Methods and Protocols最新文献

A straightforward ex vivo approach has been developed and refined to enable high-resolution imaging and quantitative assessment of motile cilia function in mouse airway epithelial tissue, allowing critical insights into cilia motility and cilia generated flow using different mouse models or following different sample treatments. In this method, freshly excised mouse trachea is cut longitudinally through the trachealis muscle which is then sandwiched between glass coverslips within a thin silicon gasket. By orienting the tissue along its longitudinal axis, the natural curling of the trachealis muscle helps maintain the sample in a configuration optimal for imaging along the full tracheal length. High-speed video microscopy, utilizing differential interference contrast (DIC) optics and a fast digital camera capturing at >200 frames per second is then used to record ciliary motion. This enables detailed measurement of both cilia beat frequency (CBF) and waveform characteristics. The application of 1 µm microspheres to the bathing media during imaging allows for additional analysis of fluid flow generated by ciliary activity. The entire procedure typically takes around 40 min to complete per animal: ~30 min for tissue harvest and sample mounting, then ~10 min for imaging samples and acquiring data.

The present study presents a comparative evaluation of two analytical deconvolution models applied to Optically Stimulated Luminescence (OSL) decay curves of zirconia-reinforced lithium silicate (ZLS), a glass-ceramic material with potential applications in accidental dosimetry. ZLS samples were subjected to beta irradiation and measured under Continuous Wave OSL (CW-OSL) protocols. A comparative analysis is conducted between two deconvolution approaches-the General Order Kinetics (GOK) model and a master analytical equation based on the Lambert W function. The results imply that both models yield a linear dose-response behavior of the fast OSL component; however, the Lambert W approach offers simpler fitting with fewer parameters. The abovementioned findings demonstrate the methodological robustness of the Lambert W formalism and also confirm that ZLS is a promising dosimetric material, aligning with the goals of protocol development in material characterization.

Background: Primary hepatocytes are excellent models for studying liver functions and liver diseases. However, obtaining high yields of viable hepatocytes remains technically challenging, limiting their broader applications. Most conventional methods rely on a two-step collagenase perfusion technique. Despite its widespread use, this approach has several limitations that reduce the success rate of hepatocyte isolation and culture. The procedure involves multiple parameters that are continually being optimized in order to obtain hepatocytes in high yield and quality that can be used to provide insights into their physiology and pathophysiology.

Aim: We aimed to enhance the success rate and reproducibility of hepatocyte isolation with high yield, enabling analysis of diverse physiological and pathophysiological aspects of lipid metabolism. It also establishes an in vitro steatosis model for evaluating therapeutic drugs and molecular interventions.

Methods: Rat liver was perfused in situ with EDTA buffer followed by collagenase IV. Liver was then isolated, and hepatocytes were mechanically liberated, filtered, and purified through density-gradient centrifugation. Viable cells were cultured at 700,000 or 1 million cells/well for 24 h. The monolayer was incubated in lipogenic media for an additional 24 or 48 h. Hepatocytes were fixed, neutral lipids were stained using Oil Red O, and the stained area was quantified using Image J software version 1.54.

Results: Yield of hepatocytes was ~75-90 million cells/liver, with viability of 86-93%. Cells seeded at 700,000 and 1 million cells/well reached confluences of 60% and 80%, respectively, after 24 h. Steatosis was then induced with lipid accumulation reaching 21% of image area after 24 h and 25% after 48 h.

Conclusions: The current protocol presents an efficient and highly reproducible method for isolating primary rat hepatocytes in high yield with high viability. Additionally, the protocol provides a foundation for studying the pathophysiology of fatty liver disease.

Lipidomics is a rapidly growing branch of metabolomics that identifies lipid compositions of samples to learn more about disease and identify potential novel therapeutic targets. In the context of ophthalmology, lipidomic research has increased our understanding of optic nerve regeneration. The diversity of experimental designs for lipidomic research and the large datasets generated are two obstacles that must be addressed by bioinformatic tools to perform statistical analysis on lipidomics data. Our study provides an objective comparison of the features in two freely accessible web-based bioinformatics tools, MetaboAnalyst 6.0 and LipidOne 2.3, for analyzing an optic nerve regeneration model lipidome. A publicly available lipidomic dataset of the optic nerve axon regeneration model, Xenopus laevis, was used to compare the analytic capabilities of both tools. Though both tools offered univariate and multivariate analysis methods, MetaboAnalyst 6.0 had advantages in customizable data processing, normalization, analysis, and image generation. It also offered consistent multiple-comparison testing correction and comprehensive results/dataset export. Meanwhile LipidOne 2.3 uniquely allowed for univariate and multivariate analysis of lipid classes and lipid building blocks.

Xenotransplantation using pig cells, tissues, or organs is advancing toward clinical application to address the shortage of human donor organs for treating organ failure. However, this emerging technology carries the risk of transmitting pathogenic porcine microorganisms, particularly viruses. The recent transmission of a porcine herpesvirus to the first human recipient of a pig heart highlights the urgent need for more rigorous screening of donor pigs. To identify potentially pathogenic porcine viruses, highly sensitive and specific detection methods are required. PCR-based techniques able to detect porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV), hepatitis E virus (HEV), porcine circoviruses (PCV1-4), porcine lymphotropic herpesviruses (PLHV-1-3), porcine endogenous retroviruses (PERVs), porcine parvovirus (PPV), Torque teno sus viruses (TTSuV1,2), atypical porcine pestivirus (APPV) and SARS-CoV-2 were established. Immunological assays that detect antibodies as indirect indicators of infection were established for PCMV/PRV, HEV, PLHVs and PERVs. Since most veterinary laboratories focus on detecting viruses that are pathogenic to pigs and cause economic losses to the swine industry, screening for viruses relevant to xenotransplantation should be conducted in specialized virological diagnostic units. In this context, we present a complete collection of the newest and detailed protocols for comprehensive viral screening, along with guidance on how to implement these methods effectively.

In the last few decades, chimeric antigen receptor (CAR) T-cell therapy has led to a paradigm shift in the treatment of hematological malignancies, including various subtypes of B-cell non-Hodgkin's lymphoma, B-cell acute lymphoblastic leukemia, and multiple myeloma. However, most patients experience refractoriness to CAR T-cells or relapse after treatment. Many efforts are underway to understand the mechanisms behind CAR T-cell failure, which are mainly related to CAR T-cell dysfunction, tumor-intrinsic resistance, an immunosuppressive tumor microenvironment, manufacturing issues, or patient-related factors. Several strategies are being developed to overcome these resistance mechanisms, including the engineering of more functional allogeneic CAR T-cell products, the targeting of alternative tumor antigens, and combination therapies with other drugs such as checkpoint inhibitors or small molecules to enhance CAR T-cell efficacy. In this review, we will provide an updated overview of the mechanisms of CAR T-cell failure and the therapeutic advances currently under development to address them.

The routine transformation and genetic modification of many plant species other than Arabidopsis thaliana is still an arduous task. In many cases, it is necessary to use special tissues or conditions performed under sterile tissue culture conditions. Nevertheless, this approach is often the most expedient one, and streamlining protocols to maximize efficiency and minimize effort without sacrificing quality is paramount to today's research agendas. The Solanaceae family tends to be amicable to tissue culture and relatively easy to transform. Here, we present our optimized, routine tissue culture protocols for the transformation of Nicotiana benthamiana, Nicotiana tabacum, Solanum tuberosum (potato), and Solanum lycopersicum (tomato). We highlight their commonalities and their differences, thus giving the researcher a framework for optimizing their own protocols in their laboratory if needed. Tissue culture transformation is still an important and dynamic field for the advancement of plant research in molecular genetics, physiology, and plant pathology, and will continue to be a viable and important resource into the future.

Biogenic amines (BAs) are nitrogenous compounds naturally present in protein-rich foods, whose accumulation may indicate spoilage and pose health risks. This study presents the development and validation of a rapid LC-MS/MS method for the simultaneous quantification of six BAs-putrescine (PUT), cadaverine (CAD), histamine (HIS), tyramine (TYR), spermidine (SPD), and spermine (SPM)-in meat products, without requiring derivatisation. Sample preparation was optimized to enhance extraction efficiency and reproducibility, using 0.5 M HCl and a double-centrifugation protocol to avoid matrix interference. Chromatographic separation was optimized using a C18 column and acidified ammonium formate/acetonitrile mobile phases. The method showed good linearity (R² > 0.99), trueness between -20% and +20%, and acceptable precision (RSD_r and RSD_R ≤ 25%). Limits of quantification were established at 10 µg/g for all analytes. The method was applied to ten commercial meat samples, where PUT, TYR, and SPD were the most frequently detected amines. Although HIS and TYR levels were below toxicological thresholds for healthy individuals, one sample showed TYR levels potentially concerning for monoamine oxidase inhibitors -treated consumers. The Biogenic Amine Index (BAI) further supported product quality assessment, identifying early spoilage in selected cases. This method offers a rapid, robust and efficient tool for routine monitoring of BAs in meat products, supporting food safety and quality control initiatives.

Human rhinovirus (RV) is the most frequent cause of the common cold, as well as severe exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. Currently, there are no effective and accurate diagnostic tools or antiviral therapies. MicroRNAs (miRNAs) are small, non-coding sections of RNA involved in the regulation of gene expression and have been shown to be associated with different pathologies. However, the precise role of miRNAs in RV infection is not yet well established. Also, no unified computational framework exists to specifically link miRNA expression with functional gene targets during RV infection. This study aimed to first analyse the impact of RV16 on miRNA expression across the viral life cycle to identify a small panel with altered expression. We then developed a novel bioinformatics pipeline that integrated time-resolved miRNA profiling with multi-database gene-phenotype mapping to identify diagnostic biomarkers and their regulatory networks. Our in-house Python-based tool, combining mirDIP, miRDB and VarElect APIs, predicted seven genes (EZH2, RARG, PTPN13, OLFML3, STAG2, SMARCA2 and CD40LG) implicated in antiviral responses and specifically targeted by RV16 and regulated by our miRNAs. This method therefore offers a scalable approach to interrogate miRNA-gene interactions for viral infections, with potential applications in rapid diagnostics and therapeutic target discovery.

SARS-CoV-2, the agent responsible for coronavirus disease in 2019 (COVID-19), has caused extensive global health and socioeconomic impact due to its transmissibility and pathology. As a result, it was classified as a Risk Group 3 human pathogen, and handling samples containing live virus requires enhanced biological containment facilities (i.e., CL3) to reduce the potential of laboratory infection to personnel and the spread of the virus into the community. While the use of an authentic live virus remains the gold standard for biological assays, alternative methods have been developed to effectively evaluate neutralization activity in the absence of a replicating viral agent. Here, we describe a cell-based spike-ACE2 binding assay as a surrogate for neutralization of SARS-CoV-2 spike to identify potential neutralizing antibodies. A main advantage of this approach is the exclusion of infectious viral particles, increasing biosafety for laboratory personnel. The interaction of recombinant SARS-CoV-2 trimeric spike protein with ACE2 is monitored and quantified by flow cytometry. Notably, our previous studies have demonstrated the utility of this assay for other viruses, beyond SARS-CoV-2. The methodology presented here has exhibited a strong correlation to other widely accepted methods, such as pseudotyped lentiviral and live virus neutralization assays, in identifying neutralizing antibodies.