Lyophilization of Molecular Biology Reactions: A Review.

Molecular biology is a widely used and widespread technique in research and as a laboratory diagnostic tool, aiming to investigate targets of interest from the obtainment, identification, and analysis of genetic material. In this context, methods, such as Polymerase Chain Reaction (PCR), Reverse Transcription Polymerase Chain Reaction (RT-PCR), real-time PCR, loopmediated isothermal amplification (LAMP), and loop-mediated isothermal amplification with reverse transcription (RT-LAMP), can be cited. Such methods use enzymes, buffers, and thermosensitive reagents, which require specific storage conditions. In an attempt to solve this problem, the lyophilization procedure (dehydration process by sublimation) can be applied, aiming to preserve and prolong the useful life of the reaction components in cases of temperature variation. In this review, we present a synthesis of the lyophilization process, describing the events of each step of the procedure and providing general information about the technique. Moreover, we selected lyophilization protocols found in the literature, paying attention to the conditions chosen by the authors for each step of the procedure, and structured the main data in tables, facilitating access to information for researchers who need material to produce new functional protocols.

Experience of quantity and quality of DNA and RNA extraction from limited pediatric blood samples: A comparative analysis of automated and manual kit-based method.

INTRODUCTION: Optimal DNA and RNA quantity and purity is essential for downstream molecular biology experimentation and to avoid re-processing of sample. Despite availability of different kits and automated systems for nucleic acid isolation there is limited data on their performance evaluation, more so with pediatric blood samples, that are usually compromised in quantity. Hence, we evaluated the performance of automated QIAcube platform using pediatric blood samples in parallel with manual Qiagen extraction kits. MATERIALS AND METHODS: : A total of 500 samples were analyzed based on groups of PBMC and direct blood input. The isolated DNA and RNA were surveyed for quantity and quality tests by spectrophotometric and downstream analysis. RESULTS: : There was no significant difference in the DNA quantity (ng/ul) between manual and automated method based on similar sample input but quality (260/280) was significantly better with the QIAcube platform when direct blood and or PBMCs were used for extraction respectively (1.82 ± 004 Vs. 1.84.002; P-0.000008 and 1.859 ± 005 Vs. 1.843 ± 0.003; P-0.02). Moreover, the standard error mean was low for both quantity and quality in the QIAcube method suggesting uniformity. Comparison of quality assessment by spectrophotometer and qubit fluorimeter showed that QIAcube sheared DNA less (P- 0.038) as compared to manual method (P-0.013). Also, time taken to process the samples in QIAcube was 23% less than the kit-based method. CONCLUSION: Overall analysis of QIAcube platform suggests that it yields more better, uniform, and less-sheared quality of nucleic acid in a relatively less time as compared to manual extraction kits.

Chromatin and transcription factor profiling in rare stem cell populations using CUT&Tag.

Muscle stem cells (MuSCs) are a rare stem cell population that provides myofibers with a remarkable capacity to regenerate after tissue injury. Here, we have adapted the Cleavage Under Target and Tagmentation technology to the mapping of the chromatin landscape and transcription factor binding in 50,000 activated MuSCs isolated from injured mouse hindlimb muscles. We have applied this same approach to human CD34+ hematopoietic stem and progenitor cells. This protocol could be adapted to any rare stem cell population. For complete details on the use and execution of this protocol, please refer to Robinson et al. (2021).

Quantification of adipocyte numbers in transgenic mice via the Cre-LoxP recombination sites.

This protocol describes a method to assess adipocyte numbers within a specific depot based on their inducible genomic label. By extracting DNA from a complete adipose tissue depot stemming from two transgenic mouse lines (Adipoq-CreERT2 x ROSA26-tdRFP and Ucp1-CreERT2 x ROSA26-tdRFP), the number of adipocytes can be determined based on the quantification of the recombined LoxPRed sites. This highly sensitive system allows for the quantification of white, brown, and brite/beige adipocytes in a spatially unbiased and size-independent manner. For complete details on the use and execution of this protocol, please refer to Moser et al. (2021).

Isolation and analysis of fractions enriched in WrappER-associated mitochondria from mouse liver.

The endoplasmic reticulum (ER) plays a central role in lipid homeostasis, but the role of individual ER subdomains in lipid biology has not been elucidated. WrappER is a curved wrapping type of rough-ER that establishes extensive contacts with almost every mitochondria of the hepatocyte in the mouse liver. Here, we describe a protocol for isolation of fractions enriched in wrappER-associated mitochondria from the mouse liver. We also provide techniques for assessing its quality by electron microscopy and biochemical/proteomic analysis. For complete information on the use and execution of this protocol, please refer to Anastasia et al. (2021).

Optimization of mito-roGFP protocol to measure mitochondrial oxidative status in human coronary artery endothelial cells.

Reactive oxygen species (ROS) are implicated in endothelial dysfunction and cardiovascular disease. Endothelial cells (ECs) produce most ATP through glycolysis rather than oxidative phosphorylation; thus mitochondrial ROS production is lower than in other cell types. This makes quantification of changes in EC mitochondrial oxidative status challenging. Here, we present an optimized protocol using mitochondrial-targeted adenovirus-based redox sensor for ratiometric quantification of specific changes in mitochondrial ROS in live human coronary artery EC. For complete details on the use and execution of this protocol, please refer to Waypa et al. (2010); Liao et al. (2020); Gao et al. (2021).

Delivery of membrane impermeable molecules to primary mouse T lymphocytes.

The pore-forming toxin streptolysin-O (SLO) enables intracellular delivery of molecules up to 100 kDa and has been used for short-term delivery of membrane-impermeable substances to assess their effects on cellular activities. A limitation of this technique is the loss of intracellular components and the potential unpredicted alterations of cellular metabolism and signaling. This protocol, optimized for primary mouse T lymphocytes, describes steps for SLO-mediated cell membrane permeabilization and substance supplementation, followed by immunoblotting and immunofluorescent microscopy for assessing cellular effects. For complete details on the use and execution of this protocol, please refer to Xu et al., 2021a, Xu et al., 2021b.

Isolation of Mitochondria from Retinal Pigment Epithelial Cell Cultures and an Application of High-Resolution Respirometric Assay (XFe96 Seahorse Assay).

Intracellular Ca2+ is strictly regulated to maintain optimal levels for function of cellular organelles as well as mitochondrial respiratory signaling at the tricarboxylic acid cycle and electron transport chain level. Optimal Ca2+ concentration for these processes vary between cell types. Furthermore, exposure of mitochondria to sustained, elevated levels of Ca2+ induces mitochondrial Ca2+ overload and damage to mitochondrial oxidative phosphorylation and ATP production. Isolated mitochondria are widely used to study mitochondrial physiology and drug effects on mitochondrial metabolism and respiratory function. However, isolated mitochondria are easily damaged during the mitochondrial isolation process. The present article describes a mitochondrial isolation method using Ca2+-chelation to minimize mitochondrial damage. We follow up the isolation process with an application that requires an optimized buffer Ca2+ concentration: the characterization of their respiratory function using a high-resolution respirometric assay.

FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices.

Fabrication and Applications of Microfluidic Devices: A Review.

Microfluidics is a relatively newly emerged field based on the combined principles of physics, chemistry, biology, fluid dynamics, microelectronics, and material science. Various materials can be processed into miniaturized chips containing channels and chambers in the microscale range. A diverse repertoire of methods can be chosen to manufacture such platforms of desired size, shape, and geometry. Whether they are used alone or in combination with other devices, microfluidic chips can be employed in nanoparticle preparation, drug encapsulation, delivery, and targeting, cell analysis, diagnosis, and cell culture. This paper presents microfluidic technology in terms of the available platform materials and fabrication techniques, also focusing on the biomedical applications of these remarkable devices.

A DNA-based voltmeter for organelles.

The role of membrane potential in most intracellular organelles remains unexplored because of the lack of suitable tools. Here, we describe Voltair, a fluorescent DNA nanodevice that reports the absolute membrane potential and can be targeted to organelles in live cells. Voltair consists of a voltage-sensitive fluorophore and a reference fluorophore for ratiometry, and acts as an endocytic tracer. Using Voltair, we could measure the membrane potential of different organelles in situ in live cells. Voltair can potentially guide the rational design of biocompatible electronics and enhance our understanding of how membrane potential regulates organelle biology.

Visualization of extracellular vesicles in the regenerating caudal fin blastema of zebrafish using in vivo electroporation.

Zebrafish have high regenerative ability in several organs including the fin. Although various mechanisms underlying fin regeneration have been revealed, some mechanisms remain to be elucidated. Recently, extracellular vesicles (EVs) have been the focus of research with regard to their role in cell-to-cell communication. It has been suggested that cells in regenerating tissues communicate using EVs. In this study, we examined the involvement of EVs in the caudal fin regeneration of zebrafish using an in vivo electroporation method. The process of regeneration appeared normal after in vivo electroporation, and the transferred plasmid showed mosaic expression in the blastema. We took advantage of this mosaic expression to observe the distribution of exosomal markers in the blastema. We transferred exosomal markers by in vivo electroporation and identified EVs in the regenerating caudal fin. The results suggest that blastemal cells communicate with other cells via EVs during caudal fin regeneration.

NAUTICA: classifying transcription factor interactions by positional and protein-protein interaction information.

BACKGROUND: Inferring the mechanisms that drive transcriptional regulation is of great interest to biologists. Generally, methods that predict physical interactions between transcription factors (TFs) based on positional information of their binding sites (e.g. chromatin immunoprecipitation followed by sequencing (ChIP-Seq) experiments) cannot distinguish between different kinds of interaction at the same binding spots, such as co-operation and competition. RESULTS: In this work, we present the Network-Augmented Transcriptional Interaction and Coregulation Analyser (NAUTICA), which employs information from protein-protein interaction (PPI) networks to assign TF-TF interaction candidates to one of three classes: competition, co-operation and non-interactions. NAUTICA filters available PPI network edges and fits a prediction model based on the number of shared partners in the PPI network between two candidate interactors. CONCLUSIONS: NAUTICA improves on existing positional information-based TF-TF interaction prediction results, demonstrating how PPI information can improve the quality of TF interaction prediction. NAUTICA predictions - both co-operations and competitions - are supported by literature investigation, providing evidence on its capability of providing novel interactions of both kinds. REVIEWERS: This article was reviewed by Zoltán Hegedüs and Endre Barta.

Cytomorphic Electronics With Memristors for Modeling Fundamental Genetic Circuits.

Cytomorphic engineering attempts to study the cellular behavior of biological systems using electronics. As such, it can be considered analogous to the study of neurobiological concepts for neuromorphic engineering applications. To date, digital and analog translinear electronics have commonly been used in the design of cytomorphic circuits; Such circuits could greatly benefit from lowering the area of the digital memory via memristive circuits. In this article, we propose a novel approach that utilizes the Boltzmann-exponential stochastic transport of ionic species through insulators to naturally model the nonlinear and stochastic behavior of biochemical reactions. We first show that two-terminal memristive devices can capture the non-linear and stochastic behavior of biochemical reactions. Then, we present the design of several building blocks based on analog memristive circuits that inherently model the biophysical mechanisms of gene expression. The circuits model induction by small molecules, activation and repression by transcription factors, biological promoters, cooperative binding, and transcriptional and translational regulation of gene expression. Finally, we utilize the building blocks to form complex mixed-signal networks that can simulate the delay-induced oscillator and the p53-mdm2 interaction in the cancer signaling pathway. Our approach can provide a fast and simple emulative framework for studying genetic circuits and arbitrary large-scale biological networks in systems and synthetic biology. Some challenges may be that memristive devices with frequent learning and programming do not have the same longevity as traditional transistor-based electron-transport devices, and operate with significantly slower time constants, which can limit emulation speed.

Native mass spectrometry: a powerful tool for structural biology?

Mass spectrometry has been used for decades and continues to be an integral part of analytical research. This feature explores its latest applications.

Precise DNA Concentration Measurements with Nanopores by Controlled Counting.

Using a solid-state nanopore to measure the concentration of clinically relevant target analytes, such as proteins or specific DNA sequences, is a major goal of nanopore research. This is usually achieved by measuring the capture rate of the target analyte through the pore. However, progress is hindered by sources of systematic error that are beyond the level of control currently achievable with state-of-the-art nanofabrication techniques. In this work, we show that the capture rate process of solid-state nanopores is subject to significant sources of variability, both within individual nanopores over time and between different nanopores of nominally identical size, which are absent from theoretical electrophoretic capture models. We experimentally reveal that these fluctuations are inherent to the nanopore itself and make nanopore-based molecular concentration determination insufficiently precise to meet the standards of most applications. In this work, we present a simple method by which to reduce this variability, increasing the reliability, accuracy, and precision of single-molecule nanopore-based concentration measurements. We demonstrate controlled counting, a concentration measurement technique, which involves measuring the simultaneous capture rates of a mixture of both the target molecule and an internal calibrator of precisely known concentration. Using this method on linear DNA fragments, we show empirically that the requirements for precisely controlling the nanopore properties, including its size, height, geometry, and surface charge density or distribution, are removed while allowing for higher-precision measurements. The quantitative tools presented herein will greatly improve the utility of solid-state nanopores as sensors of target biomolecule concentration.

Parasitological research in the molecular age.

New technological methods, such as rapidly developing molecular approaches, often provide new tools for scientific advances. However, these new tools are often not utilized equally across different research areas, possibly leading to disparities in progress between these areas. Here, we use empirical evidence from the scientific literature to test for potential discrepancies in the use of genetic tools to study parasitic vs non-parasitic organisms across three distinguishable molecular periods, the allozyme, nucleotide and genomics periods. Publications on parasites constitute only a fraction (<5%) of the total research output across all molecular periods and are dominated by medically relevant parasites (especially protists), particularly during the early phase of each period. Our analysis suggests an increasing complexity of topics and research questions being addressed with the development of more sophisticated molecular tools, with the research focus between the periods shifting from predominantly species discovery to broader theory-focused questions. We conclude that both new and older molecular methods offer powerful tools for research on parasites, including their diverse roles in ecosystems and their relevance as human pathogens. While older methods, such as barcoding approaches, will continue to feature in the molecular toolbox of parasitologists for years to come, we encourage parasitologists to be more responsive to new approaches that provide the tools to address broader questions.

Biophysical Techniques in Structural Biology.

Over the past six decades, steadily increasing progress in the application of the principles and techniques of the physical sciences to the study of biological systems has led to remarkable insights into the molecular basis of life. Of particular significance has been the way in which the determination of the structures and dynamical properties of proteins and nucleic acids has so often led directly to a profound understanding of the nature and mechanism of their functional roles. The increasing number and power of experimental and theoretical techniques that can be applied successfully to living systems is now ushering in a new era of structural biology that is leading to fundamentally new information about the maintenance of health, the origins of disease, and the development of effective strategies for therapeutic intervention. This article provides a brief overview of some of the most powerful biophysical methods in use today, along with references that provide more detailed information about recent applications of each of them. In addition, this article acts as an introduction to four authoritative reviews in this volume. The first shows the ways that a multiplicity of biophysical methods can be combined with computational techniques to define the architectures of complex biological systems, such as those involving weak interactions within ensembles of molecular components. The second illustrates one aspect of this general approach by describing how recent advances in mass spectrometry, particularly in combination with other techniques, can generate fundamentally new insights into the properties of membrane proteins and their functional interactions with lipid molecules. The third reviewdemonstrates the increasing power of rapidly evolving diffraction techniques, employing the very short bursts of X-rays of extremely high intensity that are now accessible as a result of the construction of free-electron lasers, in particular to carry out time-resolved studies of biochemical reactions. The fourth describes in detail the application of such approaches to probe the mechanism of the light-induced changes associated with bacteriorhodopsin's ability to convert light energy into chemical energy.

Simple high-resolution NMR spectroscopy as a tool in molecular biology.

NMR is one of the major techniques for investigating the structure, dynamics and interactions between biomolecules. However, non-experts often experience NMR experimentation and data analysis as intimidating. We discuss a simple yet powerful NMR technique, the so-called chemical shift perturbation (CSP) analysis, as a tool to elucidate macromolecular interactions in small- and medium-sized complexes, including protein-protein, protein-drug, and protein-DNA/RNA interactions. We discuss current software packages for NMR data analysis and present a new interactive graphical tool implemented in CcpNmr AnalysisAssign version-3, which can drastically reduce the time required for the CSP analysis. Lastly, we illustrate the usefulness of a protein three-dimensional structure for interpretation of the CSP data.

Super-resolution microscopy demystified.

Super-resolution microscopy (SRM) bypasses the diffraction limit, a physical barrier that restricts the optical resolution to roughly 250 nm and was previously thought to be impenetrable. SRM techniques allow the visualization of subcellular organization with unprecedented detail, but also confront biologists with the challenge of selecting the best-suited approach for their particular research question. Here, we provide guidance on how to use SRM techniques advantageously for investigating cellular structures and dynamics to promote new discoveries.