Current protocols最新文献

The growing utility of xeno-nucleic acids (XNAs) lies in their ability to extend the reach of genetic chemistry beyond the limits imposed by natural polymers. XNAs, with their diverse chemical backbones, resist enzymatic degradation and yet retain the capacity for sequence-defined information, and have found broad applications in biotechnology. The approach described herein provides a systematic method for the transliteration between XNAs and DNAs. This article delineates the ligase-catalyzed oligonucleotide polymerization (LOOPER) process as applied to the transcription and reverse transcription of XNA libraries using T3 DNA ligase. Two complementary procedures are presented. Basic Protocol 1 details the assembly of XNA polymers through the ligase-mediated templated ligation of 5'-phosphorylated trinucleotide anticodons bearing XNA modifications, exemplified here by locked nucleic acids (LNAs). Basic Protocol 2 describes the reverse transcription of XNA sequences into cDNA using unmodified DNA 5'-phosphorylated trinucleotide anticodons. Together, these protocols enable a bidirectional exchange between DNA and chemically diverse XNA species, a prerequisite for the application of SELEX and other evolutionary methodologies to noncanonical backbones. This ligase-based framework dispenses with substrate biases that can often be present with polymerases, allowing high-fidelity transliteration (>95%) across a variety of modified nucleotides. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Ligase-catalyzed oligonucleotide polymerization (LOOPER) for XNA synthesis Basic Protocol 2: Ligase-catalyzed oligonucleotide polymerization (LOOPER) for cDNA synthesis from XNA templates.

The scientific process is inherently iterative; we never stop at a single observation but rather conduct our research again in the same or in a different context to see if we will obtain the same results. Failure to do so makes us doubt the validity of the findings and methods of our research and the study we aimed to repeat. While this is an oversimplification of the issue, such unsuccessful attempts at replicating prior research have led to what has been globally recognized as a “Reproducibility Crisis” in research. Its many causes are deeply intertwined, and its implications are complex; yet the research community has mobilized to fight it through both formal and informal initiatives. Here we provide some tools and tips that will help make the methods, data, and code more reproducible for researchers at any stage. We hope that the uptake of these good research practices will eventually lead to positive changes within the research community. © 2025 Wiley Periodicals LLC.

Immunoheterogeneity is a potential hindrance to the maximum applicability of mesenchymal stromal cells (MSCs) in biotherapeutics and regenerative medicine. The primary causes of this heterogeneity are donor variation, diversity of tissue sources, and the culture conditions under which MSCs clonally propagate. Immunoheterogeneity could manifest as functional heterogeneity within these stem cells, affecting their ability to differentiate into adult tissue lineages. The impacted healthy human third molar tooth is a rich source of MSCs, which are isolatable both from the dental pulp and the dental follicle. Dental pulp stem cells (DPSCs), though widely characterized for their multipotential lineage differentiation, demonstrate considerable immunoheterogeneity, which demands the enrichment of these stem cells post-isolation. The aim of this study is to enrich and understand the functional differences in sub-populations of MSCs towards a specific lineage. In this protocol, we have highlighted detailed, stepwise methods for sorting and culturing post-sort and optimized methods for quantifying the differentiation of sorted DPSCs towards the osteogenic lineage. Our workflow is designed with additional considerations for cell sorter availability, maintenance of cell viability during long-distance transport to a state-of-the-art facility, and optimization of post-sorting cell culture and downstream experiments. This protocol can be universally followed to understand functional differences within MSCs isolated from different tissue sources and their implications for multi-lineage differentiation. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Assessing multiparametric expression of MSC surface markers and single cell sorting for enrichment of stem cells

Support Protocol 1: Antibody panel design

Support Protocol 2: MSC culture, preparation of cell suspension, and sample staining

Support Protocol 3: Setting up controls

Support Protocol 4: Analysis and data interpretation

Basic Protocol 2: Quantitative and qualitative assessment of osteogenic differentiation of post-sorted DPSCs

Support Protocol 5: Differentiation of MSCS (pre-sort and post-sort considerations)

Support Protocol 6: Cytochemical staining and quantification

Support Protocol 7: Immunofluorescence staining

Support Protocol 8: RNA isolation and qPCR-based characterization.

The inferior colliculus (IC) is a central hub for auditory information processing that receives widespread convergent projections. The IC comprises three main subdivisions: the central nucleus of the IC (ICC), the dorsal cortex (DC), and the lateral cortex (LC). While the ICC receives primarily ascending auditory information, DC and LC receive major cortical and multisensory projections. The LC has repeated molecular motifs that govern its input-output relationships. However, because the LC is buried deep within a sulcus, it is difficult to image in behaving animals, making it challenging to answer questions about its functional organization. Here, we describe a protocol for coupling two-photon microscopy with a microprism to obtain cellular-resolution sagittal views of functional LC maps. We employed this novel approach to investigate neuronal responses to pure tones in relation to LC motifs. This method will not only provide new insights into the auditory system but will also permit imaging of hidden brain regions previously inaccessible by conventional means. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Craniotomy and implantation of the microprism.

Basic Protocol 2: Data acquisition from sound-responsive neurons.

Basic protocol 3: Confirming the microprism location.

Basic Protocol 4: Analyzing the time traces of neuronal responses and generating a best-frequency tuning Map.

Chara braunii is an emerging model species for studying plant evolution and development. Various members of the Chara genus have been used for pioneering studies of electrophysiology, cytoplasmic streaming, and cell biology. However, many studies have been limited by challenges, such as overgrowth with epiphytes or non-continuous growth throughout the year. Here, we present protocols for the cultivation, germination, and in vivo staining of C. braunii NIES-1604 to overcome these limitations. We constructed the custom-made cultivation chamber equipped with the illumination unit that allows for the precise simulation of natural conditions and continuous cultivation of C. braunii NIES-1604 throughout the year. By determining the optimal stratification period for C. braunii NIES-1604 oospores, we achieved a germination rate of 26%. Further, we present fluorescence-based in vivo staining protocols for routine in vivo staining of cellular structures including the plasma membrane, cell wall, and nuclei. Altogether, our affordable method of controlled cultivation of C. braunii NIES-1604 and other procedures serve as a valuable resource for establishing Chara cultures in laboratory settings and advancing its use as a model organism. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Construction of a cultivation chamber for C. braunii NIES-1604 growth

Basic Protocol 2: Vegetative propagation of C. braunii NIES-1604

Basic Protocol 3: Germination of C. braunii NIES-1604 oospores

Basic Protocol 4: In vivo staining of C. braunii NIES-1604 with fluorescent dyes

Biomaterial-based bioinks are increasingly utilized in bioprinting to engineer three-dimensional (3D) constructs with living cells for tissue engineering and disease modeling. Among various bioinks explored, alginate-based formulations stand out due to their good biocompatibility, mild gelation conditions, tunable mechanical properties, and ease of crosslinking via divalent cations such as calcium. Despite their widespread use, standardized protocols for preparing alginate-based bioinks and characterizing bioprinted constructs have not been well documented. Our laboratory has developed and validated reproducible methods for preparing a variety of alginate-based bioinks and printing cell-laden constructs tailored for diverse applications. In this article, we present detailed step-by-step protocols covering bioink preparation and rheological characterization, extrusion-based bioprinting of cell-laden constructs, post-printing culture and co-culture techniques, printability assessment, and live/dead and immunofluorescence assays. These protocols serve as a standardized framework for the fabrication and characterization of 3D bioprinted alginate-based cell-laden constructs, thereby facilitating translational research in tissue engineering, disease modeling, and preclinical therapeutic development. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Bioink preparation

Basic Protocol 2: Bioink characterization using rheology

Basic Protocol 3: Scaffold design and bioprinting

Support Protocol: 3D-printing parameter determination

Basic Protocol 4: Printability and cell viability analyses, and immunofluorescence assay

This article mainly describes a fermentation and purification method for expressing recombinant collagen protein in Escherichia coli. The method comprises constructing engineered bacteria expressing human type III collagen and adopting a strategy of feeding in batches for high-density fermentation. The rapid proliferation of bacterial cells is promoted at 37°C, and then the culture is inoculated into a fermentation tank with different carbon sources for growth. When glycerol is used as the main carbon source, the yield of recombinant collagen protein can reach 0.25 to 0.40 g/L. The method allows exploration of the differences in recombinant collagen production with different carbon sources in order to identify the most suitable fermentation medium component. The human type III collagen produced by the method has the typical structure of collagen, with high cell adhesion and the stability of tissue structure. Therefore, it can be used as the raw material for various collagen products, especially facial fillers, dressings, freeze-dried fibers, and gels. © 2025 Wiley Periodicals LLC.

Basic Protocol: Fermentation and purification of recombinant human type III collagen expressed in Escherichia coli

Modern connectomics enables large-scale, comparative network neuroscience across individuals, species, development, and evolution. The field now regularly produces extensive maps of neural connectivity exceeding hundreds of millions of synapses in continuous volumes. When connectomes are deposited in central archives such as BossDB with standardized metadata, researchers can pose previously intractable questions about neuronal networks. Here, we present step-by-step protocols for connectome dataset discovery and access, scalable graph construction and analysis, and reproducible comparative connectomics using BossDB, Motif Studio, DotMotif, Neuroglancer, neuPrint, and Python-based workflows. These protocols target bench neuroscientists and computational biologists and emphasize replicability, cloud-friendly options, and publication-quality visualization. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Discovering connectome datasets and computing summary statistics with BossDB and Motif Studio

Basic Protocol 2: Writing queries with DotMotif

Basic Protocol 3: Querying known network motifs locally with DotMotif

Support Protocol 1: Provisioning ad hoc graph databases for large-scale graph analysis

Support Protocol 2: Querying structures and systems in the cloud with neuPrint

Basic Protocol 4: Viewing anatomical motif features with BossDB and Neuroglancer

Scientific progress relies on the generation, validation, and reuse of research data, yet standard practices and cultural, legal, and technological challenges have long limited data sharing. In the 21^st century, growing volumes of data, higher transparency requirements, and concerns about reproducibility have pushed research data management to the forefront. This manuscript brings together three perspectives to provide an extensive overview of data sharing: theoretical foundations, ethical and normative frameworks, and practical implementation. First, it discusses the way research data differs across fields and formats, the distinction between primary and secondary data, and how metadata helps ensure data can be reused. It emphasizes how open data fosters transparency, reproducibility, accountability, and innovation, while also acknowledging that research data has historically been viewed as private intellectual property. Second, it explores the emergence of principles and ethical standards designed to enhance data quality and promote responsible use. Documentation standards, data management plans, and sharing of code and workflows have helped the FAIR (Findability, Accessibility, Interoperability, and Reusability) principles become a cornerstone for data sharing. Regulatory frameworks, such as the General Data Protection Regulation (GDPR) and California Consumer Privacy Act (CCPA), as well as mechanisms such as de-identification and Data Trusts, address legal and ethical issues, including privacy protection, licensing, and data governance. Finally, the third major topic discusses how these principles are implemented through infrastructure, incentives, and new technologies. It addresses the significance of cultural change and recognition systems, the impact of policies by journals and funders, and the role of repositories in preservation and interoperability. It also emphasizes the emergence of novel trends, such as artificial intelligence–driven metadata generation, blockchain-based provenance, executable workflows, and privacy-preserving computation, all of which are redefining the concept of responsible and scalable data sharing. By connecting conceptual, ethical, and practical dimensions, the manuscript outlines both current challenges and realistic pathways toward transparent, collaborative, and future-oriented research. © 2025 Wiley Periodicals LLC.