Methods and Protocols最新文献

The prevalence of low-birth-weight infants has increased over the past 40 years to approximately 9-10% of Japanese live births. This study aims to identify healthy lifestyle behaviors and psychosocial factors contributing to appropriate perinatal outcomes, gestational weight gain, and postpartum weight change. The Japan Pregnancy, Eating, Activity, and Cohort study was initiated in 2020 in Tokyo, Yamagata/Miyagi, Osaka, and Fukuoka. Participants will be enrolled at approximately 12 weeks of gestation, with follow-up at 18-27 and 35-41 weeks of gestation and 1, 6, and 12 months postpartum. Approximately 3000 participants are targeted: Yamagata/Miyagi (n = 300), Tokyo (n = 1500), Osaka (n = 800), and Fukuoka (n = 400). Participants will complete questionnaires on healthy lifestyle behaviors (dietary intake, physical activity, and circadian rhythm), psychosocial factors, weight control, and behavioral intentions. Medical records will be reviewed for antenatal checkup data. The primary outcomes will include gestational weight gain, infant birth weight, perinatal complications, breastfeeding, and postpartum weight change. Demographic and psychosocial factors and lifestyle behaviors will be examined as covariates and potential confounders. Biological samples will be collected in Tokyo and Yamagata. The study's findings will inform efforts to improve perinatal care guidelines through evidence-based recommendations.

Abnormal adenylosuccinate lyase (ADSL) activity is associated with cancer and neurodevelopmental processes. However, a cell-permeable ADSL inhibitor is not yet available. Our high-throughput screen identified NF-449 as a potential lead compound. To improve cell permeability of the lead compound, fragments of NF-449 were synthesized. This fragment, 2,2'-(1,3-phenylenebis(carbonylimino))-bisbenzenesulfonate, competitively inhibits purified human ADSL with an inhibitory constant of 0.4 micromolar. Its triethylammonium salt inhibited ADSL in HeLa cells with an IC₅₀ of 0.4 micromolar. While this compound might not be ready for in vivo applications yet, further improvement in its permeability might produce useful reagents for in vivo studies of ADSL.

High variability in stem cell research is a well-known limiting phenomenon, with technical variation across experiments and laboratories often surpassing variation caused by genotypic effects of induced pluripotent stem cell (iPSC) lines. Evaluation of kidney organoid protocols and culture conditions across laboratories remains scarce in the literature. We used the original air-medium interface protocol to evaluate kidney organoid success rate and reproducibility with several human iPSC lines, including a novel patient-derived GRACILE syndrome iPSC line. Organoid morphology was assessed with light microscopy and immunofluorescence-stained maturing glomerular and tubular structures. The protocol was further adapted to four microplate-based high-throughput approaches utilizing spheroid culture steps. Quantitative high-content screening analysis of the nephrin-positive podocytes and ECAD-positive tubular cells revealed that the choice of approach and culture conditions were significantly associated with structure development. The culture approach, iPSC line, experimental replication, and initial cell number explained 35-77% of the variability in the logit-transformed proportion of nephrin and ECAD-positive area, when fitted into multiple linear models. Our study highlights the benefits of high-throughput culture and multivariate techniques to better distinguish sources of technical and biological variation in morphological analysis of organoids. Our microplate-based high-throughput approach is easily adaptable for other laboratories to combat organoid size variability.

Introducing fast, reliable, and low-input technologies that utilize wholemeal wheat is essential for efficiently screening gluten quality in wheat breeding lines. Although the GlutoPeak Tester (GPT) has been widely studied for gluten assessment, its application in breeding programs remains underexplored. This study presents a comprehensive approach to optimizing a GPT protocol using a diverse set of genotypes collected over seven harvest years and multiple environments. To improve screening capabilities, a quick and simple protein fractionation (PF) technique was integrated into the workflow. Key GPT parameters-such as peak maximum time, maximum torque, and aggregation energy-along with the newly proposed PM-AM parameter, showed strong correlations with established quality traits. PF data, especially insoluble glutenin percentage and the ratio of insoluble to soluble glutenin, provided additional insights into gluten composition. This extensive dataset supports the use of GPT and PF as a dual, high-throughput screening tool. When applied within specific wheat classes and benchmarked against established checks, this method offers a robust strategy for ranking breeding lines based on gluten performance. The use of wholemeal samples further streamlines the process by eliminating the need for milling, making this protocol particularly suitable for early-stage selection in wheat breeding programs.

Accurate quantification of immune cell subpopulations is essential for understanding immune responses in research and clinical settings. Flow cytometry (FC) is widely used for immune cell phenotyping, providing rapid and quantitative single-cell resolution. However, tissue-based pathological assessment offers additional spatial and morphological context that is often necessary for a comprehensive understanding of immune cell distribution. Traditionally, these methods are applied separately to different specimens, limiting direct comparative analysis. Here, we describe a simple combined approach to immune cell quantification that integrates both FC and pathology analysis within the same tissue specimen of colon biopsies. Tissue samples were divided into two portions: one processed into a single-cell suspension for FC-based characterization of CD45⁺, CD3⁺, CD4⁺, and CD8⁺ T cells and another formalin-fixed, paraffin-embedded (FFPE), and analyzed with hematoxylin and eosin (H&E) for eosinophils and immunohistochemistry (IHC) for CD4 and CD8. A pilot analysis of 10 samples shows high concordance of the results taken from the two methods, allowing for cross-validation between methodologies and improved diagnostic accuracy. This proof-of-principle study demonstrates the feasibility of an integrated workflow that combines FC and pathology for immune cell quantification, which provides assessment of immune cell populations from the limited material of intestinal biopsies with potential for improved diagnostic accuracy.

Background: Extensive skin injuries from severe burns or chronic non-healing ulcers overwhelm the body's natural repair mechanisms, while current therapeutic approaches relying on autologous skin grafting are limited by donor site availability. Three-dimensional epithelial spheroid cultures enhance stem cell regenerative potential, but standardized comparative methodologies are lacking. Methods: We established a comprehensive framework comparing scaffold-free and scaffold-based epithelial spheroid systems using HaCaT keratinocytes. High-throughput approaches utilized BioFloat and ELPLASIA 96-well platforms, while low-throughput 6-well ULA plates generated heterogeneous populations (holospheres, merospheres, paraspheres). Scaffold-based studies embedded spheroids in Matrigel to evaluate outgrowth capacity. ROCK1 inhibitor treatment was assessed for stemness enhancement. Results: High-throughput systems generated uniform spheroids with high reproducibility and consistent circularity. Low-throughput cultures produced heterogeneous populations with distinct size distributions (holospheres: 408.7 μm², merospheres: 99 μm², paraspheres: 14.1 μm²). In Matrigel scaffolds, merospheres and paraspheres migrated outward, forming epithelial sheets, while holospheres remained intact as BMI-1+ stem cell reservoirs. ROCK1 inhibition enhanced holosphere formation, preserved stemness markers, and reduced premature differentiation. Conclusions: This standardized toolbox demonstrates scaffold-free systems optimize scalability for screening while scaffold-based approaches enable physiologically relevant regenerative studies. Integration of both methodologies provides flexibility matching experimental design to scientific objectives, accelerating translation to clinical applications.

Patient-derived organoids (PDOs) have emerged as powerful tools in personalized medicine applicable to both non-malignant conditions and to cancer, where they are increasingly used for personalized drug screening and precision treatment strategies in part due to their ability to replicate tumor heterogeneity. They also serve as study model systems to understand disease mechanisms, pathways, and the impact of ex vivo exposures. We present a detailed step-by-step protocol for generating organoids from normal crypts, polyps, and tumors, including methods for tissue processing, crypt isolation, culture establishment, and the transition from basolateral to apical-out polarity for co-culture and exposure-based studies. The protocol also includes immunofluorescence staining procedures for cellular characterization and quality control measures. Our standardized approach successfully generates organoids from diverse colorectal tissues with high efficiency and reproducibility. This comprehensive guide addresses common technical challenges and provides troubleshooting strategies to improve success rates across different sample types. We believe that this resource will enhance reproducibility in organoid research and expand their utility in translational applications, particularly for personalized medicine approaches in colorectal cancer.

This study aimed to evaluate the placement accuracy and reproducibility of Surface Guided Radiotherapy (SGRT) compared with the conventional tattoo/laser method in patients undergoing radiotherapy for abdominal malignancies. A retrospective analysis was conducted on 43 patients treated with either SGRT (Group A) or the tattoo/laser technique (Group B). Patients in both groups underwent CBCT to quantify the positioning shifts in the vertical (Svrt), lateral (Slat) and longitudinal (Slng) axes, as well as the total shift. Statistical indicators including median, interquartile range (IQR), and range were calculated, and Mann-Whitney U tests were performed due to non-normal data distribution. Median values in all axes were same between groups: Svrt = 0.4 cm, Slat = 0.2 cm, Slng = 0.4 cm. Group A showed a higher total median shift equal to 0.8 cm versus 0.7 cm of Group B. However, IQRs were smaller in the Group B for all directions and total shift, indicating greater method consistency. Statistically significant differences (p < 0.05) were observed in all axes, except the vertical. These findings suggest that, while SGRT achieves comparable median alignment, its use in a highly variable anatomical region such as the abdomen may be associated with greater setup variability.

Maintaining the integrity of cryogenically preserved biological materials is critical, as even brief, undetected temperature excursions in storage can compromise sample viability. Existing monitoring systems may miss transient thaw-refreeze events, posing serious quality risks. To address this, we developed and validated frozen indicator tubes that visually signal deviations from the frozen state, serving as a cost-effective backup to electronic monitors. Our first method uses an aqueous dye solution that immobilizes the dye when frozen; any thawing causes the dye to disperse, providing a clear, external visual cue of a partial or complete thaw. For ultra-low-temperature storage (-80 °C), we introduced a second method using an ethanol-based solution calibrated to indicate thaw events. This system detects temperature rises of 10 °C or more sustained for at least fifteen minutes-conditions that may jeopardize sample stability. When paired with standard monitoring systems, these indicator tubes offer an added layer of protection by providing simple, reliable, and immediate visual confirmation of critical temperature breaches. This innovation enhances confidence in cryogenic storage protocols and supports the long-term preservation of sensitive biological materials.

The bioavailability of a drug is closely linked to its solubility, making its early determination essential in drug development. The saturation shake-flask (SSF) method is the gold standard protocol for this, which includes a phase separation step-either by sedimentation, filtration, or centrifugation. This step is critical, as it can directly influence the accuracy of the results. This study investigated the impact of centrifugation parameters-time and rotation speed-on solubility measurements. Additionally, we compared two sample preparation protocols: continuous stirring for 24 h versus 6 h of stirring followed by 18 h of sedimentation before centrifugation. Four model compounds were tested at three pH values using Britton-Robinson buffers. Centrifugation was conducted for 5, 10, or 20 min at either 5000 or 10,000 rpm. Results showed that pre-sedimented samples yielded solubility values closer to sedimentation-only references, while continuous stirring often led to overestimated values, particularly at higher speeds and longer durations. One such example was papaverine hydrochloride, that showed solubility values 60-70% higher than the reference after centrifugation at 10,000 rpm for 20 min without prior sedimentation. Lower standard deviations were observed with shorter, slower centrifugation, with 5 min and 5000 rpm yielding results closest to the reference values.