Showing papers in "Nature Reviews Methods Primers in 2022"

Electrochemical stripping analysis

Abstract: This PrimeView highlights the pre-concentration and stripping steps in electrochemical stripping analysis.

Mendelian randomization

Abstract: Mendelian randomization (MR) is a term that applies to the use of genetic variation to address causal questions about how modifiable exposures influence different outcomes. The principles of MR are based on Mendel’s laws of inheritance and instrumental variable estimation methods, which enable the inference of causal effects in the presence of unobserved confounding. In this Primer, we outline the principles of MR, the instrumental variable conditions underlying MR estimation and some of the methods used for estimation. We go on to discuss how the assumptions underlying an MR study can be assessed and describe methods of estimation that are robust to certain violations of these assumptions. We give examples of a range of studies in which MR has been applied, the limitations of current methods of analysis and the outlook for MR in the future. The differences between the assumptions required for MR analysis and other forms of epidemiological studies means that MR can be used as part of a triangulation across multiple sources of evidence for causal inference. Mendelian randomization is a technique for using genetic variation to examine the causal effect of a modifiable exposure on an outcome such as disease status. This Primer by Sanderson et al. explains the concepts of and the conditions required for Mendelian randomization analysis, describes key examples of its application and looks towards applying the technique to growing genomic datasets.

A guide to the organ-on-a-chip

Abstract: Organs-on-chips (OoCs) are systems containing engineered or natural miniature tissues grown inside microfluidic chips. To better mimic human physiology, the chips are designed to control cell microenvironments and maintain tissue-specific functions. Combining advances in tissue engineering and microfabrication, OoCs have gained interest as a next-generation experimental platform to investigate human pathophysiology and the effect of therapeutics in the body. There are as many examples of OoCs as there are applications, making it difficult for new researchers to understand what makes one OoC more suited to an application than another. This Primer is intended to give an introduction to the aspects of OoC that need to be considered when developing an application-specific OoC. The Primer covers guiding principles and considerations to design, fabricate and operate an OoC, as well as subsequent assaying techniques to extract biological information from OoC devices. Alongside this is a discussion of current and future applications of OoC technology, to inform design and operational decisions during the implementation of OoC systems. Organs-on-chips are microfluidic systems containing miniature tissues with the aim of mimicking human physiology for a range of biomedical and therapeutic applications. Leung, de Haan et al. report practical tips to inform design and operational decisions during the implementation of organ-on-a-chip systems.

High-content CRISPR screening

Abstract: CRISPR screens are a powerful source of biological discovery, enabling the unbiased interrogation of gene function in a wide range of applications and species. In pooled CRISPR screens, various genetically encoded perturbations are introduced into pools of cells. The targeted cells proliferate under a biological challenge such as cell competition, drug treatment or viral infection. Subsequently, the perturbation-induced effects are evaluated by sequencing-based counting of the guide RNAs that specify each perturbation. The typical results of such screens are ranked lists of genes that confer sensitivity or resistance to the biological challenge of interest. Contributing to the broad utility of CRISPR screens, adaptations of the core CRISPR technology make it possible to activate, silence or otherwise manipulate the target genes. Moreover, high-content read-outs such as single-cell RNA sequencing and spatial imaging help characterize screened cells with unprecedented detail. Dedicated software tools facilitate bioinformatic analysis and enhance reproducibility. CRISPR screening has unravelled various molecular mechanisms in basic biology, medical genetics, cancer research, immunology, infectious diseases, microbiology and other fields. This Primer describes the basic and advanced concepts of CRISPR screening and its application as a flexible and reliable method for biological discovery, biomedical research and drug development — with a special emphasis on high-content methods that make it possible to obtain detailed biological insights directly as part of the screen. CRISPR screening is a high-throughput approach for identifying genes, pathways and mechanisms involved in a given phenotype or biological process. High-content read-outs of these screens, such as imaging and single-cell sequencing techniques, have further broadened its applicability. This Primer by Bock et al. describes the main concepts of CRISPR screening and gives examples of its application as a method for biological discovery, with a focus on the use of high-content read-outs.

Nanodelivery of nucleic acids

Abstract: There is growing need for a safe, efficient, specific and non-pathogenic means for delivery of gene therapy materials. Nanomaterials for nucleic acid delivery offer an unprecedented opportunity to overcome these drawbacks; owing to their tunability with diverse physico-chemical properties, they can readily be functionalized with any type of biomolecules/moieties for selective targeting. Nucleic acid therapeutics such as antisense DNA, mRNA, small interfering RNA (siRNA) or microRNA (miRNA) have been widely explored to modulate DNA or RNA expression Strikingly, gene therapies combined with nanoscale delivery systems have broadened the therapeutic and biomedical applications of these molecules, such as bioanalysis, gene silencing, protein replacement and vaccines. Here, we overview how to design smart nucleic acid delivery methods, which provide functionality and efficacy in the layout of molecular diagnostics and therapeutic systems. It is crucial to outline some of the general design considerations of nucleic acid delivery nanoparticles, their extraordinary properties and the structure–function relationships of these nanomaterials with biological systems and diseased cells and tissues. In this Primer, Conde and colleagues explain how to design smart nucleic acid delivery methods, which provide functionality and efficacy in the layout of molecular diagnostics and therapeutic systems.

Sustainable polymers

Abstract: This PrimeView highlights the life cycle analysis and end-of-life considerations necessary for designing sustainable polymers.

Optogenetics for light control of biological systems

Abstract: Optogenetic techniques have been developed to allow control over the activity of selected cells within a highly heterogeneous tissue, using a combination of genetic engineering and light. Optogenetics employs natural and engineered photoreceptors, mostly of microbial origin, to be genetically introduced into the cells of interest. As a result, cells that are naturally light-insensitive can be made photosensitive and addressable by illumination and precisely controllable in time and space. The selectivity of expression and subcellular targeting in the host is enabled by applying control elements such as promoters, enhancers and specific targeting sequences to the employed photoreceptor-encoding DNA. This powerful approach allows precise characterization and manipulation of cellular functions and has motivated the development of advanced optical methods for patterned photostimulation. Optogenetics has revolutionized neuroscience during the past 15 years and is primed to have a similar impact in other fields, including cardiology, cell biology and plant sciences. In this Primer, we describe the principles of optogenetics, review the most commonly used optogenetic tools, illumination approaches and scientific applications and discuss the possibilities and limitations associated with optogenetic manipulations across a wide variety of optical techniques, cells, circuits and organisms. Optogenetic techniques involve the introduction of photoreceptors into selected cells to allow control over their activity using light. In this Primer, Emiliani et al. discuss the most commonly used optogenetic tools, illumination approaches and applications in medicine, cardiovascular science and plants, among many other uses.

Citizen science in environmental and ecological sciences

Abstract: Citizen science is an increasingly acknowledged approach applied in many scientific domains, and particularly within the environmental and ecological sciences, in which non-professional participants contribute to data collection to advance scientific research. We present contributory citizen science as a valuable method to scientists and practitioners within the environmental and ecological sciences, focusing on the full life cycle of citizen science practice, from design to implementation, evaluation and data management. We highlight key issues in citizen science and how to address them, such as participant engagement and retention, data quality assurance and bias correction, as well as ethical considerations regarding data sharing. We also provide a range of examples to illustrate the diversity of applications, from biodiversity research and land cover assessment to forest health monitoring and marine pollution. The aspects of reproducibility and data sharing are considered, placing citizen science within an encompassing open science perspective. Finally, we discuss its limitations and challenges and present an outlook for the application of citizen science in multiple science domains. Contributory citizen science is a method in which non-professional participants contribute to data collection in whole or in part to advance scientific research. This Primer outlines the use of citizen science in the environmental and ecological sciences, discussing participant engagement, data quality assurance and bias correction.

Optogenetics for light control of biological systems

Abstract: Optogenetic techniques have been developed to allow control over the activity of selected cells within a highly heterogeneous tissue, using a combination of genetic engineering and light. Optogenetics employs natural and engineered photoreceptors, mostly of microbial origin, to be genetically introduced into the cells of interest. As a result, cells that are naturally light-insensitive can be made photosensitive and addressable by illumination and precisely controllable in time and space. The selectivity of expression and subcellular targeting in the host is enabled by applying control elements such as promoters, enhancers and specific targeting sequences to the employed photoreceptor-encoding DNA. This powerful approach allows precise characterization and manipulation of cellular functions and has motivated the development of advanced optical methods for patterned photostimulation. Optogenetics has revolutionized neuroscience during the past 15 years and is primed to have a similar impact in other fields, including cardiology, cell biology and plant sciences. In this Primer, we describe the principles of optogenetics, review the most commonly used optogenetic tools, illumination approaches and scientific applications and discuss the possibilities and limitations associated with optogenetic manipulations across a wide variety of optical techniques, cells, circuits and organisms. Optogenetic techniques involve the introduction of photoreceptors into selected cells to allow control over their activity using light. In this Primer, Emiliani et al. discuss the most commonly used optogenetic tools, illumination approaches and applications in medicine, cardiovascular science and plants, among many other uses.

Organoids

Abstract: Organoids are simple tissue-engineered cell-based in vitro models that recapitulate many aspects of the complex structure and function of the corresponding in vivo tissue. They can be dissected and interrogated for fundamental mechanistic studies on development, regeneration and repair in human tissues, and can also be used in diagnostics, disease modelling, drug discovery and personalized medicine. Organoids are derived from either pluripotent or tissue-resident stem (embryonic or adult) or progenitor or differentiated cells from healthy or diseased tissues, such as tumours. To date, numerous organoid engineering strategies that support organoid culture and growth, proliferation, differentiation and maturation have been reported. This Primer highlights the rationale underlying the selection and development of these materials and methods to control the cellular/tissue niche; and therefore, the structure and function of the engineered organoid. We also discuss key considerations for generating robust organoids, such as those related to cell isolation and seeding, matrix and soluble factor selection, physical cues and integration. The general standards for data quality, reproducibility and deposition within the organoid community are also outlined. Lastly, we conclude by elaborating on the limitations of organoids in different applications, and the key priorities in organoid engineering for the coming years. Organoids are cell-based in vitro models derived from stem cells, reconstituting the complex structure and function of the corresponding tissue. In this Primer, Zhao, Chen, Dowbaj, Sljukic, Bratlie, Lin et al. discuss the development of organoids and methods for controlling their cellular environment.

Hydrogen generation from biomass by pyrolysis

Abstract: The growing environmental concerns associated with global warming along with the exponential rise in energy demand are boosting the production of clean energy. The combined process of biomass pyrolysis and in-line catalytic steam reforming is a promising alternative for the selective production of hydrogen from renewable sources. This Primer provides a general overview of the fundamental aspects that influence the hydrogen production potential of the process. Recent research studies and their main findings are highlighted. The current challenges and limitations of the process and ways to optimize the biomass-derived products of steam reforming are discussed. Finally, we evaluate progress toward the industrial scalability of the process. The combined process of biomass pyrolysis and in-line catalytic steam reforming is a promising alternative for the selective production of hydrogen from renewable sources. In this Primer, Lopez et al. outline the main factors influencing hydrogen production, from reactor configurations and operating conditions to product analysis and catalyst development.

Van der Waals heterostructures

Abstract: The integration of dissimilar materials into heterostructures has become a powerful tool for engineering interfaces and electronic structure. The advent of 2D materials has provided unprecedented opportunities for novel heterostructures in the form of van der Waals stacks, laterally stitched 2D layers and more complex layered and 3D architectures. This Primer provides an overview of state-of-the-art methodologies for producing such van der Waals heterostructures, focusing on the two fundamentally different strategies, top-down deterministic assembly and bottom-up synthesis. Successful techniques, advantages and limitations are discussed for both approaches. As important as the fabrication itself is the characterization of the resulting engineered materials, for which a range of analysis techniques covering structure, composition and emerging functionality are highlighted. Examples of the properties of artificial van der Waals structures include optoelectronics and plasmonics, twistronics and unique functionality arising from the generalization of van der Waals assembly from 2D to 3D crystalline components. Finally, current issues of reproducibility, limitations and opportunities for future breakthroughs in terms of enhanced homogeneity, interfacial purity, feature control and ultimately orders-of-magnitude increased complexity of van der Waals heterostructures are discussed. Van der Waals epitaxy provides numerous opportunities for materials integration in heterostructures. This Primer provides an overview of methodologies for producing van der Waals heterostructures, focusing on top-down assembly and bottom-up synthesis, and discusses future opportunities for their continued development.

Glycoproteomics

Abstract: Protein glycosylation involves the co-translational or post-translational addition of glycans to proteins and is a crucial protein modification in health and disease. The aim of glycoproteomics is to understand how glycosylation shapes biological processes by understanding peptide sequences, glycan structures and sites of modification in a system-wide context. Over the past two decades, mass spectrometry (MS) has emerged as the primary technique for studying glycoproteins, with intact glycopeptide analysis — the study of glycopeptides decorated with their native glycan structures — now a preferred approach across the community. In this Primer, we discuss glycoproteomic methods for studying glycosylation classes, including best practices and critical considerations. We summarize how glycoproteomics is used to understand glycosylation at a systems level, with a specific focus on N-linked and O-linked glycosylation (both mucin-type and O-GlcNAcylation). We cover topics that include sample selection; techniques for protein isolation, proteolytic digestion, glycopeptide enrichment and MS fragmentation; bioinformatic platforms and applications of glycoproteomics. Finally, we give a perspective on where the field is heading. Overall, this Primer outlines the current technologies, persistent challenges and recent advances in the exciting field of glycoproteomics.

Nested sampling for physical scientists

Abstract: We review Skilling's nested sampling (NS) algorithm for Bayesian inference and more broadly multi-dimensional integration. After recapitulating the principles of NS, we survey developments in implementing efficient NS algorithms in practice in high-dimensions, including methods for sampling from the so-called constrained prior. We outline the ways in which NS may be applied and describe the application of NS in three scientific fields in which the algorithm has proved to be useful: cosmology, gravitational-wave astronomy, and materials science. We close by making recommendations for best practice when using NS and by summarizing potential limitations and optimizations of NS.

Serial femtosecond crystallography

Abstract: With the advent of X-ray Free Electron Lasers (XFELs), new, high-throughput serial crystallography techniques for macromolecular structure determination have emerged. Serial femtosecond crystallography (SFX) and related methods provide possibilities beyond canonical, single-crystal rotation crystallography by mitigating radiation damage and allowing time-resolved studies with unprecedented temporal resolution. This primer aims to assist structural biology groups with little or no experience in serial crystallography planning and carrying out a successful SFX experiment. It discusses the background of serial crystallography and its possibilities. Microcrystal growth and characterization methods are discussed, alongside techniques for sample delivery and data processing. Moreover, it gives practical tips for preparing an experiment, what to consider and do during a beamtime and how to conduct the final data analysis. Finally, the Primer looks at various applications of SFX, including structure determination of membrane proteins, investigation of radiation damage-prone systems and time-resolved studies.

Electrochemical stripping analysis

Abstract: Electrochemical stripping analysis (ESA) is a trace electroanalytical technique for the determination of metal cations, inorganic ions, organic compounds and biomolecules. It is based on a pre-concentration step of the target analyte(s), or a compound of the target, on a suitable working electrode. This is followed by a stripping step of the accumulated analyte using an electroanalytical technique. Advantages of ESA include high sensitivity and low limits of detection, multi-analyte capability, low cost of instrumentation and consumables, low power requirements, potential for on-site analysis, speciation capability and scope for indirect biosensing. This Primer covers fundamental aspects of ESA and discusses methods of pre-concentration and stripping, instrumentation, types of working electrodes and sensors, guidelines for method optimization, typical applications, data interpretation and interferences, and method limitations and workarounds. Finally, the current trends and future prospects of ESA are highlighted. Trace quantities of metal cations, inorganic anions, organic compounds and biomolecules can be measured using electrochemical stripping analysis (ESA). This Primer describes the principles of ESA, including methods of performing the pre-concentration and stripping steps, instrumentation and technique optimization.

In vivo hypermutation and continuous evolution

Abstract: Directed evolution has revolutionized biomolecular engineering by applying cycles of mutation, amplification and selection to genes of interest (GOIs). However, classical directed evolution methods that rely on manually staged evolutionary cycles constrain the scale and depth of the evolutionary search that is possible. We describe genetic systems that achieve cycles of rapid mutation, amplification and selection fully inside living cells, enabling the continuous evolution of GOIs as cells grow. These systems advance the scale, evolutionary search depth, ease and overall power of directed evolution and access important new areas of protein evolution and engineering. In vivo continuous evolution is a form of directed evolution that takes advantage of cycles of rapid mutation, amplification and selection inside living cells. Molina, Rix et al. discuss best practices for designing and conducting experiments for drug discovery, enzyme engineering and fluorescence-activated cell sorting (FACS)-based evolution.