Methods and Protocols最新文献

In computed tomography (CT)-based evaluation of the extent of cystic changes in the lungs of patients with cystic lung diseases, such as Lymphangioleiomyomatosis (LAM), there is a lack of a lung phantom containing air-filled cavities that mimic pulmonary cysts to calibrate the measurement of cystic volumes from CT scans. We describe an easy-to-replicate cystic lung phantom consisting of basic lung structures of a trachea and two lung compartments. The lung compartments contain air cavities of varying sizes to mimic cystic lesions. The lung compartments are made of a foam material recommended by NIST to simulate the radiodensity of human lung parenchyma. In tests performed on a clinical scanner, various structures in the lung phantom were correctly recognized by two types of lung analysis software. The resulting cystic volume measurements revealed the relationship between the size of the cysts and the accuracy of the measurement. The significant finding was that the volumes of individual cysts were underestimated for small cysts. The error increased with decreasing cyst sizes. Such underestimation has not been mentioned previously and deserves the attention of clinicians using CT scans to assess the cyst burden in the lungs, particularly in patients presenting with numerous small pulmonary cysts.

Background: Pressure injuries (PIs) and shoulder pain (SP) are frequent problems in individuals with spinal cord injury (SCI), affecting both quality of life and healthcare use. Although pressure relief (PR) is recommended to prevent PIs, it is often not performed regularly, and its long-term benefits remain unclear. Furthermore, some PR methods may contribute to SP, resulting in conflicting clinical guidelines. This study aims to objectively measure PR performance and investigate its long-term relationship with PI and SP.

Methods: This study is a longitudinal observational study involving 70 manual wheelchair users with complete SCI. Over one year, participants attend five study visits to assess confounding factors such as comorbidities and shoulder range of motion. PR performance (technique, frequency, duration) is continuously monitored for three weeks after each of the first four visits using textile measurement mats, while SP is assessed weekly with a questionnaire. Causal associations with PI and SP will be examined using directed acyclic graphs and multivariable regression modelling.

Results: The study is ongoing. Long-term objective data on PR performance will provide insights into its relationship with PI and SP.

Conclusions: Findings will inform clinical practice and contribute to improved evidence-based PR guidelines for individuals with SCI.

Epigenetic research has made notable progress in recent years, yet our ability to explore the human brain at a cellular level remains limited. One of the main obstacles has been the difficulty of isolating specific neuronal populations from postmortem tissue-particularly interneurons, which play a central role in many psychiatric disorders. In this study, we present a practical and reproducible protocol for isolating GAD-positive interneurons from human brain samples. We isolate permeabilized cell-like structures suitable for downstream epigenetic analysis. To ensure specificity, we validated the isolated cells by comparing them with interneurons derived from human iPSCs. This approach allows for high-quality DNA extraction suitable for downstream epigenetic analysis, including methylation-specific PCR. By targeting a well-defined neuronal subtype, our method provides a solid foundation for studying the molecular changes associated with disorders such as schizophrenia and autism. This protocol opens new doors for cell-specific investigations in brain tissue, a step forward in understanding how epigenetic mechanisms contribute to neuropsychiatric pathophysiology.

The implementation of theranostics in oncologic nuclear medicine has exhibited immense potential in improving patient outcomes in prostate cancer with the implementation of [⁶⁸Ga]Ga-PSMA-11 PET and [¹⁷⁷Lu]Lu-PSMA-617 into clinical practice. However, the correlation between radiopharmaceutical biodistributions seen with [⁶⁸Ga]Ga-PSMA-11 PET imaging and downstream [¹⁷⁷Lu]Lu-PSMA-617 therapy remains imperfect. This suggests that prostate cancer theranostics could potentially be further refined through the implementation of true theranostics, tandem pairs of diagnostic and therapeutic radiopharmaceuticals that utilize the same ligand and element, thus yielding identical pharmacokinetics. The radioscandiums are one such group of true theranostic radiopharmaceuticals. The radioscandiums consist of two β+ emitting scandium isotopes (⁴³Sc/⁴⁴Sc), as well as a β^- emitting therapeutic isotope (⁴⁷Sc), which can all conjugate with PSMA-targeting PSMA-617. This potential has led to extensive investigations into the production of the radioscandiums as well as pre-clinical assessments with several ligands; however, there is a lack of literature extensively describing the complete synthesis of scandium radiopharmaceuticals. which therefore limits the accessibility of radioscandium research in theranostics. As such, this work aims to present an easily translatable protocol for the synthesis of [⁴³Sc]Sc-PSMA-617 from a [⁴²Ca]CaCO₃ starting material, including target formation, nuclear production via ⁴²Ca(d,n)⁴³Sc reaction, chemical separation, radiolabeling, solvent reformulation, and target recycling.

Human respiratory syncytial virus (RSV) causes acute respiratory illness, attributing to deaths among young children and older adults worldwide. RSV neutralization assay is an important tool to measure RSV neutralization antibody that can prevent infection and severe complication of RSV. Conventional RSV neutralization assays have some limitations of speed and cost, especially for expensive kits, reagents or instruments required for detection. To solve this problem, this paper describes an improved simple and economical RSV neutralization assay protocol using recombinant RSV (rRSV) expressing reporter fluorescent protein to measure RSV growth as reporter activity with plate reader. The condition of 3 days culture demonstrated sufficient fluorescent activity even when small amounts of rRSV were used to inoculate Hep-2 cells. In addition, white 96-well cell culture plate showed better stable reporter activities than black plate. Furthermore, RSV neutralization assay protocol using rRSV-reporter fluorescent protein demonstrated similar signal detection capacity for RSV antibody titer detection compared to other protocols, such as rRSV-Luciferase and ELISA assay. The new RSV neutralization assay protocol can be applied to RSV antibody titration of numerous samples necessary for RSV surveillance or antiviral testing.

Single-cell adhesion assays can be divided into studies on attachment and detachment events, and several methods that enable the characterization of both processes have been established in the past. Due to their low invasiveness, label-free principles, and contactless operation, optical methods are especially beneficial for this purpose. Historically, optical tweezers (OTs) have been used to explore single-cell detachment events, allowing for the precise determination of minute physical forces. However, it has been noted that OTs can also be used to study single-cell attachment dynamics, including the evaluation of minimum cell-to-cell contact times necessary to establish a stable adhesive bond. Here, we provide a step-by-step protocol to effectively evaluate minute changes in the adhesion of single leukemia-lymphoma cells using optical tweezers with low laser intensities. This serves as a valuable in vitro model to determine the effects of physical and chemical factors on the adhesive properties of leukemia-lymphoma (LL) cells.

Nile tilapia (Oreochromis niloticus) is the most widely farmed tilapia species globally, making it one of the most important aquaculture species. To meet increasing demand, hatcheries occasionally use artificial breeding techniques such as hormonal induction to synchronize breeding. Despite the common use of human chorionic gonadotropin (hCG) in fish breeding, no detailed protocol has been established specifically for Nile tilapia. The objective of this study is to establish an effective hCG-induced artificial breeding protocol for gene editing and aquaculture production, optimizing fertilization, hatching, and survival rates. We employed a single intramuscular injection of 2 IU/g hCG to induce ovulation. The protocol achieved an average fertilization rate of 88.3% and a larval survival rate of 90.5%, demonstrating its potential for obtaining high-quality embryos for functional studies and enhancing reproductive performance on a commercial scale.

Our objective was to develop a simple, low-cost colorimetric assay to detect kynurenine (L-Kyn) in human biofluids, that would be compatible with a point-of-care (POC) system being developed in our lab. Elevated L-Kyn is associated with many pathological conditions. However, current detection methods are expensive, time-consuming, and unsuitable for resource-limited settings. Existing colorimetric L-Kyn assays lack specificity, require unusual reagents, or lack sensitivity, hindering their practical application. Here we report a two-step diazotization-based colorimetric assay that produces a red chromophore upon reaction with L-Kyn. To reduce background interference, we used dilution and anion exchange chromatography for urine samples and acid precipitation for serum samples. The assay detected 5-300 μM L-Kyn in urine (lower limit of detection (LLOD) 1.34 μM) and 5-125 μM L-Kyn in serum (LLOD 1.24 μM). Correlation studies achieved strong linearity (R² = 0.98 for spiked urine, 0.99 for spiked serum) and were highly correlated (>0.95) to liquid chromatography tandem mass spectrometry (LC-MS/MS) concentrations. Bland-Altman analysis confirmed agreement between L-Kyn assay and LC-MS/MS methods. To our knowledge, this is the first application of a diazotization reaction for L-Kyn quantification at physiologically relevant levels. The assay is now being ported to a low-cost, automated POC biosensor platform.

The development of standardised, reproducible preclinical models is essential for advancing pleural mesothelioma (PM) research. Here, we present a simple and reliable minimally invasive transthoracic intrapleural injection technique that could improve the efficiency of orthotopic PM model generation. By incorporating a simple needle sleeve to control the injection depth, this method eliminates the need for surgery or general anaesthesia, reducing technical complexity and animal stress while ensuring precise delivery into the pleural cavity. We demonstrate the effectiveness of this approach by achieving a 100% tumour engraftment rate following the injection of AE17 tumour cells. Additionally, this technique has been successfully used for asbestos fibre injection in mesothelioma models, highlighting its versatility. By providing a more accessible, standardised alternative to existing methods, this protocol improves the reliability of PM models and facilitates broader adoption by researchers, including those with limited experience in invasive procedures.

Resting metabolic rate (RMR) significantly impacts total daily energy expenditure, particularly on training days, and varies among trained individuals. Studies estimating RMR in this population show notable discrepancies. This study aimed to develop and validate new bioelectrical impedance analysis-based (BIA) RMR equations for young athletes, using a calibration and a validation group of 219 and 51 participants, respectively. RMR was measured via indirect calorimetry, while body composition was assessed through DXA and BIA. Correlation and agreement were evaluated by using Pearson's correlation coefficients and Bland-Altman analysis. Multiple linear regression was applied for the estimation of RMR and a one-way ANOVA was used to compare the new BIA-based equations with other specific formulas. A significant correlation was noted between the BIA and DXA measurements. The final equation, applicable to both genders, was significantly correlated with intracellular water (ICW) and trunk fat, predicting 71.1% of RMR variance. When analyzed separately, body weight and protein displayed a moderate correlation with RMR in men (r = 0.616, p < 0.001), while ICW was correlated with the percentage of body fat in women (r = 0.579, p < 0.001). In the validation group, the values obtained through the three BIA-based equations were similar to the measured RMR, but differed significantly from those obtained through the four existing equations for trained individuals. In conclusion, the developed equations based on BIA-mediated body composition analysis provide a reliable method for estimating RMR in trained populations daily.