Experimental Considerations Research Articles

Probing and Visualizing Interfacial Charge at Surfaces in Aqueous Solution

Surface charge density and distribution play an important role in almost all interfacial processes, influencing, for example, adsorption, colloidal stability, functional material activity, electrochemical processes, corrosion, nanoparticle toxicity, and cellular processes such as signaling, absorption, and adhesion. Understanding the heterogeneity in, and distribution of, surface and interfacial charge is key to elucidating the mechanisms underlying reactivity, the stability of materials, and biophysical processes. Atomic force microscopy (AFM) and scanning ion conductance microscopy (SICM) are highly suitable for probing the material/electrolyte interface at the nanoscale through recent advances in probe design, significant instrumental (hardware and software) developments, and the evolution of multifunctional imaging protocols. Here, we assess the capability of AFM and SICM for surface charge mapping, covering the basic underpinning principles alongside experimental considerations. We illustrate and compare the use of AFM and SICM for visualizing surface and interfacial charge with examples from materials science, geochemistry, and the life sciences.

Hydrogen/Deuterium Exchange Mass Spectrometry for Weak Binders.

This note describes theoretical and experimental considerations to observe perturbation of a protein upon binding to a ligand with weak affinity by hydrogen/deuterium exchange mass spectrometry (HDX-MS). The most popular application of HDX-MS is to determine the binding site of a drug or drug lead in a protein target. However, when the affinity of a ligand is weak, driving the equilibrium to the formation of a complex is difficult, and thus, observing the perturbation upon binding is also challenging. Theoretical consideration indicates that the original concentration of a ligand over the dissociation constant ([L0]/KD) is roughly equal to the maximum protection factor expected for the experiment when the original concentration of a ligand is significantly larger than the original concentration of a protein and the dissociation constant ([L0] ≫ [P0] and [L0] ≫ KD). When HDX-MS analysis of a protein with a ligand of low affinity and low solubility is carried out, it may be challenging to achieve high enough ligand concentration to drive the equilibrium in favor of the complex due to the low solubility. There are two methods to alleviate this issue: (i) spiking a low affinity/low solubility ligand to exchange buffer to lower the required ligand concentration in aqueous protein stock solution and (ii) mixing a 1:1 ratio of aqueous protein-ligand stock solution and deuterated buffer to initiate the exchange reaction instead of the commonly used 1:9 ratio.

Epigenetic Transgenerational Modifications Induced by Xenobiotic Exposure in Zebrafish.

Zebrafish (Danio rerio) is a well-established vertebrate model in ecotoxicology research that responds to a wide range of xenobiotics such as pesticides, drugs, and endocrine-disrupting compounds. The epigenome can interact with the environment and transform internal and/or external signals into phenotypic responses through changes in gene transcription. Environmental exposures can also generate epigenetic variations in offspring even by indirect exposure. In this review, we address the advantages of using zebrafish as an experimental animal model to study transgenerational epigenetic processes upon exposure to xenobiotics. We focused mostly on DNA methylation, although studies on post-translational modifications of histones, and non-coding RNAs related to xenobiotic exposure in zebrafish are also discussed. A revision of the methods used to study epigenetic changes in zebrafish revealed the relevance and reproducibility for epigenetics-related research. PubMed and Google Scholar databases were consulted for original research articles published from 2013 to date, by using six keywords: zebrafish, epigenetics, exposure, parental, transgenerational, and F2. From 499 articles identified, 92 were considered, of which 14 were selected as included F2 and epigenetic mechanisms. Current knowledge regarding the effect of xenobiotics on DNA methylation, histone modifications, and changes in non-coding RNAs expressed in F2 is summarized, along with key experimental design considerations to characterize transgenerational effects.

Tips and traps for behavioural animal experimentation.

Behavioural animal experimentation is an inseparable part of research trying to understand the biological underpinnings of human behaviour, diseases and disorders. Working with animals comes with great responsibility to achieve reliable and reproducible results of highest scientific quality. In a simple step-by-step fashion, we highlight some common issues that may occur along the path to conducting behavioural animal experimentations and posit some solutions and grounds to ensure the excellence of work done in this research area while aspiring to improve conditions for laboratory animals. It entails topics of study design, animal and experimenter welfare, experimental considerations and frequentist biostatistics. At the end, we direct to some guidelines and manuals that may prove valuable to researchers in this field. Our ten simple tips and traps are meant for students who are learning about important concepts for the first time; graduates whose statistics training all too often has neglected the concept of power in experimental design; and researches who would like a light-hearted refresher on these topics. With this perspective, we hope that you will avoid falling into traps and find answers to what you always wanted to know about conducting behavioural animal experimentation.

Identification of cardiac liability in drug discovery using automated patch clamp: experimental and technical considerations for high throughput recordings of NaV1.5 and hERG

The Comprehensive in vitro Proarrhythmia Assay (CiPA) aim is to improve specificity compared to in vitro hERG and in vivo QT studies. Automated patch clamp (APC) instruments are increasingly adopted for cardiac safety measurements on hERG, NaV1.5 and CaV1.2, thus requiring standardized experimental protocols and technical specifications, e.g. temperature control, accessibility of recording solutions to allow for sample collection or Liquid Junction Potential (LJP) correction for accurate voltage control.

Considerations and best practices in animal science 16S ribosomal RNA gene sequencing microbiome studies.

Microbiome studies in animal science using 16S rRNA gene sequencing have become increasingly common in recent years as sequencing costs continue to fall and bioinformatic tools become more powerful and user-friendly. The combination of molecular biology, microbiology, microbial ecology, computer science, and bioinformatics—in addition to the traditional considerations when conducting an animal science study—makes microbiome studies sometimes intimidating due to the intersection of different fields. The objective of this review is to serve as a jumping-off point for those animal scientists less familiar with 16S rRNA gene sequencing and analyses and to bring up common issues and concerns that arise when planning an animal microbiome study from design through analysis. This review includes an overview of 16S rRNA gene sequencing, its advantages, and its limitations; experimental design considerations such as study design, sample size, sample pooling, and sample locations; wet lab considerations such as field handing, microbial cell lysis, low biomass samples, library preparation, and sequencing controls; and computational considerations such as identification of contamination, accounting for uneven sequencing depth, constructing diversity metrics, assigning taxonomy, differential abundance testing, and, finally, data availability. In addition to general considerations, we highlight some special considerations by species and sample type.

Convective drying of yacón (Smallanthus sonchifolius) slices: A simple physical model including shrinkage

Yacón is a tuberous root from the Andean region, and a functional food due to the presence of fructo-oligosaccharides (FOS). Due to its high moisture in natura, the product is prone to microbial degradation. The evolution of yacón cylindrical slices under convective drying is here studied from experimental work and theoretical considerations about bulk properties. The operation of a pilot-scale convective dryer allowed monitoring the moisture and dimensions of yacón slices. Evolution of moisture under controlled temperature and relative humidity conditions is described through a two-parameter drying kinetics model. A novel approach for estimating shrinkage effects is proposed, comparing measured dimensions with predictions based upon hypothetical shrinkage behaviors. The drying kinetics and the shrinkage approach are finally combined with the global energy balance for a single slice, in order to describe the evolution of surface temperature and other yacón slice properties along the drying period.

Tadpole Responses to Environments With Limited Visibility: What We (Don’t) Know and Perspectives for a Sharper Future

Amphibian larvae typically inhabit relatively shallow freshwater environments, and within these boundaries there is considerable diversity in the structure of the habitats exploited by different species. This diversity in habitat structure is usually taken into account in relation to aspects such as locomotion and feeding, and plays a fundamental role in the classification of tadpoles into ecomorphological guilds. However, its impact in shaping the sensory worlds of different species is rarely addressed, including the optical qualities of each of these types of water bodies and the challenges and limitations that they impose on the repertoire of visual abilities available for a typical vertebrate eye. In this Perspective article, we identify gaps in knowledge on (1) the role of turbidity and light-limited environments in shaping the larval visual system; and (2) the possible behavioral and phenotypic responses of larvae to such environments. We also identify relevant unaddressed study systems paying special attention to phytotelmata, whose small size allows for extensive quantification and manipulation providing a rich and relatively unexplored research model. Furthermore, we generate hypotheses ranging from proximate shifts (i.e., red-shifted spectral sensitivity peaks driven by deviations in chromophore ratios) to ultimate changes in tadpole behavior and phenotype, such as reduced foraging efficiency and the loss of antipredator signaling. Overall, amphibians provide an exciting opportunity to understand adaptations to visually limited environments, and this framework will provide novel experimental considerations and interpretations to kickstart future research based on understanding the evolution and diversity of strategies used to cope with limited visibility.

Legal Еxperiment

The goal is set: on the basis of a scientifically based concept of integrative legal understanding, synthesizing ontologically homogeneous legal regulators – principles and norms of law, to highlight the features of the legal experiment. The analysis of the prevailing doctrinal ideas about the legal experiment was carried out, which made it possible to clarify its features and social purpose. In the course of the study, various methods of cognition of legal reality were used (dialectical-materialistic, general scientific and private scientific). Consideration of the legal experiment as an effective method of cognition of the surrounding reality, a method of its transformation, allowed us to define the legal experiment as a unique type of legal activity, an integral part of the emanation of law, consisting of a number of stages, namely: definitive, law-creation, experimental implementation of the law and the final. The diversity of legal experiments and the need for their classification is emphasized. It is proposed, in particular, to distinguish legal experiments by territorial, substantive and substantive criteria. Special attention is paid to digital legal experiments in the article. The destructiveness of the proposals made in science on the administration of justice by algorithms of IT platforms is proved. The necessity of adopting a special Federal law “On Legal Experiments” as a legal act fixing all the essential conditions for conducting legal experiments, taking into account the dynamism of this phenomenon, is substantiated.

In vitro contractile studies within isolated tissue baths: Translational research from Visible Heart® Laboratories.

The isolated tissue bath research methodology was first developed in 1904. Since then, it has been recognized as an important tool in pharmacology and physiology research, including investigations into neuromuscular disorders. The tissue bath is still used routinely as the instrument for performing the "gold standard" test for clinical diagnosis of malignant hyperthermia susceptibility - the caffeine-halothane contracture test. Our research group has utilized this tool for several decades for a range of research studies, and we are currently one of four North American diagnostic centers for determining susceptibility for malignant hyperthermia. This review provides a brief summary of some of the historical uses of the tissue bath. Important experimental considerations for the operation of the tissue bath are further described. Finally, we discuss the different studies our group has performed using isolated tissue baths to highlight the broad potential applications.

Eye-Tracking in Interactive Virtual Environments: Implementation and Evaluation

Not all eye-tracking methodology and data processing are equal. While the use of eye-tracking is intricate because of its grounding in visual physiology, traditional 2D eye-tracking methods are supported by software, tools, and reference studies. This is not so true for eye-tracking methods applied in virtual reality (imaginary 3D environments). Previous research regarded the domain of eye-tracking in 3D virtual reality as an untamed realm with unaddressed issues. The present paper explores these issues, discusses possible solutions at a theoretical level, and offers example implementations. The paper also proposes a workflow and software architecture that encompasses an entire experimental scenario, including virtual scene preparation and operationalization of visual stimuli, experimental data collection and considerations for ambiguous visual stimuli, post-hoc data correction, data aggregation, and visualization. The paper is accompanied by examples of eye-tracking data collection and evaluation based on ongoing research of indoor evacuation behavior.

Towards a Comprehensive Optical Connectome at Single Synapse Resolution via Expansion Microscopy.

Mapping and determining the molecular identity of individual synapses is a crucial step towards the comprehensive reconstruction of neuronal circuits. Throughout the history of neuroscience, microscopy has been a key technology for mapping brain circuits. However, subdiffraction size and high density of synapses in brain tissue make this process extremely challenging. Electron microscopy (EM), with its nanoscale resolution, offers one approach to this challenge yet comes with many practical limitations, and to date has only been used in very small samples such as C. elegans, tadpole larvae, fruit fly brain, or very small pieces of mammalian brain tissue. Moreover, EM datasets require tedious data tracing. Light microscopy in combination with tissue expansion via physical magnification—known as expansion microscopy (ExM)—offers an alternative approach to this problem. ExM enables nanoscale imaging of large biological samples, which in combination with multicolor neuronal and synaptic labeling offers the unprecedented capability to trace and map entire neuronal circuits in fully automated mode. Recent advances in new methods for synaptic staining as well as new types of optical molecular probes with superior stability, specificity, and brightness provide new modalities for studying brain circuits. Here we review advanced methods and molecular probes for fluorescence staining of the synapses in the brain that are compatible with currently available expansion microscopy techniques. In particular, we will describe genetically encoded probes for synaptic labeling in mice, zebrafish, Drosophila fruit flies, and C. elegans, which enable the visualization of post-synaptic scaffolds and receptors, presynaptic terminals and vesicles, and even a snapshot of the synaptic activity itself. We will address current methods for applying these probes in ExM experiments, as well as appropriate vectors for the delivery of these molecular constructs. In addition, we offer experimental considerations and limitations for using each of these tools as well as our perspective on emerging tools.

GPS-Uber: a hybrid-learning framework for prediction of general and E3-specific lysine ubiquitination sites.

As an important post-translational modification, lysine ubiquitination participates in numerous biological processes and is involved in human diseases, whereas the site specificity of ubiquitination is mainly decided by ubiquitin-protein ligases (E3s). Although numerous ubiquitination predictors have been developed, computational prediction of E3-specific ubiquitination sites is still a great challenge. Here, we carefully reviewed the existing tools for the prediction of general ubiquitination sites. Also, we developed a tool named GPS-Uber for the prediction of general and E3-specific ubiquitination sites. From the literature, we manually collected 1311 experimentally identified site-specific E3-substrate relations, which were classified into different clusters based on corresponding E3s at different levels. To predict general ubiquitination sites, we integrated 10 types of sequence and structure features, as well as three types of algorithms including penalized logistic regression, deep neural network and convolutional neural network. Compared with other existing tools, the general model in GPS-Uber exhibited a highly competitive accuracy, with an area under curve values of 0.7649. Then, transfer learning was adopted for each E3 cluster to construct E3-specific models, and in total 112 individual E3-specific predictors were implemented. Using GPS-Uber, we conducted a systematic prediction of human cancer-associated ubiquitination events, which could be helpful for further experimental consideration. GPS-Uber will be regularly updated, and its online service is free for academic research at http://gpsuber.biocuckoo.cn/.

Coherent Two-Dimensional and Broadband Electronic Spectroscopies.

Over the past few decades, coherent broadband spectroscopy has been widely used to improve our understanding of ultrafast processes (e.g., photoinduced electron transfer, proton transfer, and proton-coupled electron transfer reactions) at femtosecond resolution. The advances in femtosecond laser technology along with the development of nonlinear multidimensional spectroscopy enabled further insights into ultrafast energy transfer and carrier relaxation processes in complex biological and material systems. New discoveries and interpretations have led to improved design principles for optimizing the photophysical properties of various artificial systems. In this review, we first provide a detailed theoretical framework of both coherent broadband and two-dimensional electronic spectroscopy (2DES). We then discuss a selection of experimental approaches and considerations of 2DES along with best practices for data processing and analysis. Finally, we review several examples where coherent broadband and 2DES were employed to reveal mechanisms of photoinitiated ultrafast processes in molecular, biological, and material systems. We end the review with a brief perspective on the future of the experimental techniques themselves and their potential to answer an even greater range of scientific questions.

Magnetism, symmetry and spin transport in van der Waals layered systems

The discovery of an ever-increasing family of atomic layered magnetic materials, together with the already established vast catalogue of strong spin–orbit coupling and topological systems, calls for some guiding principles to tailor and optimize novel spin transport and optical properties at their interfaces. Here, we focus on the latest developments in both fields that have brought them closer together and make them ripe for future fruitful synergy. After outlining fundamentals on van der Waals magnetism and spin–orbit coupling effects, we discuss how their coexistence, manipulation and competition could ultimately establish new ways to engineer robust spin textures and drive the generation and dynamics of spin current and magnetization switching in 2D-materials-based van der Waals heterostructures. Grounding our analysis on existing experimental results and theoretical considerations, we draw a prospective analysis about how intertwined magnetism and spin–orbit torque phenomena combine at interfaces with well-defined symmetries and how this dictates the nature and figures of merit of spin–orbit torque and angular momentum transfer. This will serve as a guiding role in designing future non-volatile memory devices that utilize the unique properties of 2D materials with the spin degree of freedom.

On the quantification of Auger recombination in crystalline silicon

Quantification of Auger recombination in crystalline silicon is usually challenging because it requires distinguishing Auger recombination from extrinsic recombination processes, especially at the surface. In this work we exploit the known injection dependence of surface recombination at surfaces passivated by highly charged dielectric films to assess the value of the ambipolar Auger coefficient Camb in crystalline silicon from carefully calibrated photoconductance decay measurements of high-lifetime Si wafers. A value of Camb = (2.11±0.02) × 10−30 cm6 s−1 is determined for both n- and p-type Si, independent of the dopant concentration for moderately doped samples near 300 K. Furthermore, by exploiting the expected equality of the surface saturation current density J0s for identically processed n- and p-type substrates, we are able to estimate the relative contributions of ehh and eeh Auger processes to Camb under the same doping and injection regime. The resulting ratio Cn/Cp=2.81 is in close agreement with the value of 2.91 derived from the data of Dziewior and Schmid at high dopant concentrations, which we reassess in light of modern mobility models. Based on our own data and literature data at higher concentrations, we offer a revised parameterisation of the Auger recombination rate in c-Si as a function of dopant concentration and injection level which is consistent with these findings. This revised parameterisation is also shown to be consistent with the most recent record lifetimes reported by other authors. Finally, we suggest experimental considerations relevant for future efforts to further refine the Auger parameterisation.

Sounding a New Era in Biomechanics with Acoustic Force Spectroscopy.

The acoustic force spectroscopy (AFS) tool was recently introduced as a novel tool for probing mechanical properties of biomolecules, expanding the application of sound waves to high-throughput quantification of the mechanical properties of single cells. By using controlled acoustic forces in the piconewton to nanonewton range, tens to hundreds of cells functionalized by attached microspheres can simultaneously be stretched and tracked in real-time with sub millisecond time response. Since its first application, several studies have demonstrated the potential and versatility of the AFS for high-throughput measurements of force-induced molecular mechanisms, revealing insight into cellular biomechanics and mechanobiology at the molecular level. In this chapter, we describe the operation of the AFS starting with the underlying physical principles, followed by a run-down of experimental considerations, and finally leading to applications in molecular and cellular biology.

CRISPR/Cas genome editing in grapevine: recent advances, challenges and future prospects

The development of clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system has revolutionized genome editing and plant breeding. Applications of CRISPR/Cas9 technology in fruit crops, including grapevine, enable precise improvement of agronomically important traits. In this review, we first describe genome editing based on the mostly used CRISPR/Cas9 system and newly developed CRISPR technologies. We then focus on applications of CRISPR/Cas9 in improvement of disease resistance, optimization of CRISPR/Cas9 system, multiplex genome editing and off-target effect analysis in grapevine. We also discuss the challenges facing genome editing that should be overcome to realize the great potential of the CRISPR technology in grapevine. Finally, we highlight possible experimental considerations for more precise and efficient genome editing in grapevine in the future.

Single-Molecule Multikilobase-Scale Profiling of Chromatin Accessibility Using m6A-SMAC-Seq and m6A-CpG-GpC-SMAC-Seq.

A hallmark feature of active cis-regulatory elements (CREs) in eukaryotes is their nucleosomal depletion and, accordingly, higher accessibility to enzymatic treatment. This property has been the basis of a number of sequencing-based assays for genome-wide identification and tracking the activity of CREs across different biological conditions, such as DNAse-seq, ATAC-seq , NOMeseq, and others. However, the fragmentation of DNA inherent to many of these assays and the limited read length of short-read sequencing platforms have so far not allowed the simultaneous measurement of the chromatin accessibility state of CREs located distally from each other. The combination of labeling accessible DNA with DNA modifications and nanopore sequencing has made it possible to develop such assays. Here, we provide a detailed protocol for carrying out the SMAC-seq assay (Single-Molecule long-read Accessible Chromatin mapping sequencing), in its m6A-SMAC-seq and m6A-CpG-GpC-SMAC-seq variants, together with methods for data processing and analysis, and discuss key experimental and analytical considerations for working with SMAC-seq datasets.

Morris water maze: a versatile and pertinent tool for assessing spatial learning and memory.

Since its development about 40 years ago (1981–2021), Morris water maze has turned into a very popular tool for assessing spatial learning and memory. Its many advantages have ensured its pertinence to date. These include its effectiveness in evaluating hippocampal-dependent learning and memory, exemption from motivational differences across diverse experimental manipulations, reliability in various cross-species studies, and adaptability to many experimental conditions with various test protocols. Nonetheless, throughout its establishment, several experimental and analysis loopholes have galvanized researchers to assess ways in which it could be improved and adapted to fill this gap. Therefore, in this review, we briefly summarize these developments since the early years of its establishment through to the most recent advancements in computerized analysis, offering more comprehensive analysis paradigms. In addition, we discuss the adaptability of the Morris water maze across different test versions and analysis paradigms, providing suggestions with regard to the best paradigms for particular experimental conditions. Hence, the proper selection of the experimental protocols, analysis paradigms, and consideration of the assay’s limitations should be carefully considered. Given that appropriate measures are taken, with various adaptations made, the Morris water maze will likely remain a relevant tool to assess the mechanisms of spatial learning and memory.