Methods and Protocols最新文献

Three-dimensional (3D) culture systems that recapitulate the tumor microenvironment are essential for studying cancer cell behavior, drug response, and cell-matrix interactions. Here, we present a detailed protocol for generating 3D spheroid cultures from murine breast cancer cells using methacrylated gelatin (GelMA)-based bioink and a CELLINK BioX bioprinter. This method enables precise deposition of spheroid-laden GelMA droplets into low-attachment plates, facilitating high-throughput and reproducible 3D culture formation. The protocol includes steps for spheroid formation, GelMA preparation, bioprinting, and post-printing analysis using a customized CellProfiler pipeline. The analysis pipeline takes advantage of the functionality of CellProfiler and ImageJ software (version 2.14.0) packages to create a versatile and accessible analysis tool. This approach provides a robust and adaptable platform for in vitro cancer research, including studies of metastasis, drug resistance, cancer cell lipid metabolism, and TME-associated hypoxia.

Introduction: Management of trochanteric fractures in revision hip surgery has a high incidence of non-union and complications. Fixation devices are often bulky, prone to breakage, and necessitate reoperation. This study describes a novel soft tissue and bony abductor repair that reduces the forces on bony fragments without the need for prominent metalwork.

Methods: This novel surgical technique involves fixation of the abductor mechanism with polyester and polyethylene sutures that are woven through the abductors and secured to the femoral shaft with a proprietary suture cerclage tape with cerclage wire supplementation in select cases. All patients undergoing fixation were retrospectively reviewed with a minimum follow-up period of 12 months. Outcomes relating to dislocation, reoperation, fracture union and the incidence of symptomatic Trendelenburg gait were recorded.

Results: A total of 17 patients underwent this novel intervention. There were no dislocations or reoperations for prominent metalwork at the last follow-up. One patient had evidence of greater trochanter (GT) non-union, and three had GT displacement of over 3 mm. Eight (47.1%) patients were independently mobile and seven (41.2%) were mobile with only one walking aid. No patients required plate or bolt fixation.

Conclusions: GT fractures and abductor deficiency are difficult to manage, with most reported methods utilising bulky metalwork to treat a soft tissue injury. We describe a novel combined soft tissue and bony fixation without the need for excessive metalwork. Our pilot study demonstrates satisfactory outcomes of this intervention that are technically reproducible and more appropriately addresses the deforming forces involved with a low complication profile.

This article describes a field- and laboratory-based framework that can be used to monitor the C balance in Mediterranean pine forest ecosystems under different management practices that determine their structure and function. By jointly monitoring stand structure, gas exchange, litter, and decomposition dynamics, this protocol enables the assessment of how management-driven changes regulate carbon uptake, turnover, and losses, thereby affecting carbon sequestration potential. As an example, we suggest the implementation of the protocol at ten (10) permanent monitoring plots across three study areas located in Greece. The first group of plots represents a post-fire chronosequence in pine stands with no management interventions. The second group includes pine stands that exhibit variation in overstory and understory density driven by differences in microclimate and management history. The third group consists of peri-urban pine stands subjected to thinning of varying intensity. The monitoring protocol is implemented across all plots and the collected data can be classified into three analytical domains: (a) demography, encompassing measurements of tree growth and mortality; (b) litter and decomposition dynamics, involving the quantification of litterfall and its seasonality and the estimation of its decomposition rates; and (c) gas exchange, focusing on measurements of leaf photosynthesis and respiration (including relevant leaf functional traits) and monitoring of soil respiration. These three data domains can be used to comparatively consider the effect of forest management on key ecosystem processes and to constrain local-scale vegetation dynamics models.

Taraxacum kok-saghyz (Tks) is a promising plant species for natural rubber (NR) production and represents a model for studying NR biosynthesis in the Asteraceae family. The generation of transgenic plants overexpressing a gene of interest is a well-established strategy to investigate gene function and potential interactions. Here, we present a comprehensive workflow-from the construction of an overexpression vector to the generation, identification, and propagation of stable transgenic Tks lines. In addition, we describe a rapid and reliable method for quantifying NR content in transformed plants, providing essential phenotypic characterisation in this species.

This study presents a standardized method for multimodal monitoring of exercise execution in isolated, confined, and extreme (ICE) environments, addressing the need for reproducible assessment of neuromuscular and cardiovascular responses under space- and equipment-limited conditions. The method integrates wearable surface electromyography (sEMG), inertial measurement units (IMU), and electrocardiography (ECG) to capture muscle activation, movement, and cardiac dynamics during space-efficient exercise. Ten exercises suitable for confined habitats were implemented during analog missions conducted in the DeepLabH03 facility, with feasibility evaluated in a seven-day campaign involving three adult participants. Signals were synchronized using video-verified repetition boundaries, sEMG was normalized to maximum voluntary contraction, and sEMG amplitude- and frequency-domain features were extracted alongside heart rate variability indices. The protocol enabled stable real-time data acquisition, reliable repetition-level segmentation, and consistent detection of muscle-specific activation patterns across exercises. While amplitude-based sEMG indices showed no uniform main effect of exercise, robust exercise-by-muscle interactions were observed, and sEMG mean frequency demonstrated sensitivity to differences in movement strategy. Cardiac measures showed limited condition-specific modulation, consistent with short exercise bouts and small sample size. As a proof-of-concept feasibility study, the proposed protocol provides a practical and reproducible framework for multimodal physiological monitoring of exercise in ICE analogs and other constrained environments, supporting future studies on exercise quality, training load, and adaptive feedback systems. The protocol is designed to support near-real-time monitoring and forms a technical basis for future exercise-quality feedback in confined habitats.

Reversed-phase high-performance liquid chromatography (RP-HPLC) remains one of the most widely applied analytical techniques in the development and quality control testing of finished pharmaceutical products. The combination of gradient chromatographic methods with diode-array detection (DAD) enhances selectivity, ensuring accuracy and reliability when testing drugs with diverse chemical properties in a single dosage form (i.e., fixed-dose combination (FDC) products). In this study, an RP-DAD-HPLC method was developed for the quantitative analysis of clofazimine (CFZ) and pyrazinamide (PZA) for inclusion in an FDC topical drug delivery system. Chromatographic separation was achieved using a C18 column (4.6 mm × 150 mm, 5 µm particle size) with gradient elution at 1 mL/min, employing 0.1% aqueous formic acid and acetonitrile (mobile phases). PZA and CFZ were detected at 254 nm and 284 nm, respectively. The method was validated in accordance with ICH Q2 guidelines, assessing specificity (considering interference from solvents, product matrix, and degradation products), linearity (7.8-500.0 µg/mL, r² = 0.9999), system repeatability (%RSD ≤ 2.7%), and intermediate precision (25-500 µg/mL, %RSD ≤ 0.85%). Method robustness was evaluated using a three-level Box-Behnken design (BBD) with response surface methodology (RSM) to assess the effects of variations in detection wavelength, mobile phase flow rate, and column temperature.

Background: Gastrointestinal discomfort affects up to 70% of individuals with spinal cord injury (SCI), largely due to gut dysbiosis caused by altered transit time and reduced gastrointestinal motility from autonomic disruption. Emerging evidence links prebiotics and probiotics to improved microbiome balance and reduced inflammation, yet data in SCI remain limited.

Methods: Individuals aged ≥ 18 years, with a chronic SCI (≥1 year) experiencing significant gastrointestinal symptoms, will be invited to participate in this single-center randomized controlled crossover trial. Persons currently taking antibiotics, who have relevant eating or digestive disorders, or who have undergone a recent diet change will be excluded from the study. Participants will be randomized (1:1) into two groups. The first group will take a probiotic (Biotics-G, Burgerstein AG, Rapperswil-Jona, Switzerland) supplement for eight weeks, then after a four-week washout period, they will take a prebiotic (Oat Bran, Naturaplan, manufactured by Swissmill, Zurich, Switzerland) supplement for another eight weeks. The second group will receive the supplements in reverse order. The primary outcome is the Gastrointestinal Quality of Life Index, a questionnaire to assess quality of life related to gastrointestinal disorders. Secondary outcomes consist of gastrointestinal transit time, inflammatory blood markers, and gut microbiome composition.

Ethics: The study will be conducted in accordance with the Declaration of Helsinki. The study was approved by the Ethics Committee for Northwest/Central Switzerland (EKNZ, ID: 2025-00238, 24.02.2025, Version 2.0). The study is registered at ClinicalTrials.gov (ID: NCT06870331, 02.04.2025). Written informed consent will be obtained from all participants involved in the study.

Although sample matrices are available for assessing cortisol output over hours/days (serum, saliva, or urine) or months (hair or nails), there is no current method for measuring integrated cortisol output over a period of 1 week. Therefore, the primary aim of this study was to develop and validate a method for collecting and measuring sweat-derived cortisol from commercially available skin patches worn for 1 week. Additional aims were to determine whether the accumulated sweat cortisol correlated with salivary cortisol measured during the same week, and whether sweat cortisol was related to psychological stress measured using two different questionnaires. After conducting preliminary in vitro validation studies, we obtained the following data from a convenience sample of university students and employees: (a) cortisol and sodium contents of patches worn for 1 week (sodium was used to correct for variation in sweating rate), (b) mean area-under-the-curve of salivary cortisol concentrations measured for 3 days during the week of patch wearing, and (c) two different measures of psychological stress. The results demonstrate that a continuously worn sweat patch can be used to collect and measure sweat cortisol over a 1-week period. However, the patch's cortisol contents did not correlate with either the salivary cortisol area under the curve or the participants' psychological stress. Because previous findings showed that sweat cortisol is significantly related to both circulating and salivary cortisol levels, we hypothesize that the lack of an observed correlation between patch and salivary cortisol may have resulted from limitations of our experimental design.

Workers' exposure to silica dust is a global occupational and public health concern and is particularly prevalent in Southern Africa, mainly because of inadequate dust control measures. It is worsened by the high prevalence of HIV/AIDS, which exacerbates tuberculosis and other occupational lung diseases. The prevalence of silicosis in the region ranges from 9 to 51%; however, silica dust exposure levels and controls, especially in the informal mining sector, particularly in artisanal small-scale mines (ASMs), leave much to be desired. This is important because silicosis is incurable and can only be eliminated by preventing worker exposure. Additionally, several studies have indicated inadequate occupational health and safety policies, weak inspection systems, inadequate monitoring and control technologies, and inadequate occupational health and hygiene skills. Furthermore, there is a near-absence of silica dust analysis laboratories in southern Africa, except in South Africa. This protocol aims to systematically evaluate the effectiveness of respirable dust and respirable crystalline silica dust exposure evaluation and control methodology for the mining industry. The study will entail testing the effectiveness of current dust control measures for controlling microscale particles using various exposure dose metrics, such as mass, number, and lung surface area concentrations. This will be achieved using a portable Fourier transform infrared spectroscope (FTIR) (Nanozen Industries Inc., Burnaby, BC, Canada), the Nanozen DustCount, which measures both the mass and particle size distribution. The surface area concentration will be analysed by inputting the particle size distribution (PSD) results into the Multiple-Path Particle Dosimetry Model (MPPD) to estimate the retained and cleared doses. The MPPD will help us understand the sub-micron dust deposition and the reduction rate using the controls. To the best of our knowledge, the proposed approach has never been used elsewhere or in our settings. The proposed approach will reduce dependence on highly skilled individuals, reduce the turnaround sampling and analysis time, and provide a reference for regional harmonised occupational exposure limit (OEL) guidelines as a guiding document on how to meet occupational health, safety and environment (OHSE) requirements in ASM settings. Therefore, the outcome of this study will influence policy reforms and protect hundreds of thousands of employees currently working without any form of exposure prevention or protection.

Multimodal methods provide valuable opportunities within Participatory Action Research (PAR), to foster meaningful participation, and amplify marginalized voices. However, conventional research approaches have not always adequately captured the complex realities of the lived experiences of families, and multimodal techniques have remained underutilized for the exploration of such experiences. This study aimed to explore the use of creative multimodal methods, within a PAR framework, grounded in care among vulnerable South African families. A qualitative design was adopted, incorporating Human-centered Design principles, within a PAR approach. The participants were recruited from the Saldanha Bay Municipality area (n = 70), as well as Mitchells Plain (n = 59). The multimodal methodology included Draw-and-Tell, painting, object and photo elicitation, I-Poems, and LEGO^®-based activities. Data were annotated and transcribed verbatim, followed by thematic analysis. A total of 42 participants contributed towards the validation of the methods. The participants described experiences of deep emotional insight, self-reflection, and self-recognition, through engagement with the multimodal activities. The findings revealed that these approaches were: (1) credible, producing internally valid and contextually rich data; (2) contributory, generating original and applicable insights into family life; (3) communicable, offering accessible and structured ways for diverse participants to express their experiences; and (4) conforming, ensuring ethical engagement through inclusive participation. These findings demonstrate the potential of creative, arts-based, and participatory approaches, to advance methodological innovation in qualitative family research.