid generation from pluripotent stem cells is a cutting-edge technique that has created new possibilities for modelling human organs in vitro, as well as opening avenues for regenerative medicine. Here, we present a protocol for generating skin organoids (SKOs) from human-induced pluripotent stem cells (hiPSCs) via direct embryoid body formation. This method provides a consistent start point for hiPSC differentiation, resulting in SKOs with complex skin architecture and appendages (e.g. hair follicles, sebaceous glands, etc.) across hiPSC lines from two different somatic sources.
PubDate: Fri, 22 Mar 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae019
Issue No: Vol. 9, No. 1 (2024)
- Protocol for preliminary, multicenteric validation of “PoCOsteo
device”: A point of care tool for proteomic and genomic study of
osteoporosis
First page: bpae006
Abstract: Abstract One of the goals of the HORIZON 2020 project PoCOsteo was to develop a medical device, which would measure and/or quantify proteomic as well as genomic factors as present in whole blood samples collected through finger prick. After validating the tool in the clinical setting, the next step would be its clinical validation based on the existing guidelines. This article presents the protocol of a validation study to be carried out independently at two different centers (Division of Endocrinology and Diabetology at the Medical University of Graz as a clinic-based cohort, and the Endocrinology and Metabolism Research Institute at the Tehran University of Medical Sciences as a population-based cohort). It aims to assess the tool according to the Clinical & Laboratory Standards Institute guidelines, confirming if the proteomics and genomics measurements provided by the tool are accurate and reproducible compared with the existing state-of-the-art tests. This is the first time that such a detailed protocol for lab validation of a medical tool for proteomics and genomic measurement is designed based on the existing guidelines and thus could be used as a template for clinical validation of future point-of-care tools. Moreover, the multicentric cohort design will allow the study of a large number of diverse individuals, which will improve the validity and generalizability of the results for different settings.
PubDate: Fri, 22 Mar 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae006
Issue No: Vol. 9, No. 1 (2024)
- A semi-automated spectral approach to analyzing cyclical growth patterns
using fish scales
First page: bpae018
Abstract: AbstractWe introduce a new semi-automated approach to analyzing growth patterns recorded on fish scales. After manually specifying the center of the scale, the algorithm radially unwraps the scale patterns along a series of transects from the center to the edge of the scale. A sliding window Fourier transform is used to produce a spectrogram for each sampled transect of the scale image. The maximum frequency over all sampled transects of the average spectrogram yields a well-discriminated peak frequency trace that can then serve as a growth template for that fish. The spectrogram patterns of individual fish scales can be adjusted to a common period accounting for differences in date of return or size of fish at return without biasing the growth profile of the scale. We apply the method to 147 Atlantic salmon scale images sampled from 3 years and contrast the information derived with this automated approach to what is obtained using classical human operator measurements. The spectrogram analysis quantifies growth patterns using the entire scale image rather than just a single transect and provides the possibility of more robustly analyzing individual scale growth patterns. This semi-automated approach that removes essentially all the human operator interventions provides an opportunity to process large datasets of fish scale images and combined with advanced analyses such as deep learning methods could lead to a greater understanding of salmon marine migration patterns and responses to variations in ecosystem conditions.
PubDate: Mon, 18 Mar 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae018
Issue No: Vol. 9, No. 1 (2024)
- TypeTaxonScript: sugarifying and enhancing data structures in biological
systematics and biodiversity research
First page: bpae017
Abstract: AbstractObject-oriented programming (OOP) embodies a software development paradigm grounded in representing real-world entities as objects, facilitating a more efficient and structured modelling approach. In this article, we explore the synergy between OOP principles and the TypeScript (TS) programming language to create a JSON-formatted database designed for storing arrays of biological features. This fusion of technologies fosters a controlled and modular code script, streamlining the integration, manipulation, expansion, and analysis of biological data, all while enhancing syntax for improved human readability, such as through the use of dot notation. We advocate for biologists to embrace Git technology, akin to the practices of programmers and coders, for initiating versioned and collaborative projects. Leveraging the widely accessible and acclaimed IDE, Visual Studio Code, provides an additional advantage. Not only does it support running a Node.js environment, which is essential for running TS, but it also efficiently manages GitHub versioning. We provide a use case involving taxonomic data structure, focusing on angiosperm legume plants. This method is characterized by its simplicity, as the tools employed are both fully accessible and free of charge, and it is widely adopted by communities of professional programmers. Moreover, we are dedicated to facilitating practical implementation and comprehension through a comprehensive tutorial, a readily available pre-built database at GitHub, and a new package at npm.
PubDate: Thu, 14 Mar 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae017
Issue No: Vol. 9, No. 1 (2024)
- Alternative community-led intervention to improve uptake of cataract
surgery services in rural Tanzania—The Dodoma Community Cataract
Acceptance Trial (DoCCAT): a protocol for intervention co-designing and
implementation in a cluster-randomized controlled trial
First page: bpae016
Abstract: AbstractAge-related lens opacification (cataract) remains the leading cause of visual impairment and blindness worldwide. In low- and middle-income countries, utilization of cataract surgical services is often limited despite community-based outreach programmes. Community-led research, whereby researchers and community members collaboratively co-design intervention is an approach that ensures the interventions are locally relevant and that their implementation is feasible and socially accepted in the targeted contexts. Community-led interventions have the potential to increase cataract surgery uptake if done appropriately.In this study, once the intervention is co-designed it will be implemented through a cluster-randomized controlled trial (cRCT) with ward as a unit of randomization.This study will utilise both the qualitative methods for co-designing the intervention and the quantitative methods for effective assessment of the developed community-led intervention through a cRCT in 80 rural wards of Dodoma region, Tanzania (40 Intervention). The ‘intervention package’ will be developed through participatory community meetings and ongoing evaluation and modification of the intervention based on its impact on service utilization. Leask’s four stages of intervention co-creation will guide the development within Rifkin’s CHOICE framework. The primary outcomes are two: the number of patients attending eye disease screening camps, and the number of patients accepting cataract surgery. NVivo version 12 will be used for qualitative data analysis and Stata version 12 for quantitative data. Independent and paired t-tests will be performed to make comparisons between and within groups. P-values less than 0.05 will be considered statistically significant.
PubDate: Fri, 08 Mar 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae016
Issue No: Vol. 9, No. 1 (2024)
- A rapid and accurate method for evaluating the degradation of pan-Akt in
cells by PROTACs using NanoLuc luciferase
First page: bpae014
Abstract: AbstractProteolysis targeting chimera (PROTAC) is a protein degradation technique that has been increasingly used in the development of new drugs in recent years. Akt is a classical serine/threonine kinase, and its role outside of the kinase has gradually gained attention in recent years, making it one of the proteins targeted by PROTACs. Currently, there are many methods used for the evaluation of intracellular protein degradation, but each has its own advantages or disadvantages. This study aimed to investigate the feasibility of evaluating the degradation of pan-Akt proteins in cells by PROTACs (MS21 and MS170) using the NanoLuc luciferase method. After conducting a thorough comparison between this method and the classical western blot assay in various cells, as well as testing the stability of the experiments between multiple batches, we found that NanoLuc luciferase is a highly accurate, stable, low-cost and easy-to-operate method for the evaluation of intracellular pan-Akt degradation by PROTACs with a short cycle time and high cellular expandability. Given the numerous advantages of this method, it is hypothesized that it could be extended to evaluate the degradation of more target proteins of PROTACs. In summary, the NanoLuc luciferase is a suitable method for early protein degradation screening of PROTAC compounds.
PubDate: Tue, 05 Mar 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae014
Issue No: Vol. 9, No. 1 (2024)
- An improved method for measuring catalase activity in biological samples
First page: bpae015
Abstract: AbstractCatalase (CAT) is an important enzyme that protects biomolecules against oxidative damage by breaking down hydrogen peroxide (H2O2) into water and oxygen. CAT is present in all aerobic microbes, animals, and plants. It is, however, absent from normal human urine but can be detected in pathological urine. CAT testing can thus help to detect such urine. This study presents a novel spectrophotometric method for determining CAT activity characterized by its simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with a carefully chosen concentration of H2O2 for a specified incubation period. Subsequently, a solution containing ferrous ammonium sulfate (FAS) and sulfosalicylic acid (SSA) is added to terminate the enzyme activity. A distinctive maroon-colored ferrisulfosalicylate complex is formed. The formation of this complex is a direct result of the reaction between FAS and any residual peroxide present. This leads to the generation of ferric ions when coordinated with SSA. The complex has a maximum absorbance of 490 nm. This advanced method eliminates the need for concentrated acids to stop CAT activity, making it safer and easier to handle. A comparative analysis against the standard ferrithiocyanate method showed a correlation coefficient of 0.99, demonstrating the new method’s comparable effectiveness and reliability. In conclusion, a simple and reliable protocol for assessing CAT activity, which utilizes a cuvette or microplate, has been demonstrated in this study. This interference-free protocol can easily be used in research and clinical analysis with considerable accuracy and precision.
PubDate: Tue, 05 Mar 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae015
Issue No: Vol. 9, No. 1 (2024)
- Pairing a bioinformatics-focused course-based undergraduate research
experience with specifications grading in an introductory biology
classroom
First page: bpae013
Abstract: AbstractIntroducing bioinformatics-focused concepts and skills in a biology classroom is difficult, especially in introductory biology classrooms. Course-based Undergraduate Research Experiences (CUREs) facilitate this process, introducing genomics and bioinformatics through authentic research experiences, but the many learning objectives needed in scientific research and communication, foundational biology concepts, and bioinformatics-focused concepts and skills can make the process challenging. Here, the pairing of specifications grading with a bioinformatics-focused CURE developed by the Genomics Education Partnership is described. The study examines how the course structure with specifications grading facilitated scaffolding of writing assignments, group work, and metacognitive activities; and describes the synergies between CUREs and specifications grading. CUREs require mastery of related concepts and skills for working through the research process, utilize common research practices of revision and iteration, and encourage a growth mindset to learning—all of which are heavily incentivized in assessment practices focused on specifications grading.
PubDate: Wed, 28 Feb 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae013
Issue No: Vol. 9, No. 1 (2024)
- Correction to: Long amplicon nanopore sequencing of Botrytis cinerea and
other fungal species present in infected grapevine leaf samples
First page: bpae010
Abstract: This is a correction to: Vladimer Baramidze, Luca Sella, Tamar Japaridze, Nino Abashidze, Daviti Lamazoshvili, Nino Dzotsenidze, Giorgi Tomashvili, Long amplicon nanopore sequencing of Botrytis cinerea and other fungal species present in infected grapevine leaf samples, Biology Methods and Protocols, Volume 9, Issue 1, 2024, https://doi.org/10.1093/biomethods/bpad042
PubDate: Fri, 23 Feb 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae010
Issue No: Vol. 9, No. 1 (2024)
- Rapid IDH1-R132 genotyping panel utilizing locked nucleic acid
loop-mediated isothermal amplification
First page: bpae012
Abstract: AbstractWhile the detection of single-nucleotide variants (SNVs) is important for evaluating human health and disease, most genotyping methods require a nucleic acid extraction step and lengthy analytical times. Here, we present a protocol which utilizes the integration of locked nucleic acids (LNAs) into self-annealing loop primers for the allelic discrimination of five isocitrate dehydrogenase 1 R132 (IDH1-R132) variants using loop-mediated isothermal amplification (LAMP). This genotyping panel was initially evaluated using purified synthetic DNA to show proof of specific SNV discrimination. Additional evaluation using glioma tumor lysates with known IDH1-R132 mutational status demonstrated specificity in approximately 35 min without the need for a nucleic acid extraction purification step. This LNA-LAMP-based genotyping assay can detect single base differences in purified nucleic acids or tissue homogenates, including instances where the variant of interest is present in an excess of background wild-type DNA. The pH-based colorimetric indicator of LNA-LAMP facilitates convenient visual interpretation of reactions, and we demonstrate successful translation to an end-point format using absorbance ratio, allowing for an alternative and objective approach for differentiating between positive and negative reactions. Importantly, the LNA-LAMP genotyping panel is highly reproducible, with no false-positive or false-negative results observed.
PubDate: Wed, 21 Feb 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae012
Issue No: Vol. 9, No. 1 (2024)
- Development of a highly sensitive TaqMan method based on multi-probe
strategy: its application in ASFV detection
First page: bpae011
Abstract: AbstractThe establishment of high sensitive detection method for various pathogenic microorganisms remains constantly concerned. In the present study, multi-probe strategy was first systematically investigated followed by establishing a highly sensitive TaqMan real-time fluorescent quantitative PCR (qPCR) method for detecting African swine fever virus (ASFV). Briefly, four probes based on the B646L gene of ASFV were designed and the effects of different combinations of the probes in a single TaqMan qPCR assay on the detection sensitivity were investigated. As less as 0.5-5 copies/μl of the ASFV gene was detected by the established TaqMan qPCR assay. Furthermore, plasmid harboring the B646L in water samples could be concentrated 1000 times by ultrafiltration to enable a highly sensitive detection of trace viral nucleic acids. Moreover, no cross-reactivity was observed with other common clinical swine viruses such as PCV2, PCV3, PCV4, PEDV, PDCoV, CSFV, PRRSV, and PRV. When detecting 173 clinical porcine serum samples, the coincidence rate between the developed method and WOAH (World Organization of Animal Health) recommended method was 100%. This study might provide an integrated strategy to achieve higher detection sensitivity of trace pathogenic microorganisms and applicably sensitive TaqMan-based qPCR assays.
PubDate: Mon, 19 Feb 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae011
Issue No: Vol. 9, No. 1 (2024)
- A methodological primer of extracellular vesicles isolation and
characterization via different techniques
First page: bpae009
Abstract: AbstractWe present four different protocols of varying complexity for the isolation of cell culture-derived extracellular vesicles (EVs)/exosome-enriched fractions with the objective of providing researchers with easily conducted methods that can be adapted for many different uses in various laboratory settings and locations. These protocols are primarily based on polymer precipitation, filtration and/or ultracentrifugation, as well as size-exclusion chromatography (SEC) and include: (i) polyethylene glycol and sodium chloride supplementation of the conditioned medium followed by low-speed centrifugation; (ii) ultracentrifugation of conditioned medium; (iii) filtration of conditioned media through a 100-kDa exclusion filter; and (iv) isolation using a standard commercial kit. These techniques can be followed by further purification by ultracentrifugation, sucrose density gradient centrifugation, or SEC if needed and the equipment is available. HEK293 and SH-SY5Y cell cultures were used to generate conditioned medium containing exosomes. This medium was then depleted of cells and debris, filtered through a 0.2-µM filter, and supplemented with protease and RNAse inhibitors prior to exosomal isolation. The purified EVs can be used immediately or stably stored at 4°C (up to a week for imaging or using intact EVS downstream) or at −80°C for extended periods and then used for biochemical study. Our aim is not to compare these methodologies but to present them with descriptors so that researchers can choose the “best method” for their work under their individual conditions.
PubDate: Tue, 13 Feb 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae009
Issue No: Vol. 9, No. 1 (2024)
- Efficient regeneration of protoplasts from Solanum lycopersicum cultivar
Micro-Tom
First page: bpae008
Abstract: AbstractProtoplast regeneration has become a key platform for genetic and genome engineering. However, we lack reliable and reproducible methods for efficient protoplast regeneration for tomato (Solanum lycopersicum) cultivars. Here, we optimized cell and tissue culture methods for protoplast isolation, microcallus proliferation, shoot regeneration, and plantlet establishment of the tomato cultivar Micro-Tom. A thin layer of alginate was applied to protoplasts isolated from third to fourth true leaves and cultured at an optimal density of 1 × 105 protoplasts/ml. We determined the optimal culture media for protoplast proliferation, callus formation, de novo shoot regeneration, and root regeneration. Regenerated plantlets exhibited morphologically normal growth and sexual reproduction. The entire regeneration process, from protoplasts to flowering plants, was accomplished within 5 months. The optimized protoplast regeneration platform enables biotechnological applications, such as genome engineering, as well as basic research on plant regeneration in Solanaceae species.
PubDate: Tue, 06 Feb 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae008
Issue No: Vol. 9, No. 1 (2024)
- An amplicon-based protocol for whole-genome sequencing of human
respiratory syncytial virus subgroup A
First page: bpae007
Abstract: Abstract It is convenient to study complete genome sequences of human respiratory syncytial virus (hRSV) for ongoing genomic characterization and identification of highly transmissible or pathogenic variants. Whole genome sequencing of hRSV has been challenging from respiratory tract specimens with low viral loads. Herein, we describe an amplicon-based protocol for whole genome sequencing of hRSV subgroup A validated with 24 isolates from nasopharyngeal swabs and infected cell cultures, which showed cycle threshold (Ct) values ranging from 10 to 31, as determined by quantitative reverse-transcription polymerase chain reaction. MinION nanopore generated 3200 to 5400 reads per sample to sequence over 93% of the hRSV-A genome. Coverage of each contig ranged from 130× to 200×. Samples with Ct values of 20.9, 25.2, 27.1, 27.7, 28.2, 28.8, and 29.6 led to the sequencing of over 99.0% of the virus genome, indicating high genome coverage even at high Ct values. This protocol enables the identification of hRSV subgroup A genotypes, as primers were designed to target highly conserved regions. Consequently, it holds potential for application in molecular epidemiology and surveillance of this hRSV subgroup.
PubDate: Tue, 06 Feb 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae007
Issue No: Vol. 9, No. 1 (2024)
- Establishment of new transurethral catheterization methods for male mice
First page: bpae005
Abstract: AbstractTransurethral catheterization in mice is multifaceted, serving essential functions such as perfusion and drug delivery, and is critical in the development of various urological animal disease models. The complex anatomy of the male mouse urethra presents significant challenges in transurethral catheterization, leading to a predominance of research focused on female specimens. This bias limits the utilization of male mice in lower urinary tract disease studies. Our research aims to develop new reliable methods for transurethral catheterization in adult male mice, thereby expanding their use in relevant disease research. Experiments were conducted on adult male C57BL/6J mice. Utilizing a PE10 catheter measuring 4.5–5 cm in length, the catheter was inserted into the bladder via the mouse’s urethra under anesthesia. The intubation technique entailed regulating the insertion force, ensuring the catheter's lubrication, using a trocar catheter, modifying the catheter’s trajectory, and accommodating the curvature of the bladder neck. Post-catheter insertion, ultrasound imaging was employed to confirm the catheter's accurate positioning within the bladder. Subsequent to catheterization, the bladder was perfused using trypan blue. This method was further validated through its successful application in establishing an acute urinary retention (AUR) model, where the mouse bladder was infused with saline to a pressure of 50 or 80 cm H2O, maintained steadily for 30 min. A thorough morphological assessment of the mouse bladder was conducted after the infusion. Our study successfully pioneered methods for transurethral catheterization in male mice. This technique not only facilitates precise transurethral catheterization but also proves applicable to male mouse models for lower urinary tract diseases, such as AUR.
PubDate: Mon, 05 Feb 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae005
Issue No: Vol. 9, No. 1 (2024)
- Identification and quantitation of multiple variants in RNA virus genomes
First page: bpae004
Abstract: AbstractThe goal of the study was to identify and characterize RNA virus variants containing mutations spread over genomic distances >5 kb. As proof of concept, high-quality viral RNA of the Dengue 2 component of Takeda’s tetravalent dengue vaccine candidate (TDV-2) was used to develop a reverse transcription–polymerase chain reaction protocol to amplify a ∼5.3 kb cDNA segment that contains the three genetic determinants of TDV-2 attenuation. Unique molecular identifiers were incorporated into each viral cDNA molecule for PacBio library preparation to improve the quantitative precision of the observed variants at the attenuation loci. Following assay optimization, PacBio long-read sequencing was validated with multiple clone-derived TDV-2 revertant variants and four complex revertant mixtures containing various compositions of TDV-2 and revertant viruses. PacBio sequencing analysis correctly identified and quantified variant composition in all tested samples, demonstrating that TDV-2 revertants could be identified and characterized and supporting the use of this method in the differentiation and quantification of complex variants of other RNA viruses. Long-read sequencing can identify complex RNA virus variants containing multiple mutations on a single-genome molecule, which is useful for in-depth genetic stability and revertant detection of live-attenuated viral vaccines, as well as research in virus evolution to reveal mechanisms of immune evasion and host cell adaption.
PubDate: Sat, 03 Feb 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae004
Issue No: Vol. 9, No. 1 (2024)
- Optimized protocol for shotgun label-free proteomic analysis of pancreatic
islets
First page: bpae003
Abstract: AbstractPancreatic islets are crucial in diabetes research. Consequently, this protocol aims at optimizing both the protein-extraction process and the proteomic analysis via shotgun methods for pancreatic islets. Six protocols were tested, combining three types of chemical extraction with two mechanical extraction methods. Furthermore, two protocols incorporated a surfactant to enhance enzymatic cleavage. The steps involved extraction and concentration of protein, protein quantification, reduction, alkylation, digestion, purification and desalination, sample concentration to ∼1 µl, and proteomic analysis using the mass spectrometer. The most effective protocol involves either a milder chemical extraction paired with a more intensive mechanical process, or a more robust chemical extraction paired with a gentle mechanical process, tailored to the sample’s characteristics. Additionally, it was observed that the use of a surfactant proved ineffective for these types of samples. Protocol 5 was recently used with success to examine metabolic changes in pancreatic islets of non-obese diabetic mice exposed to low doses of fluoride ions (F−) and the primary pathways altered by the treatment.
PubDate: Thu, 25 Jan 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae003
Issue No: Vol. 9, No. 1 (2024)
- Baricitinib statistically significantly reduced COVID-19-related
mortality: a systematic review and meta-analysis of five phase III
randomized, blinded and placebo-controlled clinical trials
First page: bpae002
Abstract: AbstractDue to high heterogeneity and risk of bias (RoB) found in previously published meta-analysis (MA), a concrete conclusion on the efficacy of baricitinib in reducing mortality in coronavirus disease 2019 (COVID-19) patients was unable to form. Hence, this systematic review and MA were conducted to analyse whether RoB, heterogeneity, and optimal sample size from placebo-controlled randomized controlled trials (RCTs) are still the problems to derive a concrete conclusion.Search engines PubMed/MEDLINE, ScienceDirect, and other sources like preprints and reference lists were searched with appropriate keywords. The RoB and MA were conducted using RevMan 5.4. The grading of the articles was conducted using the GRADEPro Guideline Development Tool.Ten RCTs were included in the current systematic review. Only five low RoB articles are Phase III placebo-controlled RCTs with a high certainty level based on the GRADE grading system. For the MA, based on five low RoB articles, baricitinib statistically significantly reduced mortality where the risk ratio (RR) = 0.68 [95% confidence interval (95% CI) 0.56–0.82; P < 0.0001; I2 = 0%; P = 0.85]. The absolute mortality effect (95% CI) based on the grading system was 35 fewer mortalities per 1000 COVID-19 patients, whereas in the baricitinib and control groups, the mortality was 7.4% and 10.9%, respectively.With the presence of an optimal sample size of 3944 from five low RoB–placebo-controlled RCTs, which represent a minimum of 300 million population of people and with the presence of 0% heterogeneity from MA, the effectiveness of baricitinib in reducing the mortality in COVID-19 patients is concretely proven.
PubDate: Tue, 23 Jan 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae002
Issue No: Vol. 9, No. 1 (2024)
- tANCHOR cell-based ELISA approach as a surrogate for antigen-coated plates
to monitor specific IgG directed to the SARS-CoV-2 receptor-binding domain
First page: bpae001
Abstract: AbstractEnzyme-linked immunosorbent assay (ELISA) systems use plates coated with peptides or expressed and purified proteins to monitor immunoglobulins derived from patient serum. However, there is currently no easy, flexible, and fast adaptive ELISA-based system for testing antibodies directed against new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. In this study, we utilized the tANCHOR protein display system that provides a cell surface decorated with the receptor-binding domain (RBD) to monitor specific antibodies derived from SARS-CoV-2 convalescent and vaccinated individuals directed against it. To test sera from vaccinees or convalescent individuals, only the RBD coding sequence needs to be cloned in the tANCHOR vector system and transfected into HeLa cells. Time-consuming protein expression, isolation, and purification followed by coating assay plates are not necessary. With this technique, the immune evasion of new SARS-CoV-2 variants from current vaccination regimes can be examined quickly and reliably.
PubDate: Fri, 19 Jan 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpae001
Issue No: Vol. 9, No. 1 (2024)
- Teaching at the intersection of science and society: An activity on
healthcare disparities
First page: bpad041
Abstract: AbstractUnderstanding the relationship between science and society is an objective of science education and is included as a core competency in the AAAS Vision and Change guidelines for biology education. However, traditional undergraduate biology instruction emphasizes scientific practice and generally avoids potentially controversial issues at the intersection of biology and society. By including these topics in biology coursework, instructors can challenge damaging ideologies and systemic inequalities that have influenced science, such as biological essentialism and health disparities. Specifically, an ideologically aware curriculum highlights how ideologies and paradigms shape our biological knowledge base and the application of that knowledge. Ideologically aware lessons emphasize the relationship between science and society with an aim to create more transparent, scientifically accurate, and inclusive postsecondary biology classrooms. Here we expand upon our ideologically aware curriculum with a new activity that challenges undergraduate biology students to consider the impacts of healthcare disparities. This lesson allows instructors to directly address systemic inequalities and allows students to connect biomedical sciences to real-world issues. Implementing an ideologically aware curriculum enables students to challenge prevailing worldviews and better address societal problems that lead to exclusion and oppression.
PubDate: Fri, 05 Jan 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpad041
Issue No: Vol. 9, No. 1 (2024)
- Long amplicon nanopore sequencing of Botrytis cinerea and other fungal
species present in infected grapevine leaf samples
First page: bpad042
Abstract: AbstractBotrytis cinerea is a well-known plant pathogen responsible for grey mould disease infecting more than 500 plant species. It is listed as the second most important plant pathogen scientifically and economically. Its impact is particularly severe in grapes since it affects both the yield of grape berries and the quality of wines. While various methods for detecting B. cinerea have been investigated, the application of Oxford Nanopore Technology (ONT) for complete ribosomal operon sequencing, which has proven effective in human and animal fungal research and diagnostics, has not yet been explored in grapevine (Vitis vinifera) disease research. In this study, we sequenced complete ribosomal operons (∼5.5 kb amplicons), which encompass the 18S, ITS1, 5.8S, ITS2, and 28S regions, from both pure cultures of B. cinerea and infected grapevine leaf samples. Minimap2, a sequence alignment tool integrated into the EPI2ME software, served as a taxonomy classifier, utilizing the custom reference database FRODO. The results demonstrate that B. cinerea was detectable when this pathogen was not the dominant fungal species in leaf samples. Additionally, the method facilitates host DNA-free sequencing and might have a good potential to distinguish other pathogenic and non-pathogenic fungal species hosted within grapevine’s infected leaves, such as Alternaria alternata, Saccharomyces cerevisiae, Saccharomyces boulardii, Mucor racemosus, and Ascochyta rabie. The sequences were uploaded to the NCBI database. Long amplicon sequencing method has the capacity to be broadened to other susceptible crops and pathogens, as a valuable tool for early grey rot detection and mycobiome research. Future large-scale studies are needed to overcome challenges, such as comprehensive reference databases for complete fungal ribosomal operons for grape mycobiome studies.
PubDate: Fri, 05 Jan 2024 00:00:00 GMT
DOI: 10.1093/biomethods/bpad042
Issue No: Vol. 9, No. 1 (2024)
