Methods and Protocols最新文献

Background: Shoulder soft tissue disorders, such as rotator cuff tears and subacromial impingement, are among the most common causes of musculoskeletal disability. Both physical examination tests and imaging techniques are routinely used in clinical settings; however, their respective contributions to patient outcomes and their potential complementarity remain underexplored.

Methods: A systematic review and meta-analysis were conducted following PRISMA 2020 guidelines. Controlled clinical studies comparing pre- and post-intervention outcomes in adults with suspected or confirmed shoulder soft tissue pathology were included. Two groups were analyzed: studies using musculoskeletal imaging (ultrasound or MRI) and studies applying orthopedic physical examination tests (e.g., Neer, Hawkins, and Jobe). Functional outcomes were converted into standardized mean differences (SMDs) and synthesized using a random-effects model. Heterogeneity was quantified using the I² statistic.

Results: In total, 11 studies met the inclusion criteria (n = 6 imaging, n = 5 orthopedic tests). Imaging-based studies showed a pooled SMD of 4.85 (95% CI: 2.77-6.93), indicating substantial clinical improvement. Orthopedic test-based studies yielded a pooled SMD of 2.34 (95% CI: 1.27-3.41). Heterogeneity was high across both groups (I² > 90%).

Conclusions: Imaging was associated with a larger overall clinical effect, while orthopedic tests provided functional insight valuable for screening and monitoring. These findings support the complementary use of both strategies to enhance diagnostic accuracy and treatment planning in shoulder care.

Background: Developing clinically relevant experimental models of the human airway can significantly advance our understanding of the mechanisms underlying airway diseases and aid in translating potential therapies to clinical settings. The aim of this study is to establish an ex vivo human airway tissue culture model.

Methods: Human donor airway tissues were obtained from clinical cases of lung transplantation. Our established method is based on the concept of scavenging metabolic activity and controlling bacterial growth and includes increased media volume, frequent media exchange, and antifungal additives to efficiently maintain the homeostatic culture environment. After a 3-day culture period, the airway was investigated, and its viability and function were compared with a standard cell culture method.

Results: Control tissue exhibited significant acidosis after 3 days, suggesting high metabolic activity of airway tissue and bacterial contamination. The airway epithelial viability-after culturing in our established method for 3 days-was better than that of the controls. We only performed an acute but early investigation of the cultures as airway complications have been known to start early at the proximal bronchus after transplantation. H&E and alcian blue staining showed intact morphology of the epithelium of airway tissue and mucus layers after 3 days in our model, while controls showed remarkable damage to the epithelial layer. Newly synthesized glycoproteins were detected in the epithelial layer using metabolic labeling and the click chemistry technique, suggesting cellular protein synthesis of the airway tissue in our established ex vivo model.

Conclusions: We successfully established a reproducible model of human ex vivo airway tissue culture (n = 3 independent biological samples) that may be useful for investigating airway complications and developing their therapies.

In the nuclear industry, the decontamination of nuclear metallic structures is an essential process to reduce radiation exposure during maintenance or dismantling. The oxide layer, such as chromium (III) oxide (Cr₂O₃), formed on stainless steel and nickel-based alloys, contributes significantly to surface radioactivity by trapping radioactive contaminants. To address this, permanganic acid (HMnO₄) has proven to be a promising oxidizing agent for dissolving these oxide layers-particularly chromium oxide-on stainless steel and nickel-based alloys. In this study, HMnO₄ was synthesized via ion exchange using AmberLite IRN97 H resin and potassium permanganate (KMnO₄). The optimized process yielded a highly acidic solution (pH~1.6) with potassium concentrations below 0.1 ppm, indicating near-complete exchange efficiency. Dissolution kinetics were investigated at HMnO₄ concentrations ranging from 240 to 1920 ppm and temperatures from 30 °C to 80 °C. At a constant temperature, increasing HMnO₄ concentration significantly improved Cr dissolution, with up to 31% of total chromium solubilized after 33 h. Lower temperatures favored higher dissolution efficiency, likely due to improved thermal stability of HMnO₄. For durations shorter than 4 h, the influence of temperature was limited compared to the effect of acid concentration. To assess post-treatment options, HMnO₄ decomposition was studied using oxalic acid (H₂C₂O₄) at 80 °C. Results showed that a minimum H₂C₂O₄/HMnO₄ molar ratio above 2.75 was necessary to achieve effective reduction while preventing MnO₂ precipitation. However, even under strongly acidic conditions and with a large excess of reductant, Mn²⁺ yields remained below 55%, suggesting that thermal degradation of oxalic acid and possible formation of undetected manganese species limited the reduction process.

Cancer development in BRCA1 carriers is a multi-step process, which is triggered by several factors and mechanisms that are not clearly understood. Most BRCA1 carriers develop triple-negative breast cancer (TNBC)-estrogen receptor (ER)-, progesterone receptor (PR)-, and HER2 -negative cancers-which originates from ER/PR/HER2-negative breast progenitor cells. Due to a lack of ER/PR/HER2-negative cell models with BRCA mutations, the processes inducing cancer development in BRCA carriers have not been comprehensively studied. Thus, studies characterizing ER/PR/HER2-negative cells carrying a BRCA1 germline mutation are needed to gain more in-depth knowledge about the steps leading to cancer initiation in BRCA1 carriers. To study the cancer development in these patients, we established a protocol for the generation of human ER/PR/HER2-negative breast organoids carrying a BRCA1 germline mutation. We confirmed that these organoids are unresponsive to estrogen, can self-renew, and express the stem/progenitor marker CD44. In addition, we observed that these organoids contain outgrowths that resemble the mature ductal and lobular units of the mammary gland, thus making it a suitable model system to study how cancer develops in ER/PR/HER2-negative mammary cells that carry a BRCA1 germline mutation.

This study discusses the challenges encountered in implementing a detailed protocol for upper abdominal imaging using magnetic resonance imaging (MRI), ranging from patient preparation and sequence selection to clinical applications. MRI is a valuable non-invasive imaging modality employed both in the early detection of diseases and as a complementary tool for the detailed characterization of various pathologies. Nevertheless, performing an abdominal MRI examination can be challenging; therefore, the understanding of sequences is particularly important, as changing the parameters can not only influence the quality of the images but also optimize scanning time improve patient experience during the examination. The methodology illustrates the purpose of each sequence and the critical role of appropriate patient preparation. Results highlighted the significance of these factors in the evaluation of hepatic lesions, showing that the proper choice of sequences and parameters is essential for distinguishing benign from malignant findings and for achieving an accurate diagnosis. It was also shown that MRI plays an important role as a complementary technique in investigation of upper abdominal pathologies in order to avoid overexposure to radiation.

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.