Methods and Protocols最新文献

Reliable characterization of circulating extracellular vesicles (EVs) in rodents may be significantly influenced by how blood is collected, yet systematic comparisons of commonly used sampling methods remain limited. Here, we directly evaluate the effects of cardiac puncture and perfusate blood collection on EV yield and surface-marker profiles in naïve mice, as well as in mice subjected to neurotrauma using a contusion spinal cord injury (SCI) model. Using matched isolation procedures and MACSPlex immunophenotyping, we analyzed newly generated cardiac puncture plasma alongside previously published perfusate-derived datasets, with both cohorts matched for age, sex, weight, injury severity, and post-injury timepoint. Cardiac puncture produced substantially higher particle concentrations due to access to undiluted blood, whereas perfusate samples exhibited modest increases in select markers, such as CD9, consistent with method-associated influences on platelet-derived vesicles. Despite these quantitative differences, both approaches yielded broadly similar EV phenotypes, and SCI-associated marker patterns remained stable across sampling methods. The consistency between cardiac puncture and perfusate datasets validates the robustness of our earlier perfusate-based findings and demonstrates that key biological signatures are preserved regardless of collection technique. These results provide practical guidance for optimizing murine EV studies and underscore the importance of methodological transparency and standardization in preclinical EV research.

During ischemia, endothelial cell integrity is compromised, as a consequence, blood barrier homeostasis is disrupted. Therefore, the structural and functional preservation of endothelial cells is paramount when trying to improve outcomes after ischemic injury. Endoplasmic reticulum (ER) stress is increasingly recognized as a key player in ischemic injury through unfolded protein response (UPR) signalling, and its crosstalk with mitochondrial death pathways. This study provides a cost-effective and straightforward method to delve into the relationship between ER stress and ischemia in human microvascular endothelial cells-1 (HMEC-1). HMEC-1 was exposed to 8 h of oxygen-glucose deprivation (OGD) in glucose-free medium with rapidly induced hypoxia. Hypoxia, oxygen consumption, cell viability, apoptosis, and ER stress markers (BiP/GRP78, PERK, ATF6, IRE1/XBP1s, CHOP) were assessed by RT-qPCR and Western blot. Cell viability decreased by approximately 33% following OGD, while CHOP expression increased ~4-fold, indicating significant ER stress induction. The model enables quantification of metabolic stress (OCR), as well as evaluation of viability loss, membrane integrity, apoptotic commitment, and discrimination between ER stress resolution versus maladaptation. Overall, GasPak EZ Pouch Systems provide a reproducible and practical in vitro platform to study ischemic injury down to the mechanistic details of ER-mitochondria signalling. They give the opportunity to evaluate therapeutic approaches that target ER homeostasis to limit apoptosis and/or recovery of metabolic function after ischemia. This method could allow rapid screening of ER stress-modulating interventions aimed at preserving endothelial barrier function, in various ischemic contexts.

The process of developing a drug is complex and involves many steps, from basic research (bench) to patient applications (bedside), which are conducted to ensure the drug is both safe and effective. In cancer research, the failure rate is high when translating basic findings to clinical trials. One of the main factors probably contributing to high failure rates is the basic quality of in vitro and in vivo study designs. Advanced basic cancer research techniques, including various types of 3D cell culture, the use of valuable organoids, organs, or tumors on chips, traditional or automated Western blots, omics research, advanced imaging techniques, usage of cutting-edge preclinical models and others, may produce inaccurate results for translational research if the basic study design is not carefully planned, especially when drugs or compounds are involved. In this manuscript, the author discussed (i) the importance of understanding and applying pharmacokinetic data in basic research, (ii) a proper comparison of the efficacy and safety of investigational drugs with the standard of care, (iii) the importance of following the actual route of drug administration as experienced by patients, the cruciality of human-to-animal dose conversion, and dose frequencies in animal models, (iv) significance of the age, gender, and strain of mice, along with adherence to the ARRIVE guidelines for ensuring transparency in conducting and reporting preclinical research, (v) benefits of having both subcutaneous and metastasis models in preclinical studies, (vi) the impact of comorbidities and related cancer drug studies in animal models and (vii) the importance of testing drug candidate/s in model mimicking acidic tumor microenvironment.

Recreational dance offers significant psychological well-being potential. However, traditional instruction emphasizes technique while limiting attention to nervous system development and embodied meaning-making. Despite empirical support for polyvagal theory, motor learning science, somatic education, and phenomenology, their systematic integration into unified structures is not clearly established in recreational dance contexts. This protocol integrates nervous system regulation, motor learning, and creative expression within structured Imperial Society of Teachers of Dancing (ISTD) modern dance syllabus for recreational adults. It presents a 12-week integrated dance-mindfulness intervention addressing this gap through a three-phase structure grounded in neuroscience and embodied pedagogy. The intervention comprises eight standardized components delivered weekly. The randomized controlled trial evaluates intervention effects using the Satisfaction With Life Scale (SWLS), Depression Anxiety Stress Scales-21 (DASS-21), the Mindful Attention Awareness Scale (MAAS), the Subjective Happiness Scale (SHS), and the Leisure Involvement Scale (LIS). Qualitative assessment via semi-structured phenomenological interviews (Weeks 8 and 12) and weekly journaling captures somatic awareness, nervous system resilience, technical confidence, creative expression, relational and social belonging, and embodied meaning-making. Intervention participants are expected to show significantly greater improvements compared to controls. Results will establish evidence-based practice standards for recreational dance and demonstrate neuroscience integration's efficacy for psychological wellbeing and embodied meaning-making.

This study aimed to establish an indirect ELISA for detecting the avian reovirus (ARV) epidemic strain xj-1.1 by using the purified recombinant protein pET-σC as the coating antigen. To optimize assay performance, key parameters were systematically evaluated, including antigen-coating concentration, serum dilution, blocking reagent and duration, serum incubation time, and the dilution and reaction time of the HRP-conjugated secondary antibody. The optimized conditions identified were a coating antigen dilution of 1:100, serum dilution of 1:1600, coating at 37 °C for 1 h followed by overnight incubation at 4 °C, and blocking with 5% skim milk for 2 h. The optimal serum incubation time was 1.5 h, with the secondary antibody diluted 1:1000 and incubated for 2 h, followed by a 20-min color development step. The cut-off value for distinguishing positive and negative samples was determined to be 0.121. Validation of the assay demonstrated favorable specificity, sensitivity, and repeatability, indicating that the developed indirect ELISA provides a reliable method for detecting ARV xj-1.1 infection.

This review evaluates the emerging paradigm of multi-agent systems (MASs) for biomedical and clinical data analysis, focusing on their ability to overcome the reasoning and reliability limitations of standalone large language models (LLMs). We synthesize findings from recent architectural frameworks, specifically LangGraph, CrewAI, and the Model Context Protocol (MCP), to examine how specialized agent teams divide labor, utilize precision tools, and cross-verify outputs. We find that MAS architectures yield significant performance gains in various domains: recent implementations improved oncology decision-making accuracy from 30.3% to 87.2% and reached a peak of 93.2% accuracy on USMLE-style benchmarks through simulated clinical evolution. In clinical trial matching, multi-agent frameworks achieved 87.3% accuracy and enhanced clinician screening efficiency by 42.6% (p < 0.001). However, we also highlight critical operational challenges, including an unreliability tax of 15-50× higher token consumption compared to standalone models and the risk of cascading errors where initial hallucinations are amplified across the agent collective. We conclude that while MAS enables a shift toward collaborative intelligence in biomedicine, its clinical and research adoption requires the development of deterministic orchestration and rigorous cost-utility frameworks to ensure safety and expert-centered oversight.

The differentiation of human embryonic stem cells (hESCs) into primordial germ cell-like cells (PGC-LCs) provides a robust in vitro model to study human germline specification. Here, we present a simple, reproducible, and cost-effective protocol for generating DEAD-box helicase 4 (DDX4)/VASA and Deleted in Azoospermia-Like (DAZL)-positive PGC-LCs from hESCs using a combination of bone morphogenetic protein 4 (BMP4) and conditioned medium (CM) derived from Stage-Specific Embryonic Antigen-4 (SSEA4)-positive human amniotic fluid stem cells (hAFSC-4). Importantly, unlike conventional protocols that rely on embryoid body formation, our method employs adherent cultures for germ cell differentiation. This approach enhances reproducibility by avoiding the spontaneous and stochastic variability inherent to embryoid body formation. This protocol provides a reproducible and physiologically relevant platform for studying human germ cell development in vitro.

Pseudomonas syringae pv. syringae (Pss) is the causal agent of bacterial canker, a disease that can result in yield losses, aerial tissue damage, and tree mortality in stone fruits worldwide. Peach, one of the major stone fruit crops, experiences significant yield losses and tree mortality attributed to bacterial canker in the United States. As the second-largest peach-producing state, South Carolina faces direct and significant impacts due to Pss. Early evaluations of peach scion responses to Pss infection have relied primarily on circumstantial field observations in rootstock trials. Although laboratory evaluations in peach have been reported, these studies primarily focused on pathogen virulence testing or small accession sets and did not establish a standardized, scalable detached twig protocol for systematic germplasm phenotyping. The absence of a clearly described laboratory assay has limited reproducible and large-scale evaluation of bacterial canker tolerance in peach. To address this gap, a detached dormant twig assay, previously developed for cherry, was adapted and optimized for peach. Dormant shoots from nine peach accessions were cut into 10 cm segments, surface-sterilized, and inoculated with a Pss suspension prepared in 10 mM MgCl₂ buffer or with the buffer alone. After six weeks of incubation, inner bark lesion size was evaluated visually and quantified using ImageJ. A newly developed visual rating scale was established and compared with quantitative lesion measurements. Spearman correlation analysis showed strong positive correlations between visual disease scores and ImageJ-based lesion measurements across two independent replicates (ρ = 0.80-1.00, p < 0.01), while shoot segment diameter showed weak-to-moderate negative correlations with disease severity. This adapted and consolidated dormant twig assay provides a practical, reproducible, and scalable method for phenotyping bacterial canker tolerance in peach and supports future germplasm screening and breeding efforts.

Gastroesophageal reflux disease (GERD) is a common chronic condition mainly caused by the dysfunction of the antireflux mechanism at the gastroesophageal junction. This is composed of the lower esophageal sphincter and the crural diaphragm. Increasing evidence suggests that diaphragmatic dysfunction and reduced inspiratory muscle strength may contribute to the persistence of GERD symptoms. Although respiratory physiotherapy has shown beneficial effects, the role of a structured inspiratory muscle training (IMT) program has not been sufficiently examined. This study aims to investigate the effects of an inspiratory muscle training program on inspiratory muscle strength and secondary clinical outcomes in individuals with GERD. A total of thirty adults with a confirmed GERD diagnosis will be enrolled in a two-arm randomized controlled trial. These volunteers will be randomly assigned either to the experimental group, which will undergo a 3-month inspiratory muscle training (IMT) using tapered flow resistive loading at 40% of maximal inspiratory pressure (MIP), or to the control group, which will receive sham IMT with a consistent low resistance. Primary outcomes will include maximal inspiratory pressure (MIP) and maximal dynamic inspiratory pressure (S-index). Secondary outcomes will assess GERD symptoms, disease-related quality of life, and pulmonary function. Measurements will be performed at baseline, at three months of intervention, and at six months from recruitment (follow-up). IMT is expected to lead to significant improvements in inspiratory muscle strength, symptom burden, and quality of life compared with sham training. This trial will provide novel evidence regarding the role of inspiratory muscle training as a non-pharmacological intervention in the management of GERD. Trial registration: ClinicalTrials.gov Identifier: NCT07131397.

Robot-assisted therapy (RT) is increasingly implemented in rehabilitation, yet evidence on its effectiveness in improving upper extremity function after cervical spinal cord injury (cSCI) remains limited. Therefore, this randomised crossover study aims to investigate the effects of unilateral RT compared to conventional unilateral occupational therapy (OT) on upper extremity function in individuals with cSCI. 40 participants with traumatic or non-traumatic cSCI (16-81 days post-injury, neurological level of injury: C1-T1) will be randomised (1:1), stratified by their predicted recovery profile, to receive 6 weeks of RT (ArmeoSpring) and 6 weeks of OT in random order, each 3 × 30 min/week in addition to the clinical routine therapy. Assessments are conducted before (t₀), between (t₁) and after both intervention blocks (t₂ and t₃). The primary outcome is the Quantitative Grasping Subtest of the Graded Redefined Assessment of Strength, Sensibility, and Prehension (GRASSP-QtG); primary analysis uses a linear mixed model to estimate the treatment effect based on change scores. Recruitment is currently ongoing. This randomised crossover study allows the collection of a comprehensive dataset to generate knowledge about treatment effectiveness, enabling future individuals with cSCI to benefit from improved and individualised therapy schedules.