Journal Of Visualized Experiments[JOURNAL]
Sun
200 papers
Biological Standardization of Human ASC-Based Biofabrication for Reproducible Macroscale Tissue Constructs.
Pimentel A, Vieira LR, Montenegro B … +7 more , Diederichs I, Fontes L, Esteves MED, Russo R, Boldrini L, Granjeiro JM, Baptista LS
J Vis Exp · 2026 Jun · PMID 42330021 · Publisher ↗
The lack of standardized protocols across laboratories poses a significant barrier to the comparability of experimental outcomes and the efficient transfer of biofabrication technologies. Rigorous biological standardizat...
The lack of standardized protocols across laboratories poses a significant barrier to the comparability of experimental outcomes and the efficient transfer of biofabrication technologies. Rigorous biological standardization not only enhances reproducibility but also facilitates alignment with national and international regulatory frameworks, which are essential for the clinical translation of tissue engineering strategies. This study introduces a standardized biofabrication workflow comprising monolayer culture of hADSCs, spheroid formation, 3D printing of Gelatin Methacryloyl (GelMA) scaffolds, and spheroid bioassembly. Quality control metrics were integrated to ensure reproducibility. The 3D-printed GelMA scaffold was designed with two central pores, each capable of housing hundreds of spheroids. Printing fidelity was confirmed by comparing scaffold dimensions to the CAD model, with minor non-significant deviations. hADSCs formed uniform spheroids within 24 h in agarose micromolds. When seeded into scaffold pores, spheroids underwent continuous fusion over 72 h, forming macroscale constructs confirmed by confocal imaging. Scaffold design and printing accuracy ensured structural support, while spheroid uniformity enabled predictable formation of the construct. The fusion of spheroids within the scaffold pores yielded tissue constructs suitable for transplantation, with or without the scaffold, highlighting the versatility of this bioassembly approach. The findings underscore the pivotal role of biological standardization in advancing reproducible methodologies for the biofabrication of living tissue constructs, with promising implications for future clinical applications in regenerative medicine.
A Randomized Controlled Trial of Multisensory Stimulation and Family-Involved Rehabilitation in Elderly Thoracic Surgery Patients.
Yuyao Z, Liangqin L, Yue Z … +1 more , Miaolin T
J Vis Exp · 2026 Jun · PMID 42330020 · Publisher ↗
Elderly patients undergoing thoracic surgery often experience postoperative pain, anxiety, and delayed functional recovery. Multisensory stimulation nursing (MSSN) and family-participatory rehabilitation training (FPRT)...
Elderly patients undergoing thoracic surgery often experience postoperative pain, anxiety, and delayed functional recovery. Multisensory stimulation nursing (MSSN) and family-participatory rehabilitation training (FPRT) are recognized non-pharmacological approaches; however, evidence for their combined use in this population remains limited. This study evaluated the effects of MSSN combined with FPRT on pain, anxiety, sleep quality, and physical function in elderly patients after thoracic surgery. In this randomized controlled trial, patients aged ≥65 years undergoing elective thoracic surgery were assigned to a control group (routine care, n = 38) or an intervention group (routine care plus MSSN and FPRT, n = 39). MSSN involved 20-min multisensory sessions five times per week for 4 weeks, while FPRT included structured family-assisted respiratory and mobility exercises. Outcomes included pain (NRS), anxiety (HADS-A), sleep quality (PSQI), grip strength, and 6-minute walking distance (6MWD), assessed at baseline, 2 weeks, and 4 weeks. The intervention group demonstrated significantly greater reductions in pain (group × time interaction, P = 0.01; week 2: B = 0.21, P = 0.007; week 4: B = 0.23, P = 0.004) and anxiety (B = -3.42, P = 0.002) compared to the control group. Significant improvements were also observed in sleep quality (PSQI change: -2.71 ± 1.13 vs. -0.94 ± 1.25, P = 0.004), grip strength (week 4: B = -0.31, P = 0.008), and 6MWD (54 m vs. 27 m improvement, P < 0.01), with the 54 m improvement exceeding the estimated minimal clinically important difference (30 m). Postoperative pulmonary complications were lower in the intervention group (7.5% vs. 22.5%), and no adverse events were reported. Combined MSSN and FPRT may safely reduce pain and anxiety while improving sleep and functional recovery in elderly thoracic surgery patients.
The Effects of The Menstrual Cycle On Growth Factors In Platelet-Rich Fibrin: A Cross-sectional Observational Study.
Yildiz HS, Ozcan M, Kayhan HG … +4 more , Ucak Turer O, Alkaya B, Yılmaz MB, Haytac MC
J Vis Exp · 2026 Jun · PMID 42330016 · Publisher ↗
Platelet-rich fibrin (PRF) is widely used in dental surgery for regenerative procedures; however, variability in its biological content remains a key limitation for standardization. This study aimed to implement a tightl...
Platelet-rich fibrin (PRF) is widely used in dental surgery for regenerative procedures; however, variability in its biological content remains a key limitation for standardization. This study aimed to implement a tightly controlled experimental workflow to evaluate how menstrual cycle phases influence the release of growth factors from PRF. Venous blood samples were obtained from 21 healthy female volunteers at three predefined menstrual phases (menstrual: days 1-5; follicular: days 6-14; luteal: days 15-28), with all collections performed within a fixed time window (11:00-11:30 a.m.) to minimize circadian variation. For each phase, separate tubes were allocated for complete blood count, hormone analysis (estrogen, progesterone, follicle-stimulating hormone [FSH], and luteinizing hormone [LH]), and PRF preparation. PRF was produced by immediate centrifugation under standardized conditions (708 × g for 12 min; fixed-angle rotor, 40 °; radius 88 mm; no anticoagulant). Clots were macroscopically verified (homogeneous, elastic structure), carefully separated from the red blood cell layer, and individually weighed. To standardize growth factor release, each clot was incubated in RPMI-1640 medium using a weight-based ratio (1 g PRF: 1 mL medium), followed by controlled incubation at 37 °C with orbital agitation (100 rpm). Supernatants were collected and stored at -80 °C prior to analysis. Growth factors (IGF-1, PDGF, FGF-2, VEGF, and TGF-β1) were quantified using enzyme-linked immunosorbent assay (ELISA) under uniform assay conditions. Within this standardized protocol, FGF-2 levels were significantly higher during the follicular phase compared to the menstrual and luteal phases. A moderate positive correlation was observed between FGF-2 and estrogen levels in this phase, whereas no statistically significant differences were detected for other growth factors. These findings indicate that menstrual cycle phase may contribute to variability in PRF-derived growth factor profiles. The outlined protocol provides a reproducible framework for minimizing pre-analytical and analytical variability in PRF studies. The clinical implications of phase-related differences warrant further investigation in studies incorporating larger cohorts and treatment outcomes.
Validation of Random Transgene Integration and Expression in Eimeria Parasites.
Sun Y, Liu Y, Liu X … +2 more , Song M, Suo X
J Vis Exp · 2026 Jun · PMID 42330014 · Publisher ↗
Validation of transgenic Eimeria parasites is essential for confirming stable genomic integration and expression of exogenous genes. This protocol describes a comprehensive workflow for verifying transgene insertion and...
Validation of transgenic Eimeria parasites is essential for confirming stable genomic integration and expression of exogenous genes. This protocol describes a comprehensive workflow for verifying transgene insertion and protein expression in Eimeria tenella. The procedure begins with genomic DNA extraction from sporulated oocysts, followed by PCR amplification to preliminarily verify the presence of the target fragment. To further confirm genomic integration, whole-genome resequencing is performed to identify insertion sites and evaluate the stability of the integrated construct in the parasite genome. Protein expression is subsequently examined by Western blotting of lysates prepared from purified sporozoites, enabling detection of the target recombinant protein. In addition, intracellular localization is visualized by indirect immunofluorescence assay (IFA) using infected host cells. Together, these assays provide a robust and reproducible framework for validating transgenic Eimeria lines. This workflow enables reliable confirmation of genomic integration and protein expression, thereby supporting downstream applications in genetic manipulation, functional genomics, and vaccine development targeting Eimeria parasites.
Cochlear Size and Shape Variation from the Russian Population and a New Cochlear Parameter for a Safe Cochlear Implant Electrode Insertion.
Sugarova S, Kuzovkov V, Schmidtleitner E … +3 more , Anaghan D, Sriperumbudur K, Dhanasingh A
J Vis Exp · 2026 Jun · PMID 42330009 · Publisher ↗
This study investigates the size and shape variation of cochleae in the Russian population. Also, introduces a new cochlear parameter that defines the longest straight segment of the basal turn, with the implication of a...
This study investigates the size and shape variation of cochleae in the Russian population. Also, introduces a new cochlear parameter that defines the longest straight segment of the basal turn, with the implication of a safe cochlear implant electrode insertion. A medical-grade, CE-marked DICOM viewer (OTOPLAN) was used to measure cochlear parameters, including A- and B-values, which define the length and width of the basal turn. The shape of the cochlear basal turn was estimated by the ratio between B-and A-values. The new cochlear parameter, I-value, defines the effective straight segment of the basal turn as the longest straight line connecting the cochlear entrance to the lateral wall at the opposite end, passing tangentially to the inner wall. Ratio between B-, and A-value determines the shape of the cochlear basal turn, with the cut-off value ≥0.75 for round-shaped and <0.75 for elliptical-shaped cochlear basal turn. CT scans of the temporal bone from 112 ears with normal inner ear anatomy were used in this study. The cochlear size of the Russian population, as measured by A-value, ranged from 8.04-10.03 mm, with a mean of 9.03 mm. The B-value ranged from 5.84-7.59 mm, with a mean of 6.70 mm. A round-shaped cochlear basal turn was observed in 48% of the population, leaving the remaining 52% of the population with an elliptical-shaped basal turn. The I-value that defines the effective straight segment of the basal turn ranged from 6.7-9.6 mm, with an average of 8.3 mm. The trend line indicates that the I-value is approximately 0.7-0.8 mm shorter than the A-value. For the first time in the literature, cochlear size, as measured by A-value, is presented for the Russian-speaking population. The round-shaped cochlear basal turn is seen in 48% of the population. The new cochlea parameter, the I-value, is approximately 0.7-0.8 mm shorter than the A-value.
Real-World M3-BREATHE: Toward Multimodal Mobile Monitoring of Behaviour, Respiration, and Exposures for Treatment and Health Evaluation.
Guo W
J Vis Exp · 2026 Jun · PMID 42330004 · Publisher ↗
The integration of mobile health (mHealth) technologies for continuous, real-time monitoring of physiological and behavioural signals, in concert with detailed measurement of environmental exposures, holds transformative...
The integration of mobile health (mHealth) technologies for continuous, real-time monitoring of physiological and behavioural signals, in concert with detailed measurement of environmental exposures, holds transformative potential for advancing personalized health risk assessment and enabling more effective treatment and intervention. In this protocol, I introduce a multimodal mobile monitoring system specifically designed to capture and integrate data on individual behaviour (daily activities and travel), respiratory function, physiological parameters and environmental exposures. The system combines mobile health devices and portable environmental sensors with GPS-enabled smartphone-based applications to generate synchronized, high-resolution data streams. These data are processed through a smart mobile phone application by incorporating physiological parameter calculation (e.g., physical activity intensity, respiratory rate) and external urban environmental data (e.g., urban green spaces, street view maps), after which a machine learning-based alert system was deployed to provide actionable insights into respiratory health, daily activities and mobility patterns, and exposure to risk factors. The protocol provides comprehensive and detailed guidance on hardware setup, software configuration, data acquisition procedures, and analytical workflows necessary for deploying the Multimodal Mobile Monitoring of Behaviour, Respiration, and Exposures (air pollution, noise, light, heat, greenness, etc.) for Treatment and Health Evaluation (M3-BREATHE) in both controlled clinical experiments and real-world contexts. This forward-looking M3-BREATHE platform establishes a scalable, non-invasive, and cost-efficient approach to longitudinal environment-mobility-health (E-M-H) monitoring, linking individual-level precision health in everyday life with long-term health intelligence at the population scale.
Diverse Methodologies Used for Preclinical Research into Prenatal Cannabis Exposure.
Davies S, Dominguez ZM, Maxwell JR
J Vis Exp · 2026 Jun · PMID 42330002 · Publisher ↗
The legalization of cannabis across the United States has led to an increase in human use, especially amongst pregnant individuals, driving the need for vigorous scientific investigation on potential health effects to th...
The legalization of cannabis across the United States has led to an increase in human use, especially amongst pregnant individuals, driving the need for vigorous scientific investigation on potential health effects to the individual and developing fetuses. Preclinical research, particularly including trans-delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), offers exciting opportunities and significant challenges. However, the variability in cannabis formulations, routes of administration (i.e., orally, inhaled, topical application), and dosing presents challenges in evaluating efficacy and safety due to differences in the pharmacokinetics and bioavailability. Understanding both the potential benefits and risks of cannabis use, including short- and long-term effects on pregnant individuals and developing fetuses, is essential for understanding neurodevelopmental effects. In this review, in vitro and in vivo methods, including cell-based assays, organoid models, animal models, artificial intelligence (AI), and machine learning (ML), are utilized for preclinical research into prenatal cannabinoid exposure (PCE) to evaluate methodological limitations and the implications for developing evidence-based treatment.
A Predictive Nursing Tool for Patient Positioning Safety in Hybrid Digital Subtraction Angiography Operating Rooms.
Jin L, Qi H
J Vis Exp · 2026 Jun · PMID 42329998 · Publisher ↗
The aim of this study was to develop and internally validate a phase-updating nursing predictive assessment tool based on baseline patient-surgical risk and modifiable nursing processes for use before surgery and after p...
The aim of this study was to develop and internally validate a phase-updating nursing predictive assessment tool based on baseline patient-surgical risk and modifiable nursing processes for use before surgery and after positioning in the hybrid digital subtraction angiography operating room. This single-center retrospective cohort included inpatient adults undergoing their first procedure in the hybrid operating room, with surgery as the unit of analysis and follow-up to 72 h postoperatively. The outcome was a composite endpoint of position-related complications. Missing predictor data were handled using multiple imputation (m = 10), and extraction consistency was assessed with Cohen κ and intraclass correlation coefficients. Candidate variables from univariable screening plus prespecified variables entered least absolute shrinkage and selection operator regression, followed by multivariable logistic modeling and nomogram construction with threshold-based risk stratification. Model performance was evaluated by area under the curve, calibration intercept and slope, Brier score, bootstrap internal validation, and decision curve analysis. A total of 1,936 cases were analyzed, and the composite outcome occurred in 10.23%. Maximum missingness of key variables was 3.25%, and extraction consistency was good (κ ≥ 0.86, intraclass correlation coefficient ≥ 0.89). Twelve predictors were retained; pressure-point protection and intraoperative position checks were protective (odds ratio 0.68-0.71). The baseline model had an area under the curve of 0.74/0.72 (apparent/corrected), and the full model achieved 0.79/0.77. The optimism-corrected calibration slope was 0.947, the intercept was 0.004, and the Brier score was 0.085. This tool showed stable discrimination, calibration, and net benefit on internal validation. Nursing process variables added value, and the tool may support preoperative assessment and post-positioning risk updating, pending external validation.
Automated Operant Intermittent Alcohol Drinking In Socially-Housed Mice via The Intellicage Testing System.
Anderson LG, Bock R, Averbeck TH … +1 more , Alvarez VA
J Vis Exp · 2026 Jun · PMID 42329995 · Publisher ↗
Despite known confounds of social isolation on rodent behavior, many investigators rely on singly housing mice to assess alcohol preference in preclinical models of alcohol use disorder (AUD). This protocol describes a s...
Despite known confounds of social isolation on rodent behavior, many investigators rely on singly housing mice to assess alcohol preference in preclinical models of alcohol use disorder (AUD). This protocol describes a social alcohol drinking task that allows for high-throughput, automated assessment of alcohol drinking behaviors in mice without the confounding impacts of stress induced by social isolation. The IntelliCage testing system enables investigators to simultaneously assess operant alcohol drinking behavior in up to 16 same-sex mice, each uniquely identified with a subcutaneously implanted radiofrequency identification (RFID) transponder. Access to sipper bottles is software-controlled and can be adjusted individually for each mouse. In this chronic, intermittent alcohol drinking paradigm, mice voluntarily engage in operant nosepokes to access sipper bottles containing 20% alcohol for six consecutive weeks. Alcohol bottles are only accessible every other day for 24 h at a time and can be optionally adulterated with quinine to assess punishment-insensitive drinking. Visits to each operant corner, nosepokes, and lick data are automatically recorded for each mouse. Detailed experimental methods and the open-source code to analyze the recorded data will allow investigators to assess alcohol drinking behaviors relevant to AUD in an increasingly efficient and ethologically relevant manner.
Generation of Bone Marrow Mesenchymal Stem Cell-Derived Islet Organoid-like Structures From Fresh Pancreatic Tissue.
Zhao W, Shan J, Wang H
J Vis Exp · 2026 Jun · PMID 42329981 · Publisher ↗
As a prevalent chronic illness across the globe, diabetes mellitus (DM) is marked by disrupted glucose balance in the body. In type 1 diabetes mellitus (T1DM), autoimmune responses destroy pancreatic β-cells entirely, re...
As a prevalent chronic illness across the globe, diabetes mellitus (DM) is marked by disrupted glucose balance in the body. In type 1 diabetes mellitus (T1DM), autoimmune responses destroy pancreatic β-cells entirely, resulting in total insulin insufficiency. Patients with this condition need lifelong supplemental insulin treatment. The present study intends to create a reliable and economical culture platform to generate pancreatic islet organoid-like structures from bone marrow mesenchymal stem cells (BMSCs). This platform can not only ease the shortage of donor islets for transplantation, but also serve as an in vitro model for diabetes-related research. By optimizing the preparation process of porcine pancreatic tissue lysate (centrifugation at 12,000 rpm for 30 min at 4 °C, followed by 0.22 µm filtration for sterilization), BMSCs were successfully induced to differentiate into pancreatic islet organoid-like structures. Morphological validation confirmed that only induced BMSCs formed compact, plump, and highly transparent islet-like aggregates, whereas noninduced BMSCs exhibited significant vacuolization and reduced transparency. Dynamic monitoring showed that cell aggregates (50-100 µm in diameter) formed on day 16 of culture and developed into capsule- like structures (200-300 µm in diameter) by day 22. This method is simple to operate, cost- effective, and does not require complex equipment. The resulting organoids are morphologically similar to pancreatic islets, providing a tool for in vitro studies and related drug screening.
Recombinant Production And Purification of Hydrophobin SC16 From Escherichia coli And Monitoring of Its Self-Assembly Using Fluorescence Assays.
Drozdowski IE, He R, Langelaan DN
J Vis Exp · 2026 Jun · PMID 42329978 · Publisher ↗
Hydrophobins are small amphipathic proteins that self-assemble at hydrophobic-hydrophilic interfaces to form stable waterproof coatings over fungal spores. Class I hydrophobins form particularly stable "rodlets" containi...
Hydrophobins are small amphipathic proteins that self-assemble at hydrophobic-hydrophilic interfaces to form stable waterproof coatings over fungal spores. Class I hydrophobins form particularly stable "rodlets" containing ordered amyloid-like structures that coat surfaces. This unusual surface chemistry gives them potential applications in drug delivery, foam stabilization, emulsification, and surface modification. To produce hydrophobins, there is a need for comprehensive, high-yield protocols from simple expression systems. Furthermore, it is necessary to provide facile methods to characterize hydrophobin function following recombinant production and purification. Herein, we describe protocols for the production and characterization of SC16, a class IB hydrophobin produced by the fungus Schizophyllum commune, which can be recombinantly expressed in Escherichia coli and subsequently purified under non-reducing, native conditions. This involves transformation of an expression plasmid encoding SC16 fused to an N-terminal fusion protein into E. coli, induction of protein expression, cell lysis under native conditions, affinity chromatography, and protease cleavage to isolate the hydrophobin at yields of ≥5 mg. The self-assembly of SC16 can then be monitored by end-point or kinetic fluorescence-based assays using thioflavin T. Both assays involve incubation of SC16 with prolonged mixing to promote self-assembly at air-water interfaces and are monitored by fluorescence measurements. Together, these approaches provide simplified protocols for purifying SC16 and monitoring its self-assembly and may be extended to other systems involving similar protein self-assembly.
Bronchoscopy-Guided Placement of Petrolatum Gauze For Closure of Peripheral Bronchopleural Fistula in Humans.
Jiang M, Zheng Y, Li Q … +2 more , Wang N, Luo Z
J Vis Exp · 2026 Jun · PMID 42329975 · Publisher ↗
Bronchopleural fistula (BPF), defined as a pathological communication between the bronchial airways and the pleural space, carries high mortality (18%-50%) due to complications like infection and respiratory failure. Cur...
Bronchopleural fistula (BPF), defined as a pathological communication between the bronchial airways and the pleural space, carries high mortality (18%-50%) due to complications like infection and respiratory failure. Current treatments, including conservative management and invasive surgery, face limitations such as variable efficacy, high trauma, and inapplicability to high-risk patients. An innovative bronchoscopy-guided petrolatum gauze occlusion protocol provides a minimally invasive, cost-effective solution for patients with small to medium-sized bronchopleural fistulas (≤5 mm) who are not candidates for surgery. Key steps included precise fistula localization via. methylene blue dilution injection, tailored gauze sizing, and bronchoscopic placement under direct visualization. Stability was ensured through forceps-guided positioning or supplemental metal stents in anatomically challenging cases. Post-procedure monitoring included chest X-ray, CT imaging, and clinical assessments at 1 day, 1 week, and 1 month. In a retrospective study of 19 patients with peripheral BPFs ≤ 5 mm who met predefined inclusion criteria (e.g., failed prior conservative therapy, high surgical risk) and exclusion criteria (e.g., untreated empyema, fistulas involving central airways), petrolatum gauze occlusion achieved 94.7% efficacy (18/19) at one week, with a low infection rate (15.8%). The observed efficacy, while promising, should be interpreted within the context of the study's limitations, including its small sample size (n = 19), single-center retrospective design, and potential for selection bias. Consequently, the generalizability of these findings to broader patient populations or other institutional settings may be limited. This cost-effective, adaptable technique demonstrates high success in sealing small-to-medium fistulas (≤5 mm), offering a safe alternative for high-risk populations, particularly in resource-limited settings. This article describes in detail the indications for this method, preoperative evaluation, intraoperative manipulation, and postoperative testing.
Staged Comprehensive Management for Necrotizing Fasciitis of the Lower Extremity with Sepsis: A Case Report.
Qu X, Liu G, Wei W … +5 more , Zhang Y, Li K, Wang X, Lang X, Jiang K
J Vis Exp · 2026 Jun · PMID 42329972 · Publisher ↗
Necrotizing fasciitis is a severe soft tissue infection characterized by rapid necrosis of the skin, subcutaneous tissue, and fascia. Its onset is sudden, and progress is rapid. Complications such as sepsis, septic shock...
Necrotizing fasciitis is a severe soft tissue infection characterized by rapid necrosis of the skin, subcutaneous tissue, and fascia. Its onset is sudden, and progress is rapid. Complications such as sepsis, septic shock, and multiple organ dysfunction syndrome (MODS) can appear early, leading to high mortality and disability. The early symptoms lack specificity, posing diagnostic challenges and a high risk of misdiagnosis. This case report describes a 37-year-old male patient who presented with progressive redness, swelling, and pain of the left lower limb. Bedside incision and exploration under local anesthesia confirmed acute necrotizing fasciitis. Staged comprehensive management included early surgical debridement, empirical antibiotics, anti‑shock therapy, vacuum sealing drainage, and eventual skin grafting. The patient's limb was salvaged, and at 1-year follow-up, he had returned to normal daily activities. This case may provide limited insights for the clinical management of similar patients, particularly regarding the importance of timely bedside exploration and a multidisciplinary, staged approach.
Green CuO/ZnO Nanoparticles: Reducing AFB1 Translocation and Accumulation in Maize.
Ngwenya SC, Sithole NJ, Mthiyane DMN … +3 more , Mwanza M, Onwudiwe D, Ramachela K
J Vis Exp · 2026 Jun · PMID 42329969 · Publisher ↗
Aflatoxin B1 (AFB1) is a potent carcinogen produced by Aspergillus species that contaminates maize crops, posing severe health risks to humans and animals through dietary exposure. Traditional post-harvest management and...
Aflatoxin B1 (AFB1) is a potent carcinogen produced by Aspergillus species that contaminates maize crops, posing severe health risks to humans and animals through dietary exposure. Traditional post-harvest management and chemical interventions are often costly or ineffective at preventing the translocation of soil-borne toxins into the plant during growth. The primary goal of this protocol was to evaluate green-synthesized copper oxide/zinc oxide (CuO/ZnO) hybrid nanoparticles (NPs) as a sustainable pre-harvest soil amendment. The study aimed to determine if these NPs could effectively inhibit the translocation and accumulation of AFB1 within maize tissues, thereby reducing toxin accumulation in edible grains. Using a randomized complete block design across two distinct field sites, maize was grown in soil inoculated with AFB1 or Aspergillus flavus spores and treated with varying concentrations of NPs (0-125 mg/kg). The protocol employed green synthesis with Pleurotus ostreatus substrate to ensure eco-friendliness. Plant tissues were analyzed at different physiological stages using High-Performance Liquid Chromatography (HPLC) to measure AFB1 levels and calculate human and livestock risk assessments. Treatment with 125 mg/kg of green hybrid NPs significantly (p < 0.05) decreased AFB1 concentrations across all growth stages. At the on-campus site, AFB1 was reduced by 68% at the vegetative stage, 82% at flowering, and 76% at maturity compared to the positive control. Crucially, grain AFB1 levels dropped to 6.91 ppb, significantly lowering the estimated daily intake for humans. However, further research is required to evaluate potential nanoparticle residues and long-term toxicity. This method demonstrates that green CuO/ZnO hybrid NPs act as an effective bioremediation agent, preventing the transport of toxins to edible parts of the plant. This approach offers a scalable, environmentally safe strategy to enhance food security and mitigate AFB1 exposure in aflatoxin-prone regions.
Mechanism and Experimental Validation of Total Flavonoids of Rhizoma Drynariae in Treating Gouty Arthritis.
Wu X, Meng L, Wei S
J Vis Exp · 2026 Jun · PMID 42329966 · Publisher ↗
Gouty arthritis (GA) is an inflammatory joint disease caused by the deposition of monosodium urate (MSU) crystals within the joint space and surrounding tissues. In traditional Chinese medicine, Rhizoma Drynariae (Gusuib...
Gouty arthritis (GA) is an inflammatory joint disease caused by the deposition of monosodium urate (MSU) crystals within the joint space and surrounding tissues. In traditional Chinese medicine, Rhizoma Drynariae (Gusuibu) has long been widely used in the clinical treatment of GA, and flavonoids are considered its key bioactive constituents. This research employed network pharmacology to construct a component-target network of total flavonoids of Rhizoma Drynariae (TFRD) against GA, thereby identifying key components, core targets, and related pathways. Rat models were established by intra-articular injection of a monosodium urate crystal suspension and treated with TFRD or the positive control, colchicine, by oral gavage. After sample collection, network pharmacology-based prediction results were subsequently validated using rat serum metabolomics, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis. Network pharmacology analysis indicated that the anti-GA effects of TFRD are mediated through key targets, including IL6, AKT1, TNF, EGFR, JUN, and PTGS2, and are mainly associated with inflammation, immune, and apoptosis-related pathways, such as the IL-17, TNF, NF-κB, MAPK, PI3K-AKT, JAK-STAT, and T-cell receptor signaling pathways. Similarly, metabolomics also uncovered the pivotal roles of the inflammatory response. Hematoxylin and eosin (H&E) staining confirmed that TFRD reduced infiltration of inflammatory cells. ELISA assay confirmed that the TFRD group significantly inhibited the expression of inflammatory factors TNF-α, IL-6, and IL-17A in synovial tissue. Western blot analysis revealed that TFRD inhibited the GA-induced hyperphosphorylation of AKT, MAPK p38, and NF-κB p65 in rat synovial tissue. TFRD can effectively ameliorate the inflammation-triggered changes in the GA rats by directly modulating related inflammatory factors and pathways.
Power Distribution Cable Defect Localization Technology Based on the Maximum Entropy Spectral Method.
Chen G, Zhu M, Lu Z … +3 more , Wen F, Li C, Zhang X
J Vis Exp · 2026 Jun · PMID 42329960 · Publisher ↗
As cable laying distances in modern urban distribution networks continue to expand, the accuracy of fault location via frequency-domain reflectometry (FDR) diminishes markedly with increasing length. To improve precision...
As cable laying distances in modern urban distribution networks continue to expand, the accuracy of fault location via frequency-domain reflectometry (FDR) diminishes markedly with increasing length. To improve precision for long cables, this study proposes a maximum-entropy spectral-estimation-based method. A high-frequency distributed parameter model for distribution cables is developed to quantitatively assess the impacts of insulation aging and mechanical damage on per-unit-length capacitance and inductance. Drawing on transmission line theory, the relationship between the reflection coefficient spectrum and the defect position is derived, along with a step-frequency optimization criterion to enhance spectral resolution. To mitigate spectral leakage and limited resolution in fast Fourier transform (FFT)-based frequency-domain analysis, the maximum entropy spectral method is employed for high-fidelity spectrum estimation, thereby elevating fault distance localization accuracy. Simulations reveal that, relative to conventional FFT, the proposed method reduces location errors by 2-4.5 times in typical long-cable cases. Experimental results confirm a relative error below 0.25% for the maximum entropy approach, surpassing the conventional method's error under 0.55%, thus validating its efficacy and superiority in engineering practice.
A Head-Mounted Optically Transparent Skull (HOTS) Window For Deep Transcranial Imaging of the Mouse Cortex.
Liu L, Gao Y, He Y … +8 more , Liao J, Ye S, Xiao N, Cao X, Wu T, Yu J, Zheng W, Li H
J Vis Exp · 2026 Jun · PMID 42329900 · Publisher ↗
High-resolution two-photon imaging of the adult mouse cerebral cortex is severely limited by light scattering from the skull, which attenuates signals and restricts imaging depth in vivo. Although skull-clearing methods...
High-resolution two-photon imaging of the adult mouse cerebral cortex is severely limited by light scattering from the skull, which attenuates signals and restricts imaging depth in vivo. Although skull-clearing methods have been developed to provide optical access to the cortex through the intact skull, their practical performance is constrained by limited clearing time and suboptimal clearing cocktails. Here, we present a detailed protocol for implementing a head-mounted optically transparent skull (HOTS) window. In this approach, a head-mounted cap was used to maintain clearing solutions over the skull, thereby avoiding prolonged anesthesia or physical restraint and enabling extended skull clearing (several hours) in awake, freely behaving mice. Additionally, a two-step clearing procedure was performed using reagents (HOTS-S1: 10% wt/v EDTA, 15% wt/v D-mannose, 10% wt/v sulfolane, 0.5% wt/v Tween 20; HOTS-S2: 70% wt/v D-mannose, 5% wt/v sulfolane, 0.5% wt/v Tween 20) optimized through systematic chemical screening. We provide a step-by-step protocol that includes skull exposure and stabilization, creation and mounting of the head-mounted cap, delivery and refreshment of clearing reagents, and subsequent imaging preparation. In 6-week-old mice (~20 g), the HOTS protocol routinely produces a highly transparent skull that supports two-photon imaging of cortical structures to depths of up to ~800 µm below the pia, approaching the performance of open-skull windows. The HOTS window enables structural imaging in Thy1-GFP-M mice and functional calcium imaging in Thy1-GCaMP6s mice. We believe that, as a convenient and minimally invasive approach, the HOTS window will significantly facilitate deep transcranial imaging and optogenetic, photopharmacological, and other light-based manipulations in vivo.
Effect of CUB Domain-containing Protein 1 On Proliferation and Epithelial-Mesenchymal Transition In Nasopharyngeal Carcinoma Cells.
Luo H, Li M, Li Y
J Vis Exp · 2026 Jun · PMID 42329893 · Publisher ↗
This protocol describes a methodological approach to investigate the expression and functional role of CUB domain-containing protein 1 in nasopharyngeal carcinoma and its potential involvement in epithelial-mesenchymal t...
This protocol describes a methodological approach to investigate the expression and functional role of CUB domain-containing protein 1 in nasopharyngeal carcinoma and its potential involvement in epithelial-mesenchymal transition. Clinical tissue samples from patients with nasopharyngeal carcinoma and rhinitis were collected to analyze CDCP1 expression using real-time quantitative PCR and immunohistochemistry. In vitro experiments were performed using CNE2 and HK1 nasopharyngeal carcinoma cell lines. CDCP1 overexpression and knockdown were achieved by transfection with a CDCP1 plasmid or specific siRNA. Cell proliferation was assessed by MTT assay, apoptosis was evaluated by Caspase-3 activity measurement, and the expression of EMT-related markers and phosphorylation levels of ERK1/2 were detected by western blot and quantitative PCR. To validate pathway involvement, rescue experiments were conducted using the ERK1/2-specific inhibitor U0126. This protocol provides a systematic in vitro and ex vivo. framework for elucidating the molecular mechanisms by which CDCP1 may regulate tumor progression in nasopharyngeal carcinoma.
Fabricating Tunable Polyacrylamide Hydrogels To Study Matrix Stiffness Effects On Stem Leydig Cells.
Li M, Chen Y, Li X … +2 more , Huang J, Wang J
J Vis Exp · 2026 Jun · PMID 42329892 · Publisher ↗
Extracellular matrix (ECM) stiffness critically regulates stem cell behavior. Previously, we demonstrated that pathological increases in matrix stiffness during aging disrupt stem Leydig cell (SLC) homeostasis, leading t...
Extracellular matrix (ECM) stiffness critically regulates stem cell behavior. Previously, we demonstrated that pathological increases in matrix stiffness during aging disrupt stem Leydig cell (SLC) homeostasis, leading to a decline in testosterone. Building on this discovery, we here present a detailed protocol-originally developed in our laboratory-for fabricating polyacrylamide (PA) hydrogels with tunable stiffness to model the testicular microenvironment in vitro. This method enables reproducible casting of gels across a stiffness range of 1-100 kPa, covering physiological to pathological conditions. Key steps include precise mixing of acrylamide/bis-acrylamide, gel swelling equilibration, surface activation with Sulfo-SANPAH, and collagen coating to support SLC adhesion and culture. We provide optimized formulations for target stiffnesses and troubleshooting guidance for common issues such as incomplete polymerization and poor cell attachment. This system allows systematic investigation of how substrate stiffness modulates SLC proliferation, differentiation, and steroidogenic function under defined 2D conditions. Beyond reproductive biology, it also serves as a valuable platform for mechanobiological studies in other cell types and for screening therapeutics targeting stiffness-related dysfunction.
A Novel k-Nearest Neighbor Method with Distribution Discrepancy and Differential Feature Importance for Rolling Bearing Fault Diagnosis.
Li Z, Zhang N, Wang Z
J Vis Exp · 2026 Jun · PMID 42329886 · Publisher ↗
Rolling bearings are among the most vulnerable components in various types of rotary machinery, and accurate fault detection and localization are essential. When a rolling bearing fails, the signal is non-stationary, and...
Rolling bearings are among the most vulnerable components in various types of rotary machinery, and accurate fault detection and localization are essential. When a rolling bearing fails, the signal is non-stationary, and the energy distribution of the vibration signal varies depending on the fault location. In traditional k-nearest neighbor (KNN) fault diagnosis algorithms, Euclidean distance is primarily used to measure the distance between sample points, which is not effective at capturing similarity across different spatial distributions. Moreover, these algorithms assume equal feature importance, which does not reflect the actual characteristics of fault vibration signals. This study proposes a KNN-based rolling bearing fault diagnosis method that incorporates distribution discrepancy and differential feature importance. First, vibration signals are decomposed using three-level wavelet packet decomposition, and the energy of each node at the third level is used as the fault feature. Then, the mean impact value (MIV) algorithm is used to determine the relative importance of each feature, and the Earth mover's distance (EMD) is applied to measure differences between spatial distributions. By integrating Euclidean distance with MIV and EMD and applying the KNN majority voting rule, fault diagnosis is performed. The experimental results indicate that this method achieves a diagnostic accuracy of 99.43%, representing a 5.97% improvement compared to traditional KNN methods. The proposed method demonstrates accurate and effective fault diagnosis performance on the rolling bearing datasets used in this study.
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