Current protocols最新文献

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.

Common problems in biological sample processing for scanning electron microscopy (SEM) include cell collapse and destruction. To overcome the challenges surrounding SEM micrograph preparation, dried rice stems were used to develop a specific set of protocols for processing dried plant samples. Dried stems are rehydrated with a glycerol solution and fixed in formalin-acetic-alcohol to avoid cell wall collapse or organ distortion. The protocols detailed here comprise the first published method for preparing SEM images of dried plant tissue. The protocols offer a cost-effective approach to obtaining high-quality micrographs, facilitating the reconstruction of growth processes and the study of plant cell wall features. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol 1: Pretreatment and preparation of rice straw samples at the heading stage

Basic Protocol 2: Paraffin infiltration and embedding

Basic Protocol 3: Preparation of microscopic sections

Basic Protocol 4: Transferring, adhering, and expanding sections on slides

Support Protocol: Preparation of gelatin slides before sectioning to affix samples

Basic Protocol 5: Preparation of samples for SEM imaging

Basic Protocol 6: SEM analysis

Basic Protocol 7: Processing and analysis of SEM images using ImageJ software

The cover image is based on the article Assays of Angiogenic Potential Using Quail and Chicken Chorioallantoic Membrane (CAM) by Ana Claudia Oliveira Carreira et al., https://doi.org/10.1002/cpz1.70223.

Targeted protein degradation using bifunctional molecules like proteolysis targeting chimeras (PROTACs) has revolutionized drug discovery but remains limited by E3 ligase availability and lack of cell selectivity. N-degron degraders provide a smaller, more drug-like alternative that harnesses endogenous N-terminal degradation signals independent of recruited ligases. However, they are prone to proteolysis and lack mechanisms for tissue-specific activation. To overcome these challenges, we developed a “caged” N-degron degrader selectively activated by the immunoproteasome (iCP), a proteasome isoform induced in cancer and inflammatory settings. By appending an iCP-recognition tetrapeptide (Ala-Thr-Met-Trp) capped with a morpholine group to the N-terminus, we shielded the degron from nonspecific cleavage and enabled selective activation in iCP-expressing cells. This design improved stability, enhanced degradation potency, and minimized activity in healthy cells. Our findings establish a generalizable strategy to spatially control N-degron activity through disease-specific proteolytic environments, advancing safer and more selective protein degradation therapeutics. © 2025 Wiley Periodicals LLC.

Basic Protocol 1: Synthesis and characterization of an N-degron degrader for Abl

Basic Protocol 2: Synthesis and characterization of an immunoproteasome N-degron prodrug

Basic Protocol 3: Assessing protein degradation in cells

DNA replication is often challenged by endogenous and exogenous sources of DNA damage, which can stall replication forks and result in single-stranded DNA (ssDNA) gaps, double-strand breaks (DSBs), and genomic instability. Detecting DNA damage specifically in newly synthesized DNA strands is essential for understanding how eukaryotic cells respond to replication stress and continue the cell cycle progression through DNA repair or DNA damage tolerance (DDT) mechanisms. Here, we present an optimized and accessible protocol for the alkaline BrdU comet assay—a single-cell technique that combines bromodeoxyuridine (BrdU; a thymidine analog) pulse-labeling of newly synthesized DNA with the alkaline comet assay (single-cell gel electrophoresis) followed by fluorescence immunodetection. This method enables the specific detection and measurement of DNA strand breaks occurring in newly replicated DNA during and immediately after the S phase, even without cell synchronization, allowing researchers to differentiate replication-associated DNA damage from overall genomic damage. We provide detailed instructions for performing the assay using human cells (RPE-1 h-TERT TP53 KO) in vitro after exposure to DNA replication-stress-inducing agents, such as hydroxyurea (HU) and ultraviolet-C (UV-C) radiation. We also demonstrate its application in translesion-synthesis-deficient (Pol eta-deficient) human fibroblasts in vitro. Importantly, this protocol supports time-course chase experiments (e.g., 0, 1, 2, and 4 hr post-treatment) to monitor the kinetics of DNA damage in nascent DNA strands. This BrdU-based protocol offers high specificity, single-cell resolution, and cost-effectiveness, making it particularly valuable for laboratories studying replication stress, post-replication DNA repair proficiency, DDT, and/or genotoxic responses in unsynchronized human cells in vitro. This protocol also adheres to the Minimum Information for Reporting Comet Assay (MIRCA) guidelines and is aligned with the objectives of the International Comet Assay Working Group (ICAW), ensuring high reproducibility and standardization. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Alkaline BrdU comet assay to assess replication-associated DNA damage in unsynchronized human cells (RPE-1 h-TERT TP53 KO) in vitro

Alternate Protocol 1: Alkaline BrdU comet assay to monitor replication-associated DNA damage dynamics in unsynchronized human cells (RPE-1 h-TERT TP53 KO) after HU and UV-C exposure and 0- to 2- hr chase

Alternate Protocol 2: Alkaline BrdU comet assay to compare replication-associated DNA damage dynamics in translesion synthesis polymerase η-deficient and complemented (XP-V comp) unsynchronized fibroblasts after UV-C exposure and 0- to 4-hr chase

To establish a standardized technical framework for large-scale production of recombinant collagen, we have employed Pichia pastoris GS115 as a host expression system and optimized the fermentation and purification processes for a transdermal peptide–type III collagen fusion protein (transdermal peptide–hCOL3A) at the 15-L bioreactor scale. A recombinant vector encoding the fusion gene was constructed through codon optimization and transformed into the GS115 strain. The seed culture was prepared via shake-flask cultivation in BMGY medium. High-cell-density fed-batch fermentation was conducted in a 15-L fermenter with an inoculum size ranging from 3% to 10%, achieving a final recombinant protein yield of 1.9 g/L. In the downstream purification, optimization of membrane-based concentration parameters and the incorporation of strong cation-exchange chromatography using an SP column enhanced the recovery rate of the target protein to 68%. Gel-filtration analysis demonstrated that transdermal peptide–hCOL3A exists as a trimer, and a CCK-8 assay revealed that it promotes proliferation of HaCaT cells in vitro, indicating biological functionality. This work successfully establishes a robust and scalable collagen production process suitable for pilot-scale manufacturing and provides a solid technical foundation for future industrial-scale translation. © 2025 Wiley Periodicals LLC.