Current protocols最新文献

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.

Although monophosphate nucleoside prodrug approaches have been extensively investigated, leading to the development of several key antiviral and anticancer drugs, less attention has been given to the design of triphosphate prodrugs for the delivery of triphosphorylated nucleotide analogues. Expanding on this strategy, we report here an efficient synthetic methodology for preparing nucleoside triphosphate prodrugs, in which the γ-phosphate of a nucleotide is masked with an amino acid ester and an aryloxy group (γ-ProTriP). This approach aims to achieve the direct intracellular release of the triphosphate nucleotide active species, circumventing metabolic bottlenecks and potential toxicity that are often associated with the accumulation of nucleoside analogues and/or their mono- and diphosphate species. This article outlines the synthetic strategy for preparing γ-ProTriP derivatives using either microwave-accelerated synthesis or conventional heating methods. The approach is exemplified by the preparation of a clofarabine γ-ProTriP, which emerges as a promising alternative to traditional monophosphate prodrug strategies. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Preparation of triphosphate aryloxy phosphoramidate of adenosine, uridine, and clofarabine with microwave heating

Alternate Protocol: Preparation of triphosphate aryloxy phosphoramidate of adenosine with conventional heating

Support Protocol 1: Cation exchange of UDP disodium salt to UDP di(triethylammonium) salt

Support Protocol 2: Synthesis of di(triethylammonium) salt of clofarabine 5′-diphosphate

Support Protocol 3: Synthesis of pentafluorophenyl phosphorylating reagents

The complex network of blood vessels plays a key role in transporting oxygen and nutrients and maintaining homeostasis in the human body. The inner walls of all blood and lymphatic vessels are lined by the endothelium, a monolayer of endothelial cells (ECs) oriented along the direction of blood flow. ECs play a pivotal role in vascular homeostasis, including regulating vascular tone, delivering oxygen and nutrients, modulating pro-inflammatory molecules and pro-inflammatory immune responses, and performing other vital functions. Therefore, the study of EC biology and vascular responses is key for a deeper understanding of vascular biology and the development of new therapeutics. Most studies in vivo and in vitro present technical challenges, either complexity or oversimplification, respectively, which slow down advances in the field. Therefore, 3D models and microfluidics offer a complementary alternative that integrates shapes similar to those observed in vivo, with the advantages of an in vitro system. Here, we present a robust and reproducible vessel-on-a-chip (VOC) composed of an EC monolayer and a microvascular microenvironment maintained by a peristaltic pump to ensure continuous media circulation and physiological levels of shear stress. In addition, we validated this model for in vitro studies of vascular inflammation by monitoring EC status. We observed cellular alignment after shear stress exposure, increased E-selectin expression, and TNF-induced morphological changes in ECs. This new VOC is a promising approach to studying EC mechanobiology and inflammation and opens new avenues for its versatile use in vascular biology, inflammation, and immune and cancer cell migration in a controlled, scalable manner. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: 3D Vessel Formation within a microfluidic organ-on-a-chip system

Basic Protocol 2: Evaluation of shear stress

Basic Protocol 3: Evaluation of inflammation

Nucleotide-sugar donors containing bioorthogonal moieties are important tools to study cellular glycosylation. Typically, the acetamide moiety in N-acetylhexosamines such as GlcNAc and GalNAc is replaced by an acylamide with a clickable tag and converted to the corresponding uridine diphosphate analogue. These probes can then be tested for acceptance by glycosyltransferase enzymes in vitro. Lengthy procedures in synthetic chemistry currently limit the availability of bioorthogonal uridine diphosphate (UDP)-sugar analogues. Chemoenzymatic synthesis has proven to be a powerful and effective alternative, and multiple approaches have been published to date. In this protocol, we describe a streamlined method for the generation of bioorthogonal UDP-GlcNAc and UDP-GalNAc analogues. We describe the chemical modification of D-glucosamine and D-galactosamine to incorporate bioorthogonal acylamides, the subsequent one-pot multienzyme conversion to the corresponding UDP-sugar analogues, and reproducible purification. Our approach features the bacterial kinase NahK and human pyrophosphorylase AGX1 as well as a recombinantly expressed AGX1 variant with an expanded substrate profile. The approach further features an inorganic pyrophosphatase and an alkaline phosphatase to improve enzymatic turnover and aid the purification process, respectively. The use of biosynthetic enzymes with substrate promiscuity extends the scope of bioorthogonal nucleotide-sugar analogue structures to aid efforts in chemical glycobiology. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Chemical synthesis of bioorthogonally tagged acylamide analogues of D-GlcNAc and D-GalNAc

Alternate Protocol 1: Chemical synthesis of bioorthogonally tagged acylamide analogues of D-GlcNAc and D-GalNAc from a protected GlcNH₂ or GalNH₂ precursor

Basic Protocol 2: Conversion of D-GlcNAc or D-GalNAc analogues to UDP-sugars using analytical- (reaction scouting) and preparative-scale enzymatic synthesis and purification

This protocol details Dataset Readiness Assessment for Training (DRAFT), a systematic method for determining if a high-dimensional biological dataset is suitable for developing reliable and equitable machine learning models. Standard model validation often fails to detect when performance is driven by spurious correlations within the dataset, leading to irreproducible findings. DRAFT provides a step-by-step methodology to perform a multiaxis assessment of a dataset's integrity before extensive modeling begins. The assessment comprises three basic protocols to probe the dataset's characteristics: (1) evaluating its potential for generalization by testing baseline model performance and stability under rigorous cross-validation; (2) auditing for inherent biases by analyzing model performance and calibration across demographic subgroups; and (3) measuring its capacity for scientific utility by assessing the stability of predictive features. The Cancer Genome Atlas Lung Adenocarcinoma (TCGA-LUAD) dataset serves as a primary case study to demonstrate DRAFT's ability to reveal a dataset's potential to produce fragile, inequitable, and scientifically uninformative models, even when preliminary modeling achieves high performance metrics. This framework provides a necessary template for vetting datasets, ensuring that subsequent computational models built upon them are robust, equitable, and capable of generating true scientific insight. © 2025 Wiley Periodicals LLC.

Support Protocol 1: Environment and software installation via Conda

Support Protocol 2: TCGA-LUAD case study: Data acquisition and preparation

Basic Protocol 1: Generalization audit: Assessing for overfitting and illusory performance

Basic Protocol 2: Equity audit: Assessing for hidden stratification and algorithmic bias

Basic Protocol 3: Stability audit: Assessing for spurious discoveries and scientific utility

The cover image is based on the article Rice straw tissue preparation for reproducible electron microscopy imaging and analysis by Mahta Mohamadiaza et al., https://doi.org/10.1002/cpz1.70262.