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CRISPR technology is a paradigm-shifting innovation in genome engineering, enabling precise gene editing in cell lines and model organisms. Robust protocols and troubleshooting strategies are essential for the successful application of CRISPR techniques in biological and clinical research. In this backstory, STAR Protocols authors and advisory board members share their advice on common questions related to CRISPR technologies, genome-editing strategies, and troubleshooting approaches.
RNA sequencing (RNA-seq) is a powerful technique that allows researchers to study gene expression at a genome-wide level in various biological systems. Advances in RNA-seq protocols and data analysis have improved the accuracy and reliability of gene expression measurements. However, RNA-seq experiments and data analysis can be technically complex and demanding, and researchers may encounter various challenges. In this backstory, STAR Protocols authors and advisory board members share their advice on common questions about RNA-seq.
Organoids can recapitulate essential physiological features of the tissue of origin, making them a promising in vitro model for studying human organ development and disease. Organoid technology can complement animal models by enabling research questions that were not addressable due to lack of tissues. However, reproducibility remains challenging, likely due to variability introduced during organoid generation and/or handling. In this backstory, STAR Protocols authors and advisory board members share their advice on common questions about organoids.
Stereotactic arrhythmia radiotherapy (STAR) is an emerging non-invasive option for refractory ventricular tachycardia (VT); yet, the underlying myocardial effects in humans remain poorly understood. Within the STOPSTORM consortium, we developed the Bio-STAR framework for standardized ex vivo assessment of STAR-treated myocardium and here report its feasibility and initial findings in human samples. Bio-STAR standardizes myocardial sampling into non-irradiated control (NFNI), minimally fibrotic irradiated, and fibrotic irradiated zones, guided by visual inspection, electroanatomical maps, and radiotherapy dose overlays. Recommended analyses span ex vivo MRI, histology, immunohistochemistry/-fluorescence, molecular panels, and live-cell assays. Application to two explanted non-ischaemic cardiomyopathy hearts (transplantation 3-5 months post-STAR) confirmed the protocol's applicability to end-stage remodelling. In exploratory analyses of irradiated regions, patient-specific patterns emerged, including stress marker upregulation, and altered NaV1.5, SERCA2a, and CaV1.2 when normalized to cardiomyocyte content. Functional analyses in a limited number of viable cardiomyocytes from the respective regions demonstrated heterogeneous excitability, supporting the feasibility of isolating live cells from STAR-treated regions in which Ca2+ signalling can be quantitatively assessed in future studies involving larger patient cohorts. Bio-STAR provides a reproducible framework for multimodal analysis of STAR-treated myocardium, enabling harmonized cross-centre research. Early human data demonstrate that cardiomyocyte viability and isolatability are preserved across all assessed regions and that STAR-exposed areas may exhibit region-specific structural features, while functional data on Ca2+ handling remain exploratory and require validation in larger datasets. Broad adoption will be the key to delineating dose-time-substrate relationships and disentangling radiation effects from underlying cardiomyopathy.
Characterizing neural connectivity at transcriptomic cell-type resolution is essential for understanding neural mechanisms of circuit function. Here, we present single transcriptome assisted rabies tracing (START), a protocol combining monosynaptic rabies tracing with single-nucleus RNA sequencing to identify transcriptomic cell types providing inputs to defined neuronal populations in the mouse cortex. We describe steps for Cre-dependent helper virus injection, EnvA-pseudotyped rabies infection, tissue microdissection, and fluorescence-activated nuclei sorting. We then detail procedures for library preparation and computational annotation of nuclei. For complete details on the use and execution of this protocol, please refer to Patiño et al.1.
To further promote continuous quality improvement of clinical practice guidelines (CPGs) and expert consensuses (ECs) in China, the Scientific, Transparent, and Applicable Rankings (STAR) Working Group conducted its 2023-2024 biennial appraisal based on its previous work. In this round, 40 specialty committees and over 1 000 experts participated, and large language model technologies were integrated to comprehensively evaluate guidelines and consensuses led by Chinese scholars and published in journals between 2023 and 2024. A total of 305 guidelines and 1 153 consensuses were included for 2023, compared to 424 guidelines and 1 391 consensuses for 2024. The average scores for guidelines and consensuses increased from 41.0 and 28.8 in 2023 to 49.2 and 33.0 in 2024, respectively, indicating a significant upward trend compared with previous years. Among the evaluation domains, "Recommendations" achieved the highest scoring rate, whereas "Protocols" and "Funding and Conflict of Interest Disclosures" recorded the lowest. In terms of publication volume, Oncology, Traditional Chinese Medicine, and Pediatrics were the top three specialties. Overall, medical guidelines and consensus documents in China are at a critical stage of transitioning from"quantity-driven"expansion to"high-quality development."In the future, guideline developers, academic journals, and the STAR Working Group should establish long-term collaborative mechanisms to jointly promote the dynamic and intelligent evolution of the ranking system. 为持续促进我国指南和共识质量的整体提升，指南科学性（Scientific）、透明性（Transparent）和适用性（Applicable）评级（Rankings）（简称“STAR”）工作组在既有基础上完成了2023—2024年双年度指南和共识评级工作。本次工作由40个专科委员会、1 000余名专家共同参与，并结合大语言模型技术，对2023—2024年由中国学者牵头在期刊发表的指南与共识进行综合评价。结果显示，2023与2024年分别纳入指南305部与424部，共识1 153部与1 391部。指南与共识平均得分从2023年的41.0分、28.8分提升至2024年的49.2分、33.0分。各评价领域中，“推荐意见”得分率最高，“计划书”与“资助作用说明”得分率最低。发表数量方面，肿瘤学、中医药学与儿科学位居前三。总体来看，我国医学指南和共识正处于由“数量驱动”向“高质量发展”转型的关键阶段。未来指南和共识制订者、期刊和STAR工作组应建立长效合作机制，共同推动评级的动态化和智能化。.
Indigenous Peoples worldwide have long understood that water is not a resource to be managed, but a living relative with spirit, agency, and sustainability. This community-led research article draws from decolonial story sharing, land-based conversations, and water ceremonies conducted with Starblanket Cree First Nation in Treaty 4 Territory, Saskatchewan, Canada. Drawing from community-led decolonial story sharing held with the Starblanket Cree First Nation in Treaty 4 Territory, Saskatchewan, Canada, the study examines how Indigenous water science contributes to relational, ethical, and community-led approaches to sustainable water governance and water healing. Focusing on relationality, ceremony, and collective responsibility, the Starblanket Cree First Nation's water activities and teachings show that sustainable water governance must be understood as an ethical and spiritual practice rooted in reciprocity, rather than as a technical exercise.
α-Gal syndrome (AGS), a tick bite-associated allergy to mammalian meat, affects an estimated 450,000 Americans, with the highest prevalence concentrated in the US South and Southeast. The lone star tick (Amblyomma americanum), ubiquitous across the region, transmits AGS sensitization, making reexposure nearly inevitable for outdoor workers and residents. Current management relies on dietary avoidance of mammalian products of an indeterminant length, a recommendation that is neither practical in a region where mammalian meat is central to cuisine and culture nor evidence based regarding duration or necessity. In the absence of treatment options, patients are turning to alternative interventions such as acupuncture protocols that lack validation from controlled trials. Meanwhile, oral immunotherapy has demonstrated safety and efficacy for other immunoglobulin E-mediated food allergies, and individual case reports suggest desensitization may be feasible for AGS syndrome. The US South, with its high disease burden, clinical expertise, and patient populations, is uniquely positioned to lead the rigorous phase 1 and phase 2 trials needed to develop evidence-based treatment alternatives. Without such research, patients have only dietary restriction as an evidence-based option and may seek purported treatments that lack rigorous validation.
Ovarian stimulation using exogenous follicle-stimulating hormone may enhance follicular recruitment; however, in the context of artificial insemination, it does not substantially improve pregnancy rates. Moreover, such physiological stimulation may impair oocyte developmental competence. To investigate the underlying metabolic mechanisms, the present study employed targeted metabolomics to compare the amino acid profiles in the follicular fluid of dairy cows during the natural estrous cycle (control group) and following ovarian stimulation (treated group). In total, 21 differentially abundant amino acids were identified: 7 were significantly upregulated, and 14 were significantly downregulated in the treatment group. Among the downregulated amino acids, L-serine was identified as a candidate hub metabolite via network analysis. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that these differentially abundant amino acids were primarily enriched in the metabolic pathways of glycine, serine, and threonine, as well as in monocyclic lactam and arginine biosynthesis. Supplementation with 25-μM L-serine in the in vitro maturation medium significantly improved the oocyte maturation rate, cleavage rate, and blastocyst rate of oocytes from dairy cows undergoing ovarian stimulation. Additionally, supplementation promoted cumulus cell expansion and reduced the apoptosis rate of the formed blastocyst cells. The in vitro culture of bovine granulosa cells further demonstrated that L-serine could significantly promote cell proliferation and increase the estradiol to progesterone ratio (E2/P4). Real-time quantitative PCR analysis revealed that L-serine significantly upregulated the mRNA expression of key functional genes, including aromatase (CYP19A1), steroidogenic acute regulatory protein (STAR), and proliferating cell nuclear antigen (PCNA). These findings suggest a potential role for L-serine in supporting oocyte and embryo development, offering metabolic insights for further optimization of ovarian stimulation protocols.
Fast estimations of direct oral anticoagulant (DOAC) plasma concentrations are useful in ruling out clinically significant concentrations before urgent surgical interventions. DOAC-specific anti-factor Xa (anti-Xa) assays are time consuming and not available 24/7 in most hospitals. Anti-Xa assays calibrated with low-molecular weight heparins (LMWHs) are widely available 24/7 and could give concentration estimations for anti-Xa DOACs (DXaIs). This study aimed to determine whether LMWH-calibrated anti-Xa assays could establish thresholds corresponding to DXaI concentrations associated with non-increased bleeding risk. Omniplasma was spiked with rivaroxaban, apixaban, or edoxaban in a 5-604 ng/mL range. One pooled patient sample was included per DXaI. Samples were analysed in 18 hospital laboratories, using 5 different analyser-reagent combinations (Stago Liquid anti-Xa on STA-R Max2/3, HemosIL Liquid anti-Xa on ACL-TOP350/550, Innovance Heparin on CS2500/CN3000, Biophen Heparin LRT on CS2500/CS5100, Roche anti-Xa on Cobas t511 analysers). Samples were analysed using local LMWH anti-Xa assay protocols using local reagent lots. Delta optical density and "LMWH" anti-Xa-activity were reported. DXaI concentrations were confirmed using liquid chromatography with tandem mass spectrometry detection. Large differences in DXaI sensitivity between reagent groups were observed, which prevent formulation of universal threshold values for non-increased risk of bleeding. However, reagent-specific threshold values can be established with the exception of the Hyphen reagent group, due to high interlaboratory variability. Within analyser-reagent groups, a good correlation between spiked DXaI concentrations and "LMWH" activity assay readout is observed, permitting conversion between the two units up to 75 ng/mL for all DXaIs. Widely available rapid low-to-moderate DXaI concentration estimation can be achieved using LMWH-calibrated anti-Xa assays. Potential applications include ruling out clinically relevant DXaI concentrations before surgery and screening for the presence of a factor Xa inhibitor, but not specific DXaI monitoring.
Unconscious bias influences clinical judgment and communication across healthcare, but its effects in obstetric practice are particularly consequential due to time-sensitive decision making, high emotional vulnerability, and longstanding maternal health inequities. When bias-driven interactions escalate into coercion, non-consented procedures, or normalized mistreatment, they constitute forms of gender-based violence embedded within routine clinical care. This review synthesizes evidence on how unconscious bias shapes provider-patient communication, shared decision making, autonomy, and obstetric outcomes. A systematic review with narrative synthesis was conducted using PubMed Central and Google Scholar from inception to August 2025. English-language studies examining unconscious or implicit bias in obstetric communication or decision making were eligible. Dual screening, structured data extraction, and thematic synthesis were performed. A total of 89 studies met inclusion criteria, including qualitative, quantitative, mixed-methods, and conceptual designs. Across settings, unconscious bias manifested through: (1) Dismissal of symptoms and credibility discounting; (2) selective or incomplete information exchange; (3) stereotyping and moral judgment; (4) differential clinical discretion, surveillance, and escalation; and (5) normalized mistreatment and non-consented care. These mechanisms systematically undermined communication quality, restricted shared decision making, and reduced autonomy, particularly for Black, Indigenous, migrant, low-income, uninsured, adolescent, and disabled patients. Quantitative and vignette studies demonstrated measurable effects on clinical decisions, including biased cesarean recommendations, disparities in pain management, and delays in recognizing deterioration. These processes contributed to documented inequities in severe maternal morbidity, intervention rates, and maternal near-miss events. Notably, coercive consent practices, non-consented procedures, and normalized disrespect emerged as manifestations of gender-based violence within obstetric settings. Unconscious bias shapes obstetric interactions through interconnected communication and decision making pathways that constrain autonomy, reduce quality of care, and reinforce structural inequities. At its most severe, bias-driven care constitutes a form of gender-based violence that disproportionately affects racialised and socially marginalized women. Effective mitigation requires multi-level strategies combining communication-focused interventions, standardized protocols, interpreter support, reflective practice, and institutional accountability frameworks.
To assess the knowledge, attitudes, and practices (KAP) regarding herpes zoster (HZ) and its vaccination among rheumatologists in China. A nationwide cross-sectional survey was conducted between April 1 and May 31, 2025, using the Questionnaire Star online platform. The questionnaire assessed sociodemographic characteristics, knowledge of HZ and its vaccine (via a 13-item scale), attitudes and behaviors regarding vaccination, and willingness to disseminate information. Ordinal logistic regression was applied to identify associated factors of knowledge scores, while subgroup analyses were conducted to evaluate attitude-related factors. A total of 1,772 rheumatologists from a wide range of provinces participated (mean age: 41.4 y; 67.3% female). Regarding professional background, most held a master's degree (44.1%), followed by a bachelor's (38.9%) and doctoral degree (17.0%). Clinical titles were distributed as associate chief (31.3%), chief (26.4%), attending (26.2%), and junior physicians (16.0%). The median knowledge score was 7 (IQR: 6-9). Multivariable analysis revealed that higher regional GDP per capita (β = 0.020, p = .040), advanced education (β = 0.234, p < .001), and greater clinical seniority (β = 0.296, p < .001) were independently associated with higher knowledge scores. Clinically, physicians expressed significant concern primarily in three areas: HZ's potential to interfere with the underlying autoimmune inflammatory rheumatic diseases (AIIRDs) (89.0%), HZ-related complications (81.5%), and deterioration of patient quality of life (73.0%). Notably, 65.6% of respondents were concerned about all three aspects simultaneously. Regarding vaccination attitudes, 96.4% of respondents reported conditional support for vaccinating patients with AIIRDs, contingent upon pre-vaccination risk assessment and adherence to recommended protocols. Additionally, 80.0% favored prioritizing recombinant vaccines. Subgroup analyses indicated that higher clinical seniority was significantly associated with more supportive attitudes (p < .001). In clinical practice, 76.5% of physicians reported actively recommending HZ vaccination, although several barriers remained. The main reasons for not recommending included limited consultation time (52.2%), perception of low HZ incidence (42.8%), and lack of patient concern (31.3%). Targeted education and strengthened rheumatologist-led counseling may help address existing barriers, particularly among junior physicians in under-resourced settings. Such measures could contribute to improving HZ vaccination uptake and protection in patients with AIIRDs.
Placental growth factor (PLGF) drives cardiomyogenesis and vasculogenesis during cardiogenesis. Here, we present a protocol for enhancing production of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) and endothelial cells (ECs). We describe steps for CM-/EC-directed hPSC differentiation with PLGF. Our approaches can achieve production of hPSC-CMs/-ECs by 2- to 2.5-fold compared with those in traditionally established protocols, through promoting proliferation and differentiation of cardiac and vascular progenitors. For complete details on the use and execution of this protocol, please refer to Witman et al.1.
Global warming is increasing extreme heat events, directly impacting labour health and economic productivity. High-resolution global reanalysis datasets, such as ERA5-Land, are enabling worldwide and regional assessments of heat-related labour impacts. This study systematically examines the reliability and bias of ERA5-Land-derived labour loss estimates across diverse Köppen-Geiger climate zones (A, C, D) and land cover types (urban and natural land cover) at 37 sites (including 36 WMO stations) in advanced-economy regions of Pacific Asia (Japan, Korea, Taiwan, and Singapore) from 2014 to 2023. Using the physics-based Liljegren wet-bulb globe temperature (WBGT) model and established exposure-response functions, we quantified annual cumulative labour loss and productivity loss while accounting for spatial labour distribution. A comparison of WBGT‑derived labour‑loss estimates from 37 ground‑station datasets with ERA5‑Land-driven estimates indicates strong concordance in temperate zones (R2 ≈ 0.85) and urban areas (R2 ≈ 0.94), contrasted by a systematic underestimation in tropical regions (up to ~ 18%, R2 ≈ 0.27). Incorporating a weighting approach that integrates population density and labour force participation rate (LFPR) significantly improves the assessment of the effective labour force scale, with ERA5-Land's R2 in tropical regions increasing from 0.27 to 0.70. Based on our findings, we recommend (1) application of ERA5-Land in temperate and urban local-climate-zone (LCZ)-dominant dominant areas with standard caution, (2) for macro-level, climate- or land-cover-aggregated assessments, a 20% safety margin for tropical climate class (Köppen-Geiger Type A) and a 15% adjustment for natural environments to address underestimation uncertainty; and (3) for fine-scale, coupled climate × land-cover applications, context-specific correction (e.g., 31.55% for tropical natural areas) where localised land-cover data is available. These guidelines refine the operational use of reanalysis data for precise heat-risk assessments, and inclusion of population-density weighting in standard assessments, which improves index performance by 15-50%. While these guidelines refine the use of reanalysis data and offset policy under-protection risks, future applications must still account for microclimatic heterogeneity and the inherent assumptions of uniform sector-specific labour distributions. Ultimately, integrating these safety margins and weighting protocols provides a practical framework for informed public health interventions.
Single-nuclei multiomic profiling of clinical biopsies is often hindered by limited tumor mass and high debris content. Here, we present a protocol for the dissociation of head and neck squamous cell carcinoma (HNSCC) and matched-normal tissues using a mechanical-enzymatic workflow. We describe steps for minimizing ambient nucleic acid background by red blood cell (RBC) lysis and DNase-I treatment and for removing cellular debris using density gradient centrifugation. We then detail procedures for isolating intact nuclei and modified single-nuclei assay for transposase-accessible chromatin sequencing (sn-ATAC) and 3'-GEX (gene expression) indexing PCR and size selection to maximize library complexity.
Cerebral organoids (COs) are valuable for studying neurodegenerative diseases and pathogens, but their limited microglia pose challenges. Here, we present a protocol to generate organoids with physiologically relevant microglia (CO-iMs). We describe steps for differentiating induced pluripotent stem cells (iPSCs) into mesoderm, seeding embryoid bodies (EBs), and harvesting hematopoietic progenitor cells (HPCs). We detail procedures for co-culturing HPCs and iPSCs to generate CO-iMs, followed by their infection and treatment. The model provides a platform to study viral infections and neuroinflammation. For complete details on the use and execution of this protocol, please refer to Narasipura et al.1.
Here, we present a protocol for total internal reflection fluorescence (TIRF) microscopy image preprocessing and radial analysis of lymphocyte function-associated antigen-1 (LFA-1) in T cell immune synapses. We describe steps for combining customizable Fiji/ImageJ macros for image background subtraction and segmentation with flexible R-based scripts for peripheral supramolecular activation cluster (pSMAC) center identification, center-based cropping, and radial analysis of synaptic LFA-1 in T cells. This workflow supports established analyses, including radial averaging/spinning and fluorescence intensity measurements across the synapse.
We present a protocol for modeling early tumorigenesis using air-liquid interface (ALI) organoids derived from oral, esophageal, and lung epithelia bearing first-hit oncogenic mutations. We describe steps for identifying candidate amplified oncogenes through outlier analysis, establishing ALI organoids, and inducing oncogenic activation. We then perform pooled lentiviral open reading frame (ORF) library delivery, selection, and barcode sequencing for functional screening. This approach enables validation of candidate drivers of neoplastic transformations, addressing a gap between in vitro and in vivo tumorigenesis studies. For complete details on the use and execution of this protocol, please refer to Salahudeen et al.1.
Disruption of mitochondrial morphology occurs during various diseases, but the biological significance is not entirely clear. Here, we describe a detailed step-by-step protocol for a chemically inducible dimerization (CID) system-based synthetic protein device, termed inducible counter mitochondrial morphology (iCMM). This system allows artificial manipulation of mitochondrial morphology on a timescale of minutes in living mammalian cells. We also describe an AI-assisted image processing approach. For complete details on the use and execution of this protocol, please refer to Miyamoto et al.1 This protocol provides an updated version of the method described by Miyamoto et al.2.
Protein phosphorylation regulates essential cellular processes, yet probing kinase and phosphatase activities in native contexts remains challenging. Here, we present a protocol to study direct (de)phosphorylation events on a proteome-wide scale using on-bead in vitro kinase and phosphatase assays (OBIKA/OBIPhA). We describe steps for employing active enzyme complexes and identifying direct substrates and regulated phosphosites using mass spectrometry. Compared to traditional in vitro approaches, this protocol operates under native conditions to preserve protein interactions and modifications in eukaryotic cell systems. For complete details on the use and execution of this protocol, please refer to Brunner et al.1 and Hu et al.2.