Protocol for Seahorse analysis of ex vivo mouse brown and white adipose tissues.

The mitochondrial stress test is a gold-standard approach for assessing adipose tissue physiological functions and pathological changes. Here, we present a protocol for conducting Seahorse assays using ex vivo mouse brown and white adipose depots. We describe steps for rehydrating the cartridge, preparing freshly harvested fat depots, placing them onto an islet capture plate, and incubating them in a non-CO2 incubator. We then detail procedures for adding mitochondrial stressor solutions and conducting the mitochondrial stress test using the Seahorse XFe24 Analyzer. For complete details on the use and execution of this protocol, please refer to An et al.1.

Protocol for detecting micro(nano)plastics released from intravenous infusion products.

Approaches for detecting micro(nano)plastics (MNPs) released from intravenous infusion products (IVIPs) are vital for evaluating the safety of both IVIPs and their derived MNPs on human health, yet current understanding is limited. Here, we present a protocol for detecting polyvinyl chloride (PVC) MNPs by combining Raman spectroscopy, scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy (SEM-EDS), and pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS). We describe steps for collecting, pretreating, and measuring PVC MNPs released from IVIPs. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Protocol to optimize the Rice-Vannucci rat pup model of perinatal asphyxia to ensure predictable hypoxic-ischemic cerebral lesions.

The Rice-Vannucci model in rodent pups is subject to substantial loss of animals, result inconsistency, and high lab-to-lab variability in extent and composition of induced injury. This protocol allows for highly predictable and reproducible hypoxic-ischemic cerebral injury lesions in post-natal day 10 Wistar rat pups with no mortality. We describe steps for common carotid artery ligation, brief post-operative normothermia, exposure to hypoxia, and post-hypoxic normothermia. Precise timing and temperature control in each step are crucial for a successful procedure. For complete details on the use and execution of this protocol, please refer to Hartman et al.1.

Human SIRT5 variants with reduced stability and activity do not cause neuropathology in mice.

SIRT5 is a sirtuin deacylase that removes negatively charged lysine modifications, in the mitochondrial matrix and elsewhere in the cell. In benign cells and mouse models, under basal conditions, the phenotypes of SIRT5 deficiency are quite subtle. Here, we identify two homozygous SIRT5 variants in patients suspected to have mitochondrial disease. Both variants, P114T and L128V, are associated with reduced SIRT5 protein stability and impaired biochemical activity, with no evidence of neomorphic or dominant negative properties. The crystal structure of the P114T enzyme was solved and shows only subtle deviations from wild-type. Via CRISPR-Cas9, we generated a mouse model that recapitulates the human P114T mutation; homozygotes show reduced SIRT5 levels and activity, but no obvious metabolic abnormalities, neuropathology, or other gross phenotypes. We conclude that these human SIRT5 variants most likely represent severe hypomorphs, but are likely not by themselves the primary pathogenic cause of the neuropathology observed in the patients.

Bibliometric analysis on physical ergonomics: A visual mapping approach.

BACKGROUND: The concept of ergonomics in health services is attracting significant attention in the scientific community. There is a need for an integrated study presenting a summary of the published literature backed by detailed bibliometric characteristics. OBJECTIVE: The aim of this study is to provide a summary of the published literature supported by detailed bibliometric properties. METHODS: Within the scope of this study, a total of 3008 articles on ergonomics in the health field were reviewed and analyzed using a bibliometric method. RESULTS: It reveals the trends of the publications conducted between 1999-2023, and defines the common citation structure between the articles, bibliographic coupling, and keyword co-occurrences. This study presents a knowledge map of ergonomics studies conducted in the health field using a bibliometric analysis method. CONCLUSION: The research results provide comprehensive information to the relevant literature, and define global research focuses and future scopes. This serves as a guide for academics to understand developments in the field of ergonomics and health more easily and quickly.

ESR1 and p53 interactome alteration defines mechanisms of tamoxifen response in luminal breast cancer.

The canonical mechanism behind tamoxifen's therapeutic effect on estrogen receptor α/ESR1+ breast cancers is inhibition of ESR1-dependent estrogen signaling. Although ESR1+ tumors expressing wild-type p53 were reported to be more responsive to tamoxifen (Tam) therapy, p53 has not been factored into choice of this therapy and the mechanism underlying the role of p53 in Tam response remains unclear. In a window-of-opportunity trial on patients with newly diagnosed stage I-III ESR1+/HER2/wild-type p53 breast cancer who were randomized to arms with or without Tam prior to surgery, we reveal that the ESR1-p53 interaction in tumors was inhibited by Tam. This resulted in functional reactivation of p53 leading to transcriptional reprogramming that favors tumor-suppressive signaling, as well as downregulation of oncogenic pathways. These findings illustrating the convergence of ESR1 and p53 signaling during Tam therapy enrich mechanistic understanding of the impact of p53 on the response to Tam therapy.

Bradykinin induces acute kidney injury after hypothermic circulatory arrest through the repression of the Nrf2-xCT pathway.

Postoperative acute kidney injury (AKI) is a common complication in patients undergoing deep hypothermic circulatory arrest (HCA); however, its underlying pathogenesis is unclear. In this study, we established a rat cardiopulmonary bypass model and demonstrated that hypothermia during HCA, rather than circulatory arrest, was responsible for the occurrence of AKI. By recruiting 56 patients who underwent surgery with HCA and analyzing the blood samples, we found that post-HCA AKI was associated with an increase in bradykinin. Animal experiments confirmed this and showed that hypothermia during HCA increased bradykinin levels by increasing kallikrein expression. Mechanistically, bradykinin inhibited the Nrf2-xCT pathway through B2R and caused renal oxidative stress damage. Application of Icatibant, a B2R inhibitor, reversed changes in the Nrf2-xCT pathway and oxidative stress damage. Finally, Icatibant reversed hypothermia-induced AKI in vivo. This finding reveals the pathogenesis of AKI after HCA and helps to provide therapeutic strategy for patients with post-HCA AKI.

Lack of memory recall in human CD4 T cells elicited by the first encounter with SARS-CoV-2.

The studies reported here focus on the impact of pre-existing CD4 T cell immunity on the first encounter with SARS-CoV-2. They leverage PBMC samples from plasma donors collected after a first SARS-CoV-2 infection, prior to vaccine availability and compared to samples collected prior to the emergence of SARS-CoV-2. Analysis of CD4 T cell specificity across the entire SARS-CoV-2 proteome revealed that the recognition of SARS-CoV-2-derived epitopes by CD4 memory cells prior to the pandemic are enriched for reactivity toward non-structural proteins conserved across endemic CoV strains. However, CD4 T cells after primary infection with SARS-CoV-2 focus on epitopes from structural proteins. We observed little evidence for preferential recall to epitopes conserved between SARS-CoV-2 and seasonal CoV, a finding confirmed through use of selectively curated conserved and SARS-unique peptides. Our data suggest that SARS-CoV-2 CD4 T cells elicited by the first infection are primarily established from the naive CD4 T cell pool.

Hallmarks of cancer resistance.

This review explores the hallmarks of cancer resistance, including drug efflux mediated by ATP-binding cassette (ABC) transporters, metabolic reprogramming characterized by the Warburg effect, and the dynamic interplay between cancer cells and mitochondria. The role of cancer stem cells (CSCs) in treatment resistance and the regulatory influence of non-coding RNAs, such as long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), are studied. The chapter emphasizes future directions, encompassing advancements in immunotherapy, strategies to counter adaptive resistance, integration of artificial intelligence for predictive modeling, and the identification of biomarkers for personalized treatment. The comprehensive exploration of these hallmarks provides a foundation for innovative therapeutic approaches, aiming to navigate the complex landscape of cancer resistance and enhance patient outcomes.

Construction and validation of serum Metabolic Risk Score for early warning of malignancy in esophagus.

Using noninvasive biomarkers to identify high-risk individuals prior to endoscopic examination is crucial for optimization of screening strategies for esophageal squamous cell carcinoma (ESCC). We conducted a nested case-control study based on two community-based screening cohorts to evaluate the warning value of serum metabolites for esophageal malignancy. The serum samples were collected at enrollment when the cases had not been diagnosed. We identified 74 differential metabolites and two prominent perturbed metabolic pathways, and constructed Metabolic Risk Score (MRS) based on 22 selected metabolic predictors. The MRS generated an area under the receiver operating characteristics curve (AUC) of 0.815. The model performed well for the within-1-year interval (AUC: 0.868) and 1-to-5-year interval (AUC: 0.845) from blood draw to diagnosis, but showed limited ability in predicting long-term cases (>5 years). In summary, the MRS could serve as a potential early warning and risk stratification tool for establishing a precision strategy of ESCC screening.

Molecular maps of synovial cells in inflammatory arthritis using an optimized synovial tissue dissociation protocol.

In this study, we optimized the dissociation of synovial tissue biopsies for single-cell omics studies and created a single-cell atlas of human synovium in inflammatory arthritis. The optimized protocol allowed consistent isolation of highly viable cells from tiny fresh synovial biopsies, minimizing the synovial biopsy drop-out rate. The synovium scRNA-seq atlas contained over 100,000 unsorted synovial cells from 25 synovial tissues affected by inflammatory arthritis, including 16 structural, 11 lymphoid, and 15 myeloid cell clusters. This synovial cell map expanded the diversity of synovial cell types/states, detected synovial neutrophils, and broadened synovial endothelial cell classification. We revealed tissue-resident macrophage subsets with proposed matrix-sensing (FOLR2+COLEC12high) and iron-recycling (LYVE1+SLC40A1+) activities and identified fibroblast subsets with proposed functions in cartilage breakdown (SOD2highSAA1+SAA2+SDC4+) and extracellular matrix remodeling (SERPINE1+COL5A3+LOXL2+). Our study offers an efficient synovium dissociation method and a reference scRNA-seq resource, that advances the current understanding of synovial cell heterogeneity in inflammatory arthritis.

Immortalized human myotonic dystrophy type 1 muscle cell lines to address patient heterogeneity.

Historically, cellular models have been used as a tool to study myotonic dystrophy type 1 (DM1) and the validation of therapies in said pathology. However, there is a need for in vitro models that represent the clinical heterogeneity observed in patients with DM1 that is lacking in classical models. In this study, we immortalized three DM1 muscle lines derived from patients with different DM1 subtypes and clinical backgrounds and characterized them at the genetic, epigenetic, and molecular levels. All three cell lines display DM1 hallmarks, such as the accumulation of RNA foci, MBNL1 sequestration, splicing alterations, and reduced fusion. In addition, alterations in early myogenic markers, myotube diameter and CTCF1 DNA methylation were also found in DM1 cells. Notably, the new lines show a high level of heterogeneity in both the size of the CTG expansion and the aforementioned molecular alterations. Importantly, these immortalized cells also responded to previously tested therapeutics. Altogether, our results show that these three human DM1 cellular models are suitable to study the pathophysiological heterogeneity of DM1 and to test future therapeutic options.

Protocol for fiber photometry recording from deep brain regions in head-fixed mice.

To exclude the influence of motion on in vivo calcium imaging, animals usually need to be fixed. However, the whole-body restraint can cause stress in animals, affecting experimental results. In addition, some brain regions are prone to bleeding during surgery, which lowers the success rate of calcium imaging. Here, we present a protocol for calcium imaging using heparin-treated fiber in head-fixed mice. We describe steps for stereotaxic surgery, including virus injection and optic fiber implantation, fiber photometry, and data analysis. For complete details on the use and execution of this protocol, please refer to Du et al.1.

High-throughput screening identifies broad-spectrum Coronavirus entry inhibitors.

The COVID-19 pandemic highlighted the need for antivirals against emerging coronaviruses (CoV). Inhibiting spike (S) glycoprotein-mediated viral entry is a promising strategy. To identify small molecule inhibitors that block entry downstream of receptor binding, we established a high-throughput screening (HTS) platform based on pseudoviruses. We employed a three-step process to screen nearly 200,000 small molecules. First, we identified hits that inhibit pseudoviruses bearing the SARS-CoV-2 S glycoprotein. Counter-screening against pseudoviruses with the vesicular stomatitis virus glycoprotein (VSV-G), yielded sixty-five SARS-CoV-2 S-specific inhibitors. These were further tested against pseudoviruses bearing the MERS-CoV S glycoprotein, which uses a different receptor. Out of these, five compounds, which included the known broad-spectrum inhibitor Nafamostat, were subjected to further validation and tested against pseudoviruses bearing the S glycoprotein of the Alpha, Delta, and Omicron variants as well as bona fide SARS-CoV-2. This rigorous approach revealed an unreported inhibitor and its derivative as potential broad-spectrum antivirals.

Integrative neurovascular coupling and neurotransmitter analyses in anisometropic and visual deprivation amblyopia children.

The association between visual abnormalities and impairments in cerebral blood flow and brain region potentially results in neural dysfunction of amblyopia. Nevertheless, the differences in the complex mechanisms of brain neural network coupling and its relationship with neurotransmitters remain unclear. Here, the neurovascular coupling mechanism and neurotransmitter activity in children with anisometropic amblyopia (AA) and visual deprivation amblyopia (VDA) was explored. The neurovascular coupling of 17 brain regions in amblyopia children was significantly abnormal than in normal controls. The classification abilities of coupling units in brain regions differed between two types of amblyopia. Correlations between different coupling effects and neurotransmitters were different. The findings of this study demonstrate a correlation between the neurovascular coupling and neurotransmitter in children with AA and VDA, implying their impaired neurovascular coupling function and potential molecular underpinnings. The neuroimaging evidence revealed herein offers potential for the development of neural therapies for amblyopia.

Cerebrospinal fluid and plasma concentrations of the inflammatory marker soluble CD27 in a large surgical population.

Soluble CD27 (sCD27) is a potential biomarker for diseases involving immune dysfunction. As there is currently little data on cerebrospinal fluid (CSF) sCD27 concentrations in the general population we measured CSF and plasma concentrations in 486 patients (age range 18-92 years, 57% male) undergoing spinal anesthesia for elective surgery. Across the complete cohort the median [range] sCD27 concentrations were 163 [<50 to 7474] pg/mL in CSF and 4624 [1830 to >400,000] pg/mL in plasma. Plasma sCD27, age and Qalb were the factors most strongly associated with CSF sCD27 levels. Reference sCD27 concentration intervals (central 95% of values) in a sub-group without the indication of neuropsychiatric, inflammatory or systemic disease (158 patients) were <50 pg/mL - 419 pg/mL for CSF and 2344-36422 pg/mL for plasma. These data provide preliminary reference ranges that could inform future studies of the validity of sCD27 as a biomarker for neuro- and systemic inflammatory disorders.

Usability of Standards for Scaffolding in a Health Sciences Programme: A feasibility Study.

BACKGROUND: Standards contribute to comprehensive and programmatic implementation of educational strategies, such as scaffolding. Although the development of educational standards follows a rigorous consensus approach, they are socially constructed and could result in varied interpretations by users. Reports of varied implementation of standards in health professions education underscore the need to test the developed standards for scaffolding in health sciences programmes. Usability entails determining whether a product like standards works as intended under the expected conditions and contexts. This study aimed to describe the usability of standards for scaffolding in a health sciences programme through a pilot study. METHODS: A multi-method design employing user and expert-based usability evaluation techniques sought to describe the usability of the standards for scaffolding in a three-year pre-registration nursing programme. The user sample of nurse educators drawn from the programme, conducted a self-assessment on scaffolding practices in the programme using a developed standards checklist. For the expert sample, three-panel members with an understanding of the discipline and programme context were purposively sampled. These panelists studied the users' self-assessment reports before completing an author-generated heuristics checklist to support or refute any of the standards. Descriptive statistics, comparative and content analysis were applied to analyse data from users' interviews and expert's completed heuristics checklist, determining the standards' usability, and identifying the usability flaws or strengths. RESULTS: The users had three or more years of teaching experience in the competency-based curriculum for nursing. The experts shared an average of 16 years of experience in teaching in higher education, and seven years of experience in quality assurance and programme accreditation. The four standards had a usability score of above average (68%). Seven usability strengths and four usability flaws were identified. Usability flaws related to misinterpretation of some criteria statements and terminologies, multiple meanings, and users' challenges in generating evidence for some criteria. CONCLUSIONS: The pilot study revealed the context-based 'truth' regarding the fidelity of a health sciences programme evaluation on scaffolding, as well as identifying the ideal contextual conditions in which the standards for scaffolding health sciences programmes would work best. The identified usability flaws highlighted the need for further revisions of the standards. Future research on the feasibility of the standards in other health sciences programmes and contexts is recommended.

Position statement of the GMA committee "teaching evaluation".

The evaluation of teaching can be an essential driver for curriculum development. Instruments for teaching evaluation are not only used for the purpose of quality assurance but also in the context of medical education research. Therefore, they must meet the common requirements for reliability and validity. This position paper from the GMA Teaching Evaluation Committee discusses strategic and methodological aspects of evaluation in the context of undergraduate medical education and related courses; and formulates recommendations for the further development of evaluation. First, a four-step approach to the design and implementation of evaluations is presented, then methodological and practical aspects are discussed in more detail. The focus here is on target and confounding variables, survey instruments as well as aspects of implementation and data protection. Finally, possible consequences from evaluation data for the four dimensions of teaching quality (structural and procedural aspects, teachers and outcomes) are discussed.

The Relation Between Theoretical and Practical Exams for Health Sciences Students at King Saud Bin Abdulaziz University for Health Sciences- Jeddah.

Introduction: A comprehensive approach to assessment is essential to ensure that all students' learning competencies are measured accurately. Therefore, multiple methods of assessment have been developed to address this matter. This Study aims to assess the correlation between health sciences students' performance on theoretical and practical exams. Methods: A correlational study design was conducted. The academic performance of 352 students across theoretical/practical courses was tested. SPSS version 29.0 was used for analysis. Spearman's rho correlation (Rs), Wilcoxon, and Mann Whitney were computed at p<0.05. Results: The theoretical performance was strongly correlated with the practical performance of all programs pooled together (Rs (352) = 0.67, p<0.001). Also, there was a strong correlation between theoretical and practical performance for male students (Rs (181) = 0.72, p<0.001), while a moderate correlation for female students (Rs (171) = 0.53, p<0.001). Mann-Whitney test revealed significant mean performance difference by gender both at theoretical (U = 9284, p<0.0001) and practical (U = 11,373, p < 0.0001) levels. Conclusion: There were significant correlations between theoretical knowledge and practical skills across the selected four programs.; The mean student's performance was better in the practical skills than in the theoretical knowledge assessment, and female students surpassed male students in both practical and theoretical assessments in the four programs offered to both genders.

Increased innate immune responses in adolescents with obesity and its relation to subclinical cardiovascular measures: An exploratory study.

Cardiometabolic risk accrues across the life course and childhood and adolescence are key periods for effective prevention. Obesity is associated with inflammation in adults, but pediatric data are scarce. In a cross-sectional and longitudinal study, we investigated immune cell composition and activation in 31 adolescents with obesity (41.9% male, BMIz>2.5, 14.4 years) and 22 controls with healthy weight (45.1% male, -1.5