JMJD3 downregulates IL4i1 aggravating lipopolysaccharide-induced acute lung injury via H3K27 and H3K4 demethylation.

The pro-inflammation M1 to anti-inflammation M2 macrophage ratio contribute to the severity of lipopolysaccharide (LPS)-induced acute lung injury (ALI). JMJD3 aggravates the inflammatory reaction through affecting epigenetic modification and macrophage's phenotype to deteriorate ALI. To explore the mechanism underlying the upregulation of the macrophage M1/M2 ratio through JMJD3, we developed an ALI mouse model using intratracheal LPS, LPS-stimulated RAW 264.7 cells, and inhibited JMJD3 using GSK-J4. H3K27me3 and H3K4me3 were investigated as JMJD3-mediated epigenetic alteration sites in vivo and in vitro. C/EBPß and KDM5A were validated as linking factors between H3K27 and H3K4. IL4i1 was investigated as a JMJD3-mediated targeted gene to regulate the macrophage M1/M2 ratio. Chromatin immunoprecipitation was used to evaluate the relationship between H3K27me3 and C/ebpß, C/EBPß and Kdm5a, H3K4me3 and Il4i1. Inhibiting JMJD3 with GSK-J4 can relieve inflammation and pathological performance in ALI. JMJD3 can reduce IL4i1 expression to increase the macrophage M1/M2 ratio and aggravated ALI which process was mediated via JMJD3-indcued H3K27me3 and H3K4me3 demethylation, latter H3K4me3 demethylation inhibited IL4i1 transcription. Inhibiting JMJD3 with GSK-J4 can increase IL4i1 expression, subsequently decreasing the expressions of M1 and increasing of M2 in vivo. The over-expression IL4i1 in LPS-stimulated macrophage or inhibiting JMJD3 with GSK-J4 can both reverse the increase of the macrophage M1/M2 ratio in vitro. C/EBPß and KDM5A were upregulated by LPS simulation, which linked JMJD3-induced H3K27-H3K4 demethylation. JMJD3 inhibited IL4i1 to increase the macrophage M1/M2 phenotype ratio and aggravate LPS-induced ALI. Using GSK-J4 to inhibit JMJD3 may facilitate the treatment of LPS-induced ALI.

Large scale genotype- and phenotype-driven machine learning in Von Hippel-Lindau disease.

Von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome where individuals are predisposed to tumor development in the brain, adrenal gland, kidney, and other organs. It is caused by pathogenic variants in the VHL tumor suppressor gene. Standardized disease information has been difficult to collect due to the rarity and diversity of VHL patients. Over 4100 unique articles published until October 2019 were screened for germline genotype-phenotype data. Patient data were translated into standardized descriptions using Human Genome Variation Society gene variant nomenclature and Human Phenotype Ontology terms and has been manually curated into an open-access knowledgebase called Clinical Interpretation of Variants in Cancer. In total, 634 unique VHL variants, 2882 patients, and 1991 families from 427 papers were captured. We identified relationship trends between phenotype and genotype data using classic statistical methods and spectral clustering unsupervised learning. Our analyses reveal earlier onset of pheochromocytoma/paraganglioma and retinal angiomas, phenotype co-occurrences and genotype-phenotype correlations including hotspots. It confirms existing VHL associations and can be used to identify new patterns and associations in VHL disease. Our database serves as an aggregate knowledge translation tool to facilitate sharing information about the pathogenicity of VHL variants.

Development of a career questionnaire for medical undergraduates using Mokken scale analysis.

BACKGROUND: Individual-centred career questionnaires are important for understanding the motivations of medical students. This study aimed to collect validity evidence of a questionnaire to measure the career choice of medical undergraduates. METHODS: A cross-sectional survey was sent to third-year undergraduate students at a Chinese university-affiliated hospital. The questionnaire was formed using items that were selected after a systematic literature review. Item reduction was conducted using Mokken scale analysis, followed by reliability and validity testing, which described the validity evidence of the content, response process and internal structure. RESULTS: The preliminary 20-item questionnaire was returned by 213 undergraduate students (response rate: 86.59%). To construct a monotone homogeneity model, 6 items were removed after testing for unidimensionality, local independence, and latent monotonicity according to the sequence. The final questionnaire included 14 items in two subscales: a 10-item 'career advantage' subscale and a 4-item 'career disadvantage' subscale. The questionnaire was judged to be acceptably reliable (Molenaar-Sijtsma method: 0.87 and 0.75, Cronbach's alpha: 0.87 and 0.74) and to have good construct validity (χ2/df: 1.748, normed fit index: > 0.9, comparative fit index: > 0.9, root mean square error of approximation: 0.05-0.08). Male and female undergraduates had different responses regarding their salary, subspecialty, career prospects, and ability to serve their relatives. Male undergraduates might be more willing to accept on-call positions and have subspecialties with greater likelihoods of patient-physician conflict. CONCLUSION: We used Mokken scale analysis to develop and collect evidence of the validity of a 14-item questionnaire regarding career preferences among Chinese medical undergraduate students. This short and simple questionnaire may provide a suitable tool for exploring insights regarding the motivations of Chinese medical students.

Treatment Patterns and Outcomes of Women with Symptomatic and Asymptomatic Breast Cancer Brain Metastases: A Single-Center Retrospective Study.

BACKGROUND: Breast cancer is the most common cancer among women worldwide and the second leading cause of brain metastases (BrM). We assessed the treatment patterns and outcomes of women treated for breast cancer BrM at our institution in the modern era of stereotactic radiosurgery (SRS). MATERIALS AND METHODS: We conducted a retrospective analysis of women (≥18 years of age) with metastatic breast cancer who were treated with surgery, whole brain radiotherapy (WBRT), or SRS to the brain at the Sunnybrook Odette Cancer Centre, Toronto, Canada, between 2008 and 2018. Patients with a history of other malignancies and those with an uncertain date of diagnosis of BrM were excluded. Descriptive statistics were generated and survival analyses were performed with subgroup analyses by breast cancer subtype. RESULTS: Among 683 eligible patients, 153 (22.4%) had triple-negative breast cancer, 188 (27.5%) had HER2+, 246 (36.0%) had hormone receptor (HR)+/HER2-, and 61 (13.3%) had breast cancer of an unknown subtype. The majority of patients received first-line WBRT (n = 459, 67.2%) or SRS (n = 126, 18.4%). The median brain-specific progression-free survival and median overall survival (OS) were 4.1 months (interquartile range [IQR] 1.0-9.6 months) and 5.1 months (IQR 2.0-11.7 months) in the overall patent population, respectively. Age >60 years, presence of neurological symptoms at BrM diagnosis, first-line WBRT, and HER2- subtype were independently prognostic for shorter OS. CONCLUSION: Despite the use of SRS, outcomes among patients with breast cancer BrM remain poor. Strategies for early detection of BrM and central nervous system-active systemic therapies warrant further investigation. IMPLICATIONS FOR PRACTICE: Although triple-negative breast cancer and HER2+ breast cancer have a predilection for metastasis to the central nervous system (CNS), patients with hormone receptor-positive/HER2- breast cancer represent a high proportion of patients with breast cancer brain metastases (BrM). Hence, clinical trials should include patients with BrM and evaluate CNS-specific activity of novel systemic therapies when feasible, irrespective of breast cancer subtype. In addition, given that symptomatic BrM are associated with shorter survival, this study suggests that screening programs for the early detection and treatment of breast cancer BrM warrant further investigation in an era of minimally toxic stereotactic radiosurgery.

Developing a scoring tool to estimate the risk of deterioration for normotensive patients with acute pulmonary embolism on admission.

BACKGROUND: It is important to identify deterioration in normotensive patients with acute pulmonary embolism (PE). This study aimed to develop a tool for predicting deterioration among normotensive patients with acute PE on admission. METHODS: Clinical, laboratory, and computed tomography parameters were retrospectively collected for normotensive patients with acute PE who were treated at a Chinese center from January 2011 to May 2020 on admission into the hospital. The endpoint of the deterioration was any adverse outcome within 30 days. Eligible patients were randomized 2:1 to derivation and validation cohorts, and a nomogram was developed and validated by the aforementioned cohorts, respectively. The areas under the curves (AUCs) with 95% confidence intervals (CIs) were calculated. A risk-scoring tool for predicting deterioration was applied as a web-based calculator. RESULTS: The 845 eligible patients (420 men, 425 women) had an average age of 60.05 ± 15.43 years. Adverse outcomes were identified for 81 patients (9.6%). The nomogram for adverse outcomes included heart rate, systolic pressure, N-terminal-pro brain natriuretic peptide, and ventricle/atrial diameter ratios at 4-chamber view, which provided AUC values of 0.925 in the derivation cohort (95% CI 0.900-0.946, p < 0.001) and 0.900 in the validation cohort (95% CI 0.883-0.948, p < 0.001). A risk-scoring tool was published as a web-based calculator ( https://gaoyzcmu.shinyapps.io/APE9AD/ ). CONCLUSIONS: We developed a web-based scoring tool that may help predict deterioration in normotensive patients with acute PE.

Decreased size of the left anterior descending coronary artery is an independent predictor of deterioration in non-high-risk patients with acute pulmonary embolism.

To evaluate the efficacy of measuring left coronary artery size to predict deterioration in non-high-risk acute pulmonary embolism (PE) patients. This retrospective study enrolled non-high-risk acute PE patients from January 2011 to December 2019. Patient deterioration was defined as the occurrence of adverse events within 30 days of hospital admission. Patients with adverse events were sex- and age-matched to patients without adverse events. Risk stratification was performed. Cross-sectional areas (CSAs) of the left main and left anterior descending (LAD) coronary artery inlets were measured. The main pulmonary artery (MPA) inlet and outlet and MPA LAD plane, which adjoined the LAD in the MPA, were reconstructed. CSAs, perimeters, and hydraulic diameters were measured to evaluate MPA size and deformation. Cardiac volume was also measured. Quantitative parameters were divided into tertiles. After adjustment by risk stratification, univariate and multivariate analyses were performed. Correlations between different parameters were analysed. Seventy-three patients with adverse events were matched to 73 patients without adverse events. The results of the univariate and multivariate analyses revealed that LAD inlet CSAs (middle and high) predicted adverse events (odds ratio: 0.28 and 0.07, 95% confidence interval: 0.10-0.77 and 0.02-0.22, p = 0.013 and < 0.0001). LAD inlet CSA was strongly and negatively correlated with MPA LAD hydraulic diameter and CSA (correlation coefficients: - 0.643 and - 0.604, p < 0.001). LAD inlet CSA measurement would facilitate adverse event prediction in non-high-risk acute PE patients on the basis of risk stratification. The dilated MPA may involve the decrease in LAD inlet CSA.

A predictive tool for the assessment of right ventricular dysfunction in non-high-risk patients with acute pulmonary embolism.

BACKGROUND: Rapid and accurate identification of right ventricular (RV) dysfunction is essential for decreasing mortality associated with acute pulmonary embolism (PE), particularly for non-high-risk patients without hypotension on admission. This study aimed to develop a rapid and accurate tool for predicting the risk of RV dysfunction in non-high-risk patients with acute PE. METHODS: The medical records of non-high-risk patients with acute PE admitted to Shengjing Hospital of China Medical University between January 2011 and May 2020 were retrospectively analysed. The primary outcome of this study was RV dysfunction within 24 h after admission. The enrolled patients were randomized into training or validation sets as a ratio of 2:1. In the training set, a nomogram was developed, and the consistency was corroborated in the validation set. The areas under the receiver operating characteristic curves (AUCs) and 95% confidence intervals (CIs) were calculated. RESULTS: A total of 845 patients were enrolled, including 420 men and 425 women with an average age of 60.05 ± 15.43 years. Right ventricular dysfunction was identified in 240 patients (28.40%). The nomogram for RV dysfunction included N-terminal pro-brain natriuretic peptide, cardiac troponin I, and ventricular diameter ratios, which provided AUC values of 0.881 in the training dataset (95% confidence interval (CI): 0.868-0.898, p < 0.001) and 0.839 in the validation set (95% CI: 0.780-0.897, p < 0.001). The predictive tool was published as a web-based calculato ( https://gaoyzcmu.shinyapps.io/APERVD/ ). CONCLUSIONS: The combination of CT and laboratory parameters forms a predictive tool that may facilitate the identification of RV dysfunction in non-high-risk patients with acute PE.

Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase.

Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts.

Effects of the seedling tray overlapping for seed emergence mode on emergence characteristics and growth of rice seedlings.

Seedling mode plays a crucial role in the rice production process, as it significantly affects the growth and development of seedlings. Among the various seedling modes, the seedling tray overlapping for seed emergence mode (STOSE mode) has been demonstrated to be effective in enhancing seedling quality. However, the impact of this mode on the germination and growth of seeds with varying plumpness remains uncertain. To investigate the effect of the STOSE mode on seedling emergence characteristics, growth uniformity, and nutrient uptake of seeds with varying plumpness levels, we conducted a study using super early rice Zhongzao 39 (ZZ39) as the test material. The seeds were categorized into three groups: plumped, mixed, and unplumped. The results indicated that the STOSE mode significantly improved the seedling rate for all types of seeds in comparison to the seedling tray nonoverlapping for seed emergence mode (TSR mode). Notably, the unplumped seeds exhibited the most pronounced enhancement effect. The soluble sugar content of the seeds increased significantly after 2 days of sowing under the STOSE mode, whereas the starch content exhibited a significant decrease. Furthermore, the STOSE mode outperformed the TSR mode in several aspects including seedling growth uniformity, aboveground dry matter mass, root traits, and nutrient uptake. Overall, the STOSE mode not only promoted the germination and growth of plumped and mixed seeds but also had a more pronounced impact on unplumped seeds.

H3K27 tri-demethylase JMJD3 inhibits macrophage apoptosis by promoting ADORA2A in lipopolysaccharide-induced acute lung injury.

Acute lung injury (ALI) is a common critical disease, which is characterized by an uncontrolled, acute inflammatory response, diffuse lung damage and ultimately directly deteriorates into acute respiratory distress syndrome. The number of pro-inflammatory macrophages is related to the severity of ALI. Up-regulation of lipopolysaccharide (LPS)-activated macrophage apoptosis can reduce the pro-inflammatory reactions. Jumonji domain-containing protein D3 (JMJD3)-mediated histone 3 lysine 27 trimethylation (H3K27me3) demethylation may promote the pro-inflammatory response of macrophages under LPS stimulation. However, the mechanism of JMJD3 affecting macrophage apoptosis is still not clear. To explore this gap in knowledge, the ALI mice model with intratracheal administration of LPS and RAW264.7 cells with LPS stimulation were used as in vivo and in vitro experiments. The expression of JMJD3 and H3K27me3 and their cellular localization were analysed in lung tissue. Apoptosis was evaluated using TUNEL staining and flow cytometry. Expression of H3K27me3, ADORA2A and C/EBPß were compared among different treatments and chromatin immunoprecipitation was performed to investigate the regulatory relationship. Our study showed that JMJD3 expression was upregulated in LPS-induced ALI mice and RAW264.7 cells. JMJD3-indued H3K27me3 demethylation inhibited caspase-3 cleavage by upregulating ADORA2A to decrease LPS-stimulated macrophage apoptosis and promoted the inflammatory reaction. This H3K27me3 demethylation also increased C/EBPß expression, which may enhance ADORA2A expression further. Besides, inhibiting ADORA2A can also promote LPS-limited macrophage apoptosis. Moreover, the inhibition of JMJD3 in vivo and in vitro relieved the inhibition of macrophage apoptosis thus leading to the resolution of the inflammation. JMJD3 might inhibit macrophage apoptosis by promoting ADORA2A expression in LPS-induced ALI.

Rapid prediction of deterioration risk among non-high-risk patients with acute pulmonary embolism at admission: An imaging tool.

BACKGROUND: Computed tomography (CT) pulmonary angiography as the first-line diagnosis tool of acute pulmonary embolism (PE), might improve this discriminatory power. We aimed to developed a simply tool combining multi-CT parameters to complete individualized risk assessment of deterioration in non-high-risk patients with acute PE at admission. METHOD: Consecutive non-high-risk patients with acute PE who were treated in a Chinese center during 2010-2021, were collected.Prognosis-related CT parameters were reviewed. Deterioration was defined as any adverse event within 30 day after admission. Eligible patients were randomized into derivation and validation cohorts. In the derivation cohort, CT parameters were screened for importance using classification tree methodology and enrolled variables was partitioned via curve-fitting and dose-response analysis. A nomogram was developed and the predictive power in both cohorts was evaluated based on the area under the receiver operating characteristic curve (AUROC) and the corresponding 95% confidence interval (CI). RESULT: A total of 1001 patients were included. The preliminary analyses revealed that deterioration risk was related to the right-to-left ventricular diameter ratio at 4-chamber view, pulmonary vein filling abnormality. After a curve-fitting to deterioration risk, these parameters were partitioned and used to develop a nomogram, which had AUROC values of 0.91 (95% CI: 0.87-0.96) in the derivation cohort and 0.89 (95% CI: 0.81-0.97) in the validation cohort. A web-based version of the radiomics scoring tool was published online for use in clinical practice (https://acutepeprediction.shinyapps.io/Radiomics_Predictive_Tool/). CONCLUSION: This simply tool can complete rapid estimation of deterioration risk among non-high-risk acute PE patients at admission.

The incidence of brain metastases among patients with metastatic breast cancer: a systematic review and meta-analysis.

BACKGROUND: Patients with metastatic breast cancer (MBC) are living longer, but the development of brain metastases often limits their survival. We conducted a systematic review and meta-analysis to determine the incidence of brain metastases in this patient population. METHODS: Articles published from January 2000 to January 2020 were compiled from four databases using search terms related to breast cancer, brain metastasis, and incidence. The overall and per patient-year incidence of brain metastases were extracted from studies including patients with human epidermal growth factor receptor-2 positive (HER2+), triple negative, and hormone receptor (HR)+/hormone receptor negative (HER2-) MBC; pooled overall estimates for incidence were calculated using random effects models. RESULTS: 937 articles were compiled, and 25 were included in the meta-analysis. Incidence of brain metastases in patients with HER2+ MBC, triple negative MBC, and HR+/HER2- MBC was reported in 17, 6, and 4 studies, respectively. The pooled cumulative incidence of brain metastases was 31% for the HER2+ subgroup (median follow-up: 30.7 months, IQR: 24.0-34.0), 32% for the triple negative subgroup (median follow-up: 32.8 months, IQR: 18.5-40.6), and 15% among patients with HR+/HER2- MBC (median follow-up: 33.0 months, IQR: 31.9-36.2). The corresponding incidences per patient-year were 0.13 (95% CI: 0.10-0.16) for the HER2+ subgroup, 0.13 (95%CI: 0.09-0.20) for the triple negative subgroup, and only 0.05 (95%CI: 0.03-0.08) for patients with HR+/HER2- MBC. CONCLUSION: There is a high incidence of brain metastases among patients with HER2+ and triple negative MBC. The utility of a brain metastases screening program warrants investigation in these populations.

Immunogenicity, safety, and compliance of high- and standard-strength four-dose hepatitis B vaccination regimens in patients receiving methadone maintenance therapy in China: a randomized, parallel-arm controlled trial.

BACKGROUND: We evaluated the safety and immunogenicity of four doses of 20 or 60 µg, and the immunogenicity and compliance of the short-term vaccination regimen (0, 1, and 2 months) among patients receiving MMT. RESEARCH DESIGN AND METHODS: We conducted a randomized controlled trial among 303 patients receiving MMT who were randomized to receive 3 or 4 doses of 20 or 60 µg of recombinant hepatitis B vaccine. RESULTS: At month 7, the seroconversion rates in both IM20 × 4 and IM60 × 4 groups were numerically higher than the IM20 × 3 group (P > 0.05). The high-level responses and geometric mean concentration (GMC) of anti-HBs in both IM20 × 4 and IM60 × 4 groups were significantly higher than the IM20 × 3 group (P < 0.05). The completion rate of the short-term high-strength vaccination group was significantly higher than the standard vaccination group (P < 0.05), with similar immunogenicity (P > 0.05). CONCLUSIONS: Both the high-strength and standard-strength four-dose hepatitis B vaccine regimens could improve the immune response for patients receiving MMT. The high-strength short-term vaccination regimen could improve compliance and attain comparable immunogenicity with the standard vaccination regimen. The high-strength short-term vaccination regimen is recommended and the fourth dose is encouraged for this population considering the compliance and immunogenicity. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (NCT03962816).

Shallow periorbital injection of triamcinolone acetonide in treatment of lower eyelid entropion related to thyroid-associated ophthalmopathy: Three case reports.

INTRODUCTION: Entropion and secondary trichiasis can lead to irritative symptoms and essential damage of ocular surface. There is no literature reporting the lower eyelid entropion related to thyroid-associated ophthalmopathy (TAO), let alone the treatment. Treatment based on etiology may yield effective and sustained results. We report 3 case reports of lower eyelid entropion associated with TAO, and provide an effective and persistent alternative to cure this entropion via the administration of shallow periorbital injections of triamcinolone acetonide (TA). PATIENT CONCERNS: Three patients presented irritative symptoms of ocular surface and diplopia. DIAGNOSIS: According to thyroid dysfunction, physical examination, and imaging findings of extraocular muscle involvement, TAO and unilateral or bilateral lower eyelid entropion were diagnosed. INTERVENTIONS: We administered shallow periorbital injections of TA to the affected eye at 3- to 4-week intervals depending on clinical response. OUTCOMES: All patients underwent complete correction of the lower eyelid entropion and no recurrence was found. CONCLUSION: The cause of lower eyelid entropion related to TAO might be the immunoinflammatory reaction of the lower eyelid retractors, enhancing the traction of pulling the lower eyelid inferoposteriorly. This condition can be treated with shallow periorbital injections of TA. Histopathological evidence and randomized controlled trials are expected to confirm our hypothesis.

Cancer Patient-Reported Preferences and Knowledge for Liquid Biopsies and Blood Biomarkers at a Comprehensive Cancer Center.

BACKGROUND: Blood-based biomarkers (liquid biopsy) are increasingly used in precision oncology. Yet, little is known about cancer patients' perspectives in clinical practice. We explored patients' depth of preferences for liquid vs tissue biopsies and knowledge regarding the role of blood biomarkers on their cancer. METHODS: Three interviewer-administered trade-off scenarios and a 54-item self-administered questionnaire were completed by cancer outpatients across all disease sites at the Princess Margaret Cancer Centre. RESULTS: Of 413 patients, 54% were female; median age was 61 (range 18-101) years. In trade-off scenario preference testing, 90% (n=372) preferred liquid over tissue biopsy at baseline; when wait times for their preferred test were increased from 2 weeks, patients tolerated an additional mean of 1.8 weeks (SD 2.1) for liquid biopsy before switching to tissue biopsy (with wait time 2 weeks). Patients also tolerated a 6.2% decrease (SD 8.8) in the chance that their preferred test would conclusively determine optimal treatment before switching from the baseline of 80%. 216 patients (58%) preferred liquid biopsy even with no chance of adverse events from tissue biopsy. Patients' knowledge of blood-based biomarkers related to their cancer was low (mean 23%); however, the majority viewed development of blood biomarkers as important. CONCLUSION: Patients had limited understanding of cancer-specific blood-based biomarkers, but 90% preferred liquid over tissue biopsies to assess biomarkers. There was little tolerance to wait longer for results, or for decreased test-conclusiveness. Developing accurate, low-risk tests for cancer diagnosis and management for blood biomarkers is therefore desirable to patients.

A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis.

AIMS: Retention of low-density lipoprotein (LDL) cholesterol beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. Since the overlying endothelium is healthy and intact early on, it is likely that LDL passes through endothelial cells by transcytosis. However, technical challenges have made confirming this notion and elucidating the mechanisms of transcytosis difficult. We developed a novel assay for measuring LDL transcytosis in real time across coronary endothelial cell monolayers; we used this approach to identify the receptor involved. METHODS AND RESULTS: Murine aortas were perfused ex vivo with LDL and dextran of a smaller molecular radius. LDL (but not dextran) accumulated under the endothelium, indicating that LDL transcytosis occurs in intact vessels. We then confirmed that LDL transcytosis occurs in vitro using human coronary artery endothelial cells. An assay was developed to quantify transcytosis of DiI-LDL in real time using total internal reflection fluorescence microscopy. DiI-LDL transcytosis was inhibited by excess unlabelled LDL, while degradation of the LDL receptor by PCSK9 had no effect. Instead, LDL colocalized partially with the scavenger receptor SR-BI and overexpression of SR-BI increased LDL transcytosis; knockdown by siRNA significantly reduced it. Excess HDL, the canonical SR-BI ligand, significantly decreased LDL transcytosis. Aortas from SR-BI-deficient mice were perfused ex vivo with LDL and accumulated significantly less sub-endothelial LDL compared with wild-type littermates. CONCLUSION: We developed an assay to quantify LDL transcytosis across endothelial cells and discovered an unexpected role for SR-BI. Elucidating the mechanisms of LDL transcytosis may identify novel targets for the prevention or therapy of atherosclerosis.