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PubMed2026

A Standardized Nursing-Led Protocol Integrated Pain, Sleep, Medication Adherence, and Symptom Management in Postherpetic Neuralgia.

Postherpetic neuralgia (PHN) is a persistent neuropathic pain condition after herpes zoster that frequently coexists with sleep disturbance, medication-related problems, and fluctuating symptoms. This study evaluated whether a standardized nursing-led protocol could improve multidimensional short-term outcomes beyond usual care. In this prospective, parallel-group randomized controlled trial, 128 adults with PHN were allocated 1:1 to usual care or usual care plus an eight-week protocol integrating structured pain assessment, sleep monitoring, medication-adherence support, and rule-based digital symptom monitoring. The primary outcome was the between-group difference in change in Numeric Rating Scale (NRS) pain score from baseline to Week 8. Secondary outcomes included Pittsburgh Sleep Quality Index (PSQI), MMAS-8 medication adherence, symptom burden, pain-related nocturnal awakenings, breakthrough pain, rescue analgesic use, adverse events, rule-based alerts, and nursing satisfaction. Week-8 data were available for 116 participants (57 usual care; 59 protocol). Mean NRS scores decreased from 7.19 ± 1.08 to 4.82 ± 1.53 in the usual-care group and from 7.28 ± 1.05 to 3.24 ± 1.28 in the protocol group. An NRS reduction of at least 2 points occurred in 49.1% and 76.3% of participants, respectively. The protocol group also showed larger improvements in PSQI, MMAS-8, symptom burden, and nocturnal awakenings, with fewer breakthrough-pain episodes and less rescue-analgesic use. These findings support further evaluation of the standardized nursing-led protocol in preregistered multicenter trials with intention-to-treat analyses and longer follow-up.

Humans↗

PubMed2026

The Case for Master Protocols for Rare Neurological Diseases.

Master protocol trials allow for simultaneous multiple hypothesis testing within a common framework and might be applicable for rare diseases. In May 2025, the Network for Excellence in Neuroscience Clinical Trials convened a multistakeholder conference to discuss master protocol trials in rare neurological disorders. In this paper, we explore how master protocol trial designs may apply to rare neurological disorders, using the neuronal ceroid lipofuscinoses as an example. Through shared protocol elements and trial infrastructure, master protocols may decrease cost and improve efficiency in testing potential therapeutics in rare disease, accelerating the delivery of urgently needed therapies to patients. ANN NEUROL 2026;100:477-486.

Humans↗

PubMed2026

DNA Extraction Optimisation for Minute Land Snails of Vertigo Müller, 1773 (Gastropoda: Vertiginidae): A Comparative Evaluation of Six Methods, Including a Non-Destructive Shell-Preserving Protocol.

No systematic comparison of DNA extraction strategies exists for minute Vertiginidae (shell height < 3 mm), a group posing a dual analytical challenge: extremely low tissue input and co-purified PCR-inhibitory mucus. For legally protected species, an additional requirement to preserve the shell voucher further constrains available protocols. Using Vertigo antivertigo as the model species, we compared six approaches applied to specimens preserved in 96% ethanol (n = 10 per method): two HotSHOT alkaline-lysis protocols (destructive and non-destructive shell-preserving variants), a modified CTAB protocol supplemented with PVP-40 and DTT, and three commercial silica-column kits (GeneJET Genomic, DNeasy Blood & Tissue, QIAamp DNA Micro). DNA yields were quantified by QuantiFluor fluorometry, and PCR performance was subsequently assessed across four loci (COI barcode, COI mini-barcode, ITS1, ITS2). DNeasy Blood & Tissue produced the highest fluorometric concentrations; QIAamp DNA Micro and CTAB + PVP-40 gave intermediate values. The shell-preserving HotSHOT variant yielded lower concentrations but improved A260/230 ratios. BSA and trehalose supplementation increased PCR success in inhibition-prone HotSHOT extracts from 70% to 100%. ITS1 Sanger sequencing of three Vertigo species listed in Annex II of the EU Habitats Directive, all extracted with the shell-preserving protocol, confirmed species-level identification (99.8%-100% BLASTn identity; mean Phred Q > 51). The shell-preserving non-destructive HotSHOT protocol yields sequenceable DNA from protected Vertiginidae while retaining the morphological voucher, making it the preferred option for conservation-genetic monitoring. The practical decision framework documented here-integrating voucher preservation, amplification robustness and per-sample cost-has broad applicability to other minute terrestrial gastropods processed in large-scale biodiversity surveys.

Habitats Directive↗

PubMed2026

A User-Friendly Protocol for Microinjection into Teleost Embryos to Study Gene Function.

Zebrafish (Danio rerio) and medaka (Oryzias latipes) are popular teleost models used in developmental biology and functional genomics. To achieve high-quality and reproducible microinjections, it is essential to have robust protocols for breeding, egg collection, and the precise delivery of genetic material. In this protocol, we present a comprehensive and optimized methodology for setting up breeding tanks under controlled photoperiod conditions to maximize egg yield while minimizing contamination. We provide detailed procedures for sex identification, pair selection, the use of grated breeding inserts, and methods to increase egg collection efficiency. We outline procedures for making injection gel beds, pulling needles, and calibration using one-microliter microcapillaries to achieve consistent nanoliter-scale injections. Our protocol outlines settings for the pico-liter injector that are optimized to deliver a precise amount per pulse with minimal variability. Finally, we demonstrate the application of these methods for gene knockdown using morpholino antisense oligonucleotides, gene knockout using CRISPR-Cas9, and gain-of-function mRNA overexpression experiments. Phenotypic assessments conducted at various developmental stages to evaluate gene-specific effects reveal consistent phenotypic outcomes between the morpholino and CRISPR-Cas9 approaches. This easy and comprehensive protocol enables efficient, precise, and scalable genetic manipulation of zebrafish and medaka embryos, thereby supporting advanced functional studies in developmental biology and disease modeling. To our knowledge, this is the first unified protocol for both zebrafish and medaka microinjection systems achieving 97.7% phenotype penetrance in CRISPR-Cas9 knockouts with precision together with a triple validation approach that confirms gene function across multiple techniques.

Animals↗

PubMed2026

Mapping and measuring addiction recovery: Recommended protocols through the PHENX toolkit.

BACKGROUND: Addiction "recovery" has served as a positive conceptual basis for major public health frameworks, communication strategies, and policies. With greater research interest and increasing explication of the recovery construct a multitude of measures have emerged, prompting a need to reach consensus on recommendations to enable better data harmonization and cross-study syntheses. The PhenX (consensus measures for Phenotypes and eXposures) Toolkit (www.phenxtoolkit.org) is a freely accessible catalog of validated protocols designed to promote data comparability across clinical, epidemiological, and genomic research, yet contained no recovery-specific measures. METHOD: In 2024, a Substance Use and Recovery Working Group (SURWG) followed a well-established and detailed PhenX consensus process to identify and recommend protocols suitable for recovery research. Protocols were chosen based on criteria including recovery specificity, brevity, psychometrics, scoring simplicity, and non-proprietary access. The broader scientific community (12 national/regional organizations) provided feedback (respondent N = 86) on the SURWG's preliminary recommendation which was incorporated into final decisions. RESULTS: In 2025, the PhenX Toolkit released 15 new recommended protocols across three broad recovery elements: 1. Biopsychological (post-acute withdrawal; craving), 2. Socio-ecological (social relationships;social network characteristics; substance use goal; recovery identity; recovery capital), 3. Treatment and recovery services (Treatment and Recovery Services Use; Satisfaction; and Happiness in Recovery; Mutual-Help). CONCLUSIONS: This PhenX Toolkit SURWG process resulted in a new Substance Use Recovery Specialty Collection of recommended protocols with specific multidimensional applicability. As such, they provide the field with pre-vetted tools that researchers can employ with some confidence to enhance empirical efficiency and return on national research investment.

Humans↗

PubMed2025

Development and Validation of Amplicon-Based Protocol for Sequencing of Respiratory Syncytial Virus Genome.

The most prevalent cause of severe respiratory infections in children is the human respiratory syncytial virus (RSV). The advent of next-generation sequencing (NGS) has made it possible to incorporate this technology into pathogen monitoring and surveillance. Whole-genome sequencing (WGS) of RSV has now become a relatively widely used method for tracking viral evolution. Here we report an improved high-throughput RSV-WGS assay performed directly on clinical samples that is suitable for short-read sequencing platforms. A total of 100 RSV-positive samples collected between November 2022 and March 2024 fulfilled the inclusion cycle quantification criteria and were randomly included in the validation process. The WGS protocol was designed to amplify three distinct amplicons to cover the entire RSV genome. The protocol described here can be successfully replicated in several instances (approximately 95%) in samples with a relatively low viral load, typically corresponding to cycle of quantification values of 27-32. The amplicon-based protocol produced meaningful sequencing results in terms of median depth of coverage (more than 12000×) and median of mapped reads (> 1 × 106 reads). The sequences that had passed the filters showed a coverage of at least 98% across the entire genome, with cycle quantification values of 32. Based on the obtained data resulting in an easy-to-perform protocol helpful for the molecular epidemiology surveillance of RSV.

Humans↗

PubMed2026

Therapeutic Exercise Protocol During Hospitalization in Pediatric Oncohematological Patients: Randomized Clinical Trial.

BACKGROUND: Leukemias, lymphomas, and central nervous system tumors are among the most common pediatric cancers and may lead to motor deficits, impaired balance, reduced muscle strength, fatigue, and decreased functional capacity. Early physiotherapy during hospitalization may help prevent inactivity and support functional preservation in this population. OBJECTIVE: To evaluate the effects of a therapeutic exercise program on quality of life, muscle strength, fatigue, and functional capacity in hospitalized pediatric oncohematological patients. METHODS: Thirty participants aged 8-17 years with oncohematological diseases were randomized to an intervention group (IG) or a minimal active physiotherapy comparator group (CG). Assessments included the 6-min walk test, handgrip dynamometry, the PedsQL Multidimensional Fatigue Scale, and the PedsQL Cancer Module at admission and discharge. The IG performed daily 25-min supervised sessions including aerobic, resistance, and breathing exercises with ambulation guidance, whereas the CG received breathing exercises and ambulation guidance. RESULTS: No significant group × time interactions were observed for total fatigue or its domains, overall quality of life or its assessed domains, handgrip strength, or six-minute walk test distance. Time-related changes were observed for some outcomes, but these occurred without evidence of differential change between groups and were not interpreted as effects of the structured exercise protocol. No intervention-related adverse events requiring permanent protocol discontinuation were recorded. CONCLUSION: The structured in-hospital therapeutic exercise protocol could be delivered under close clinical supervision without recorded intervention-related adverse events requiring permanent discontinuation. However, the structured protocol did not demonstrate superiority over the minimal active physiotherapy comparator for fatigue, quality of life, muscle strength, or functional capacity. These findings should be interpreted cautiously because of the small sample size, clinical heterogeneity, variable intervention exposure, and limited intervention-fidelity data. TRIAL REGISTRATION: Brazilian Registry of Clinical Trials (ReBEC), RBR-8sxnfyd.

Humans↗

PubMed2025

Dual RNA isolation from blood: an optimized protocol for host and bacterial RNA purification for dual RNA-sequencing analysis in whole blood sepsis samples.

Dual RNA-sequencing (dual RNA-seq) holds significant promise for deciphering bacterial virulence mechanisms during systemic infections. However, its application in sepsis research is hindered by technical challenges, including a low bacterial burden in blood and limited sample volumes and RNA yield from vulnerable populations, such as neonates. We developed an optimized protocol [dual RNA isolation from blood (DRIB)] for simultaneous stabilization, isolation and purification of high-quality host leukocyte and bacterial RNA from low-volume whole blood samples (0.5 ml). This protocol is compatible with clinical sample collection workflows and high-throughput RNA sequencing. The feasibility of DRIB for dual RNA-seq was validated using a pilot cohort of clinical adult sepsis samples, enabling the investigation of host-bacterial gene expression during sepsis. The DRIB protocol yielded 2.10-6.91 µg of total RNA per clinical sample in our pilot cohort. Dual-species ribosomal RNA (rRNA) depletion and RNA-seq generated 16.6-24.8 million filtered reads per sample, with 63±7% of reads uniquely mapped to host or bacterial sequences. Host genes accounted for 51-68% (8.4-10.9 million) reads, while 0.5-6.7% (79,496-789,808 reads) mapped to bacterial genomes. Bioinformatic analysis revealed that both shared and individual transcriptional patterns were identified in host and bacterial responses, including pathways related to immune metabolism and metal-ion binding. Our optimized DRIB protocol and RNA-seq pipeline effectively captured both host and bacterial RNA transcription in clinical sepsis samples. Expanding this approach to larger cohorts and varying disease timepoints will provide crucial new insights into host-bacterial gene co-expression dynamics in sepsis progression and outcomes.

Humans↗

PubMed2026

Comparative performance of portable DNA extraction protocols and bioinformatics workflows for rapid detection of gram-negative bacteria and antimicrobial resistance using Oxford Nanopore sequencing.

Oxford Nanopore Technology (ONT) enables rapid, portable pathogen identification and antimicrobial resistance (AMR) detection, but the reliability of downstream genomic analyses is highly dependent on DNA extraction quality, particularly in resource-limited settings. This study comparatively evaluated four portable bacterial DNA extraction protocols derived from three commercial kits to determine their impact on nanopore sequencing performance, bioinformatics workflow completion, and field deployability. Six gram-negative bacterial isolates (Escherichia coli, n = 4; Pseudomonas sp., n = 1; and Salmonella sp., n = 1) were processed using four extraction protocols: SwiftX DNA, SwiftX DNA with proteinase K (ProtK), SwiftX ParaBact, and NucleoSpin Microbial. Twenty-four resulting DNA extracts were sequenced on a single multiplexed MinION R10.4.1 flow cell. Sequencing data were analyzed using validated Galaxy-based generic and species-specific pipelines. Workflow completion was defined as successful progression through quality control, assembly, virulence, plasmid, and AMR detection modules. DNA purity varied substantially by extraction protocol and was strongly associated with successful workflow completion (Kruskal-Wallis, P = 0.0006). Accordingly, NucleoSpin Microbial achieved 100% workflow completion, and SwiftX ParaBact achieved 83%, while both SwiftX DNA-based protocols failed to complete full workflows. Importantly, key AMR genes required to classify isolates as multidrug-resistant were consistently detected using both NucleoSpin Microbial and SwiftX ParaBact extractions. However, NucleoSpin Microbial assemblies showed significantly higher contiguity and enabled a broader, more complete detection of virulence factors, pathogenicity islands, plasmid replicons, and accessory AMR genes, reflecting enhanced genomic resolution.IMPORTANCERapid whole-genome sequencing is increasingly used to detect antimicrobial resistance and guide public health responses, but its reliability depends strongly on how bacterial DNA is extracted. In this study, we have shown that DNA extraction method choice has a major impact on Oxford Nanopore sequencing performance across clinically relevant gram-negative bacteria. While silica column-based extraction maximized genomic completeness and analytical depth, paramagnetic bead-based reverse purification offered superior portability with sufficient resolution for frontline AMR surveillance. These findings highlight a practical trade-off between field deployability and high-resolution genomic characterization in low-resource settings.

DNA extraction↗

PubMed2026

Trans-Mitochondrial Cybrid Generation from mtDNA Patient Platelets: An Efficient Protocol Optimizing Colony Selection and Functional Validation.

Trans-mitochondrial cybrid cell line generation represents the gold-standard method for determining pathogenicity by enabling biochemical analyses of a specific mitochondrial DNA (mtDNA) variant of interest at high and low percentages (heteroplasmy levels) within an otherwise identical mtDNA and nuclear genome background. Historically, the cybrid generation process has been tedious and poorly efficient. Here, we describe a highly efficient and effective protocol for generating trans-mitochondrial cybrid cell lines by fusing human platelets with a standard osteosarcoma 143B cell line to provide an isogenic nuclear background depleted of mtDNA (Rho0 cells). Cell isolates capture a given mtDNA genome of interest to establish stable cell lines harboring different degrees of heteroplasmy, or to compare divergent effects of distinct mitochondrial haplogroups. Because cybrids from mitochondrial patients may be more difficult to establish with standard protocols, this current methodology focuses on isolating mtDNA variants where the electron transport chain activity is affected. We here demonstrate that colony selection techniques reduce time and improve the yield of generating high-level heteroplasmy mtDNA mutant cybrid lines. A case study is provided of cybrid generation for a variant of unknown significance in MT-ND1, m.3985G>A (p.E227K). We analyze the efficiency of the cybrid generation process using this protocol and run functional studies performed by high-resolution respirometry. High-level heteroplasmy MT-ND1 m.3985G>A cybrid mutants generated by this protocol are shown to have impaired complex I-dependent mitochondrial respiration relative to wild-type control, demonstrating m.3985G>A is likely pathogenic.

Humans↗

PubMed2027

Protocol for Detecting and Sequencing Chikungunya Virus from Field-Collected Mosquitoes.

Arboviral diseases represent a major public health challenge, especially in tropical regions where environmental conditions may favor the proliferation and spread of mosquito vectors. Thus, early and accurate detection of chikungunya virus (CHIKV) in mosquito populations can be a valuable tool for effective surveillance of circulating variants and for identifying new viral introductions. Given the challenges of detecting arboviruses in field-captured mosquitoes, we describe an integrated workflow for CHIKV molecular detection and whole-genome sequencing. This protocol includes mosquito homogenization using a bead-based mechanical disruptor, RNA extraction using TRIzol reagent with minor modifications, molecular screening using CHIKV-specific RT-qPCR, and whole-genome amplification followed by sequencing on Illumina platforms. Despite the protocol being optimized for individual mosquitoes, it results in high-quality RNA suitable for both entomological surveillance and genomic analysis. As this protocol allows recovery of complete CHIKV genomes from mosquito specimens, it can serve as a basis for genomic epidemiology studies, enabling monitoring of viral diversity and lineage dynamics, and facilitating early detection of emerging variants to support timely and targeted public health interventions in endemic and at-risk regions.

Animals↗

PubMed2026

Next-Generation Sequencing Completion and Timeliness Using a Reflex Testing Protocol for Patients with Stage II to IV Nonsquamous Non-Small Cell Lung Cancer.

BACKGROUND: Next-generation Sequencing (NGS) is critical for providing treatment recommendations across multiple stages of non-small cell lung cancer (NSCLC). However, a substantial proportion of patients do not undergo testing. This study evaluated the completion rates and timeliness of NGS in patients with stage II to IV NSCLC at a single academic institution with a reflex NGS testing protocol. METHODS: Patients with stage II to IV nonsquamous NSCLC (ns-NSCLC) diagnosed between 2015 and 2022 were identified retrospectively. A reflex, tissue-based testing protocol was initiated in 2015 using in-house NGS. Pyrosequencing was performed if NGS failed. RESULTS: 501 patients were included: 75 (15.0%) with stage II, 82 (16.4%) with stage III, and 344 (68.6%) with stage IV ns-NSCLC. Tissue NGS was completed in 380 (75.8%) patients and 465 (92.8%) completed some tissue-based genomic testing when including pyrosequencing. Median time from biopsy to NGS was 17.0 days (range, 6-61 days). 61.0% of patients had NGS results prior to a first treatment of any type and 88.4% had tissue NGS results prior to systemic therapy. Among stage IV patients with completed NGS, median overall survival was 2.27 years for patients with NGS results prior to first treatment compared to 1.08 years for patients without NGS results prior to treatment initiation (P = .04). CONCLUSIONS: Implementation of an in-house, reflex NGS testing protocol enabled rapid genomic profiling in a high proportion of patients with stage II to IV ns-NSCLC. NGS completion prior to receiving first-line therapy was associated with improved survival compared to completion after first line treatment in stage IV patients.

Humans↗

PubMed2026

A STORM-based protocol for nanoscale imaging and quantitative analysis of protein-associated and phospholipid-associated structures in natural rubber.

Stochastic Optical Reconstruction Microscopy (STORM) enables nanoscale mapping of molecular components beyond the diffraction limit; however, its reproducible implementation in hydrophobic polymer matrices remains challenging because fluorescence-labeling specificity, fluorophore photoswitching, three-dimensional localization, chromatic registration, and quantitative image analysis must be carefully controlled. This protocol presents a standardized experimental workflow for dual-color labeling, astigmatism-based three-dimensional STORM acquisition, and quantitative analysis of protein-associated and phospholipid-associated structures in natural rubber (NR). The workflow covers sample pretreatment, Cy5 NHS ester labeling of protein-associated primary amines, DiI labeling of phospholipid-rich domains, STORM imaging-buffer preparation, three-dimensional single-molecule localization, dual-channel registration, generation of standardized xy projections, aggregate-size analysis, and projected lateral spatial correlation assessment. Reproducibility is supported by defined acquisition and localization criteria, three independent sample preparations with at least five fields of view analyzed per condition, and unlabeled, single-color, dye-only matrix, and processing-associated Cy5 controls. Mean lateral localization precisions of 11.8 ± 2.3 nm for Cy5 and 13.5 ± 2.9 nm for DiI were obtained, while two-dimensional Fourier ring correlation analysis of the xy projections yielded effective lateral image resolutions of approximately 25 and 28 nm, respectively. Image-based particle segmentation and localization-coordinate-based density-based spatial clustering of applications with noise (DBSCAN) were applied to standardized xy projections as complementary quantitative approaches. Application of the protocol to untreated, centrifuged, and protease-treated NR samples demonstrated treatment-associated changes in the detected abundance and projected size distributions of protein- and phospholipid-associated aggregates, together with a non-monotonic change in their projected lateral spatial correlation. These observations describe alterations in nanoscale organization but do not, by themselves, establish stable protein-phospholipid complex formation. Unlike previous studies that primarily demonstrated the feasibility of STORM imaging in rubber materials, the principal contribution of this work is an end-to-end, step-by-step protocol incorporating defined controls, three-dimensional localization, image-quality metrics, chromatic-registration procedures, and complementary quantitative-analysis pipelines for non-expert users. The workflow may be adaptable to other hydrophobic polymers and soft-material systems after appropriate optimization and validation.

Rubber↗

PubMed2024

Protocol to decode the role of transcriptionally active microbes in SARS-CoV-2-positive patients using an RNA-seq-based approach.

The elucidation of the role of microorganisms in human infections has been hindered by difficulties using conventional culture-based techniques. Here, we present a protocol for the investigation of transcriptionally active microbes (TAMs) using an RNA sequencing (RNA-seq)-based approach. We describe the steps for RNA isolation, viral genome sequencing, RNA-seq library preparation, and metatranscriptomic and transcriptomic analysis. This protocol permits a comprehensive evaluation of TAMs' contributions to the differential severity of infectious diseases, with a particular focus on diseases such as COVID-19. For complete details on the use and execution of this protocol, please refer to Devi et al.1.

Humans↗

PubMed2025

A xeno-free protocol for rapid differentiation of human iPSC-derived microglia from the KOLF2.1J reference line.

We present a detailed, xeno-free protocol for the rapid differentiation of human induced pluripotent stem cells (hiPSCs) into microglia using the well-characterized KOLF2.1J reference line. This system employs doxycycline-inducible expression of six transcription factors (6-TF), stably integrated into the CLYBL safe harbor locus, to drive uniform microglial differentiation within two weeks. Adapted from Dräger et al. (1), our protocol includes key optimizations for KOLF2.1J, including culture on Laminin-521 to support xeno-free conditions. The resulting i-Microglia exhibit hallmark features of mature microglia, including expression of P2RY12, loss of the pluripotency marker SSEA4, phagocytic activity, and upregulation of immune markers (e.g., CD80, CD83) upon LPS stimulation. We also demonstrate compatibility with co-culture systems using iPSC-derived neurons. Additionally, we describe a modification of the line to include a constitutive mCherry reporter integrated into the SH4-2 safe harbor locus, enabling fluorescent tracking of microglia in mixed cultures or in vivo. This protocol provides a reproducible and scalable platform for generating functional human microglia from a widely used hiPSC line, supporting applications in disease modeling, neuroinflammation research, and therapeutic screening.

Journal Article↗

PubMed2026

ChromID: A Protocol for Mapping Protein Chromatin Interactions in Living Cells.

Chromatin modifications regulate genome function by recruiting proteins that control transcription, genome organization, and DNA repair. Identifying the proteins associated with specific chromatin modifications is therefore essential for understanding how these regulatory processes operate. Traditional approaches, including chromatin immunoprecipitation and affinity purification coupled to mass spectrometry, have uncovered many chromatin-associated proteins. However, they often rely on crosslinking and chromatin fragmentation, which can disrupt native chromatin architecture and limit the detection of transient interactions. Here, we describe a proximity-labeling protocol for identifying the chromatin-dependent protein interactome associated with specific chromatin marks, termed ChromID. ChromID uses engineered chromatin readers (eCRs) fused to a promiscuous biotin ligase, which labels proteins in the immediate vicinity of the targeted chromatin mark. The protocol includes in vivo biotin labeling, nuclear extract preparation, streptavidin-based enrichment, and tryptic digestion for downstream LC-MS/MS analysis. The protocol has been validated across multiple cell types and chromatin contexts and can be extended to other chromatin-associated proteins, providing a versatile approach to profile chromatin-associated proteomes within their native cellular environment. Key features • Maps proteins associated with different chromatin modifications in living cells using engineered chromatin readers fused to TurboID, BASU, or other promiscuous biotin ligases. • Preserves native chromatin organization and captures transient chromatin-associated interactions that are often lost during conventional affinity purification workflows. • Validated across multiple chromatin contexts, including histone modifications, DNA methylation, transcription factors, RNA polymerase II, and DNA damage-associated chromatin states. • Applicable to diverse cell types and organisms and adaptable to other chromatin-associated proteins, including transcription factors and chromatin regulators.

Biotin proximity labeling↗

PubMed2025

Development of a highly efficient protoplast regeneration and transfection protocol for enhancing CRISPR genome editing of Brassica carinata.

Brassica carinata is an important oil crop with significant potential for food and industrial production. The application of the CRISPR/Cas9 genome editing tool in B. carinata could accelerate its breeding cycle. However, no efficient DNA-free gene editing method currently exists for this species. Protoplast-based CRISPR editing presents a promising solution, though it is often challenging for many crop species. In this study, we investigated several critical factors influencing in vitro shoot regeneration, including genotype, sugar type, selection and combination of plant growth regulators (PGRs), and culture duration on different media throughout various stages of protoplast development. As a result, we developed a highly efficient, five-stage protoplast regeneration protocol for B. carinata based on specific stages of protoplast development. Key findings of this study include the requirement for high concentrations of NAA and 2,4-D in the initial medium (MI) for cell wall formation, while a lower auxin concentration relative to cytokinin was necessary for active cell division (MII). For callus growth and shoot induction, a high cytokinin-to-auxin ratio was essential (MIII), and an even higher cytokinin-to-auxin ratio was optimal for shoot regeneration (MIV). For shoot elongation, low levels of BAP and GA3 were sufficient (MV). Our results also demonstrated that the duration of culture on different media and maintaining appropriate osmotic pressure at the early stages were crucial for successful protoplast regeneration. With this optimized protocol, we achieved an average regeneration frequency of up to 64% and a transfection efficiency of 40% using the GFP marker gene. This efficient protoplast regeneration protocol is now being employed for genome editing in our lab and is expected to significantly enhance the application of the CRISPR system in both basic research and the genetic improvement of B. carinata over the long term.

Brassica carinata↗

PubMed2026

Establishment of an efficient Agrobacterium-mediated genetic transformation protocol for Saccharum officinarum using Black Cheribon as a model genotype.

Efficient Agrobacterium-mediated transformation (AMT) is vital for the biotechnological improvement of sugarcane (Saccharum spp.). Saccharum officinarum is the main ancestor of all modern cultivars, yet little research has been conducted on its AMT system. In this work, an efficient AMT protocol for S. officinarum was developed, with Black Cheribon as the model genotype owing to its superior tissue culture performance and regeneration capacity. The optimized agro-infection protocol comprised the following main parameters: concentration of acetosyringone (AS) in Agrobacterium culture, concentration of AS for infection, Agrobacterium concentration at OD600 = 0.4, infection time of 30 minutes, vacuum infiltration time of 10 minutes and co-cultivation time of 3 days. To further improve transformation efficiency, 0.5 mg/L thidiazuron and 200 mg/L citric acid were added to the regeneration medium, which enhanced the regeneration of shoots. A modified stage-dependent selection strategy (FlexII) was established by using glufosinate-ammonium at concentrations of 2.0, 1.0, and 0.75 mg/L in the callus proliferation, shoot regeneration, and rooting stages, respectively. This strategy was more successful than the minimum inhibitory concentration-based strategy in S. officinarum transformation. The optimized protocol further boosted the transformation efficiency of Black Cheribon from 1.12% to 7.17%. The resulting transgenic lines were confirmed by PCR amplification of T-DNA regions and immunochromatographic detection of Bar protein expression in primary transformants, respectively. These results provide a sound technical foundation for the functional genomics and biotechnological optimization of S. officinarum germplasm, and may serve as a reference for future transformation studies in other sugarcane germplasm.

Agrobacterium↗