Integration of single-cell transcriptomes and biological function reveals distinct behavioral patterns in bone marrow endothelium.

Heterogeneity of endothelial cell (EC) populations reflects their diverse functions in maintaining tissue's homeostasis. However, their phenotypic, molecular, and functional properties are not entirely mapped. We use the Tie2-CreERT2;Rosa26-tdTomato reporter mouse to trace, profile, and cultivate primary ECs from different organs. As paradigm platform, we use this strategy to study bone marrow endothelial cells (BMECs). Single-cell mRNA sequencing of primary BMECs reveals that their diversity and native molecular signatures is transitorily preserved in an ex vivo culture that conserves key cell-to-cell microenvironment interactions. Macrophages sustain BMEC cellular diversity and expansion and preserve sinusoidal-like BMECs ex vivo. Endomucin expression discriminates BMECs in populations exhibiting mutually exclusive properties and distinct sinusoidal/arterial and tip/stalk signatures. In contrast to arterial-like, sinusoidal-like BMECs are short-lived, form 2D-networks, contribute to in vivo angiogenesis, and support hematopoietic stem/progenitor cells in vitro. This platform can be extended to other organs' ECs to decode mechanistic information and explore therapeutics.

Effect of wrinkles on extreme ultraviolet pellicle reflectivity and local critical dimension.

Extreme ultraviolet (EUV) pellicles must have an EUV reflectance (EUVR) below 0.04% to prevent the reduction of critical dimension (CD). However, pellicle wrinkles cause localized CD variation by locally amplifying the EUVR. This study demonstrates that wrinkles can increase the pellicle's EUVR by approximately four times, and the CD drop depends on the relative position of the reflected light from the wrinkle to the 0th- or 1st-order diffracted light. The CD decreases by 6 nm. Therefore, even if the pellicle satisfies the requirement for the EUVR, we need to tightly control the generation of wrinkles to suppress CD variation during the entire exposure process.

The Transmembrane Adaptor Protein LIME Is Essential for Chemokine-Mediated Migration of Effector T Cells to Inflammatiory Sites.

Lck-interacting transmembrane adaptor 1 (LIME) has been previously identified as a raft-associated transmembrane protein expressed predominantly in T and B lymphocytes. Although LIME is shown to transduce the immunoreceptor signaling and immunological synapse formation via its tyrosine phosphorylation by Lck, a Src-family kinase, the in vivo function of LIME has remained elusive in the previous studies. Here we report that LIME is preferentially expressed in effector T cells and mediates chemokine-mediated T cell migration. Interestingly, in LIME-/- mice, while T cell receptor stimulation-dependent proliferation, differentiation to effector T cells, cytotoxic T lymphocyte (CTL) function and regulatory T lymphocyte (Treg) function were normal, only T cell-mediated inflammatory response was significantly defective. The reduced inflammation was accompanied by the impaired infiltration of leukocytes and T cells to the inflammatory sites of LIME-/- mice. More specifically, the absence of LIME in effector T cells resulted in the reduced migration and defective morphological polarization in response to inflammatory chemokines such as CCL5 and CXCL10. Consistently, LIME-/- effector T cells were found to be defective in chemokine-mediated activation of Rac1 and Rap1, and dysregulated phosphorylation of Pyk2 and Cas. Taken together, the present findings show that LIME is a critical regulator of inflammatory chemokine-mediated signaling and the subsequent migration of effector T cells to inflammatory sites.

Performance of Extreme Ultraviolet Coherent Scattering Microscope.

For the successful implementation of extreme ultraviolet lithography (EUVL) into high-volume manufacturing, the development of a novel structure mask for resolution improvement is essential. In this paper, coherent scattering microscopy (CSM) is introduced as an actinic metrology technique based on coherent diffractive imaging (CDI) for EUV mask development. CDI reconstructs the mask image using diffraction patterns from the mask through mathematical calculations. CSM can analyze details of an EUV mask such as its diffraction efficiency and phase information.

BAF53A regulates androgen receptor-mediated gene expression and proliferation in LNCaP cells.

The actin-like protein of the SWI/SNF complex, BAF53A, regulates gene expression by the gene-specific chromatin remodeling of target genes. However, the function of BAF53A in the androgen receptor pathway in prostate cancer cells remains unclear. Here, we demonstrated that BAF53A positively regulates the expression of endogenous AR target genes (e.g. PSA, TMPRSS2, FKBP5, and KLK2) in LNCaP cells. It functions as a coactivator in AR-mediated transcription by interacting with other nuclear receptor coactivators, such as p300 and FLII, and is associated with AR in the presence of dihydrotestosterone (DHT). The DHT-induced recruitment of BAF53A to the proximal and distal androgen response elements (AREs) of the PSA gene in the presence of BRG1 (but not BRM) was inhibited by an AR antagonist, suggesting the coactivator function of BAF53A in the SWI/SNF complex. Depletion of BAF53A in LNCaP cells resulted in a significant decrease in growth rate. Furthermore, the expression of BAF53A in prostate cancer tissue was significantly elevated, compared to that in normal prostate tissue, and correlated with the expression of AR, and BRG1, but not BRM. Therefore, our results suggested that BAF53A plays an important role in the expression of AR target genes in prostate cancer, and can be used clinically for the treatment of prostate cancer.

Aberrant expression of SETD1A promotes survival and migration of estrogen receptor α-positive breast cancer cells.

The histone H3 lysine 4-specific methyltransferase SETD1A is associated with transcription activation and is considered a key epigenetic regulator that modulates the cell cycle and metastasis in triple-negative breast cancer cells. However, the clinical role of SETD1A in estrogen receptor (ER)-positive breast cancer cells remains unclear. Here, we examined whether SETD1A is a potential target for ERα-positive breast cancer therapy. SETD1A expression was upregulated in breast tumor tissue compared to that in normal breast tissue. Moreover, ER-target genes regulated by SETD1A were particularly enriched in cell cycle and cancer pathways. SETD1A is involved in histone H3K4 methylation, subsequent recruitment of ERα, and the establishment of accessible chromatin structure at the enhancer region of ERα target genes. In addition to ERα target genes, other cell survival genes were also downregulated by SETD1A depletion in MCF-7 cells, leading to significant decrease in cell proliferation and migration, and spontaneous induction of apoptosis. We also found that miR-1915-3p functioned as a novel regulator of SETD1A expression in breast cells. Importantly, the growth of tamoxifen-resistant MCF-7 cells was effectively repressed by SETD1A knockdown. These results indicate that SETD1A may serve as a molecular target and prognostic indicator in ERα-positive breast cancer.

Erdheim-Chester Disease Presenting as an Anterior Mediastinal Tumor without Skeletal Involvement.

Erdheim-Chester disease (ECD) is a form of non-Langerhans cell histiocytosis that most commonly involves the skeletal system. We report an unusual case of ECD presenting as an anterior mediastinal tumor without skeletal involvement. A 60-year-old man with no remarkable medical history was referred for evaluation of a mediastinal mass. The patient underwent surgical excision of the tumor via video-assisted thoracoscopic surgery. Histologic examination revealed marked proliferation of atypical histiocytes with sclerosis, and the results of immunohistochemical staining were suggestive of ECD.

The Treatment of Left Atrial Appendage Aneurysm by a Minimally Invasive Approach.

Left atrial appendage (LAA) aneurysm is a rare, pathologic condition that may lead to atrial tachyarrhythmia or thromboembolic events. A 49-year-old man presented with aggravated palpitation and dizziness. He suffered from refractory atrial fibrillation despite a previous history of radiofrequency catheter ablation. Echocardiography revealed a 57-mm LAA aneurysm. Surgical ablation was performed through a right mini-thoracotomy, and the LAA aneurysm was obliterated with a 50-mm AtriClip (Atricure Inc., Westchester, OH, USA). However, follow-up computed tomography showed residual communication, so the patient is still taking warfarin. We report that a minimally invasive strategy for treating LAA aneurysm can be considered, but incomplete closure may occur; thus, caution is needed.

Outcomes of Reoperative Valve Replacement in Patients with Prosthetic Valve Endocarditis: A 20-Year Experience.

BACKGROUND: Prosthetic valve endocarditis (PVE) is a serious complication of cardiac valve replacement, and many patients with PVE require reoperation. The aim of this study was to review our institutional 20-year experience of surgical reoperative valve replacement in patients with PVE. METHODS: A retrospective study was performed on 84 patients (mean age, 54.8±12.7 years; 51 males) who were diagnosed with PVE and underwent reoperative valve replacement from January 1995 to December 2016. RESULTS: PVE was found in 1 valve in 61 cases (72.6%), and in 2 or more valves in 23 cases (27.4%). The median follow-up duration was 47.3 months (range, 0 to 250 months). Postoperative complications occurred in 39 patients (46.4%). Reinfection occurred in 6 cases, all within 1 year. The freedom from reinfection rate at 5 years was 91.0%±3.5%. The overall survival rates at 5 and 10 years were 64.4%±5.8% and 54.3%±7.3%, respectively. In stepwise multivariable Cox proportional hazard models, older age (hazard ratio [HR], 1.48; 95% confidence interval [CI], 1.05 to 2.10; p=0.027) and cardiopulmonary bypass (CPB) time (HR, 1.03; 95% CI, 1.00 to 1.01; p=0.033) emerged as independent risk factors for death. CONCLUSION: Older age and a longer CPB time were associated with an increased risk of overall mortality in PVE patients.

Novel Blood-based Transcriptional Biomarker Panels Predict the Late-Phase Asthmatic Response.

RATIONALE: The allergen inhalation challenge is used in clinical trials to test the efficacy of new treatments in attenuating the late-phase asthmatic response (LAR) and associated airway inflammation in subjects with allergic asthma. However, not all subjects with allergic asthma develop the LAR after allergen inhalation. Blood-based transcriptional biomarkers that can identify such individuals may help in subject recruitment for clinical trials as well as provide novel molecular insights. OBJECTIVES: To identify blood-based transcriptional biomarker panels that can predict an individual's response to allergen inhalation challenge. METHODS: We applied RNA sequencing to total RNA from whole blood (n = 36) collected before and after allergen challenge and generated both genome-guided and de novo datasets: genes, gene-isoforms (University of California, Santa Cruz, UCSC Genome Browser), Ensembl, and Trinity. Candidate biomarker panels were validated using the NanoString platform in an independent cohort of 33 subjects. MEASUREMENTS AND MAIN RESULTS: The Trinity biomarker panel consisting of known and novel biomarker transcripts had an area under the receiver operating characteristic curve of greater than 0.70 in both the discovery and validation cohorts. The Trinity biomarker panel was useful in predicting the response of subjects that elicited different responses (accuracy between 0.65 and 0.71) and subjects that elicit a dual response (accuracy between 0.70 and 0.75) upon repeated allergen inhalation challenges. CONCLUSIONS: Interestingly, the biomarker panel containing novel transcripts successfully validated compared with panels with known, well-characterized genes. These biomarker-blood tests may be used to identify subjects with asthma who develop the LAR, and may also represent members of novel molecular mechanisms that can be targeted for therapy.

Investigating Immune Gene Signatures in Peripheral Blood from Subjects with Allergic Rhinitis Undergoing Nasal Allergen Challenge.

Nasal allergen challenge (NAC) is a human model of allergic rhinitis (AR) that delivers standardized allergens locally to the nasal mucosa allowing clinical symptoms and biospecimens such as peripheral blood to be collected. Although many studies have focused on local inflammatory sites, peripheral blood, an important mediator and a component of the systemic immune response, has not been well studied in the setting of AR. We sought to investigate immune gene signatures in peripheral blood collected after NAC under the setting of AR. Clinical symptoms and peripheral blood samples from AR subjects were collected during NAC. Fuzzy c-means clustering method was used to identify immune gene expression patterns in blood over time points (before NAC and 1, 2, and 6 h after NAC). We identified and validated seven clusters of differentially expressed immune genes after NAC onset. Clusters 2, 3, and 4 were associated with neutrophil and lymphocyte frequencies and neutrophil/lymphocyte ratio after the allergen challenge. The patterns of the clusters and immune cell frequencies were associated with the clinical symptoms of the AR subjects and were significantly different from healthy nonallergic subjects who had also undergone NAC. Our approach identified dynamic signatures of immune gene expression in blood as a systemic immune response associated with clinical symptoms after NAC. The immune gene signatures may allow cross-sectional investigation of the pathophysiology of AR and may also be useful as a potential objective measurement for diagnosis and treatment of AR combined with the NAC model.

Identifying Molecular Mechanisms of the Late-Phase Asthmatic Response by Integrating Cellular, Gene, and Metabolite Levels in Blood.

RATIONALE: Individuals with allergic asthma respond differently, but reproducibly, to allergen inhalation challenge. Some individuals develop an isolated early response (early responders) (ERs), whereas others go on to develop a late response (dual responders) (DRs). It is not understood why late responses do not develop in all sensitized individuals. OBJECTIVES: The aim of this study was to identify blood biomarkers that can discriminate ERs and DRs using cellular frequencies and gene and metabolite expression from whole blood. METHODS: Thirty-two individuals participated in the allergen inhalation challenge as part of the AllerGen Clinical Investigator Collaborative. Fifteen participants were classified as ERs and 17 as DRs. Blood samples were collected before (pre) and 2 hours after (post) the allergen challenge. Cell counts were obtained using a hematolyzer, gene transcript relative levels using RNA sequencing, and metabolite concentrations using tandem mass spectrometry. An integrative ensemble algorithm that was based on canonical correlation analysis was used to classify ERs and DRs using all three data sets, adjusting for age and sex. The objective of this algorithm was to identify a correlated subset of molecules from each data set that best discriminated ERs from DRs. Gene set enrichment analysis was performed using Enrichr (Chen et al., BMC Bioinformatics 2013;128). MEASUREMENTS AND MAIN RESULTS: The pre-challenge multisignature classifier (error = 30%) outperformed the post-challenge multisignature classifier (error = 50%) in separating ERs from DRs. The cells selected in the prechallenge multisignature panel included eosinophils, lymphocytes, and neutrophils. The selected metabolites were enriched for glycerophospholipids. The subset of gene transcripts in the multisignature panel was enriched for the T-cell receptor and costimulatory signaling pathway (P = 3.4 × 10(-6)) (Wikipathways) and positive regulation of antigen receptor-mediated signaling pathway (P = 5.7 × 10(-4)) (GO Ontology). CONCLUSIONS: This study provides a systems perspective on the deregulated molecular processes between early and dual responses in whole blood. The integrative biomarker analysis suggests that a molecular signature that is predictive of the late-phase response can be identified. The variability in the onset of the late response may explain the poor predictive performance of the postchallenge multiomic biomarker signature. Replication of the prechallenge biomarker signature in additional independent samples is required to validate this panel.

Stability and function of adult vasculature is sustained by Akt/Jagged1 signalling axis in endothelium.

The signalling pathways operational in quiescent, post-development vasculature remain enigmatic. Here we show that unlike neovascularization, endothelial Akt signalling in established vasculature is crucial not for endothelial cell (EC) survival, but for sustained interactions with pericytes and vascular smooth muscle cells (VSMCs) regulating vascular stability and function. Inducible endothelial-specific Akt1 deletion in adult global Akt2KO mice triggers progressive VSMC apoptosis. In hearts, this causes a loss of arteries and arterioles and, despite a high capillary density, diminished vascular patency and severe cardiac dysfunction. Similarly, endothelial Akt deletion induces retinal VSMC loss and basement membrane deterioration resulting in vascular regression and retinal atrophy. Mechanistically, the Akt/mTOR axis controls endothelial Jagged1 expression and, thereby, Notch signalling regulating VSMC maintenance. Jagged1 peptide treatment of Akt1ΔEC;Akt2KO mice and Jagged1 re-expression in Akt-deficient endothelium restores VSMC coverage. Thus, sustained endothelial Akt1/2 signalling is critical in maintaining vascular stability and homeostasis, thereby preserving tissue and organ function.

Receptor-Mediated Mechanism Controlling Tissue Levels of Bioactive Lipid Oxidation Products.

RATIONALE: Oxidative stress is an important contributing factor in several human pathologies ranging from atherosclerosis to cancer progression; however, the mechanisms underlying tissue protection from oxidation products are poorly understood. Oxidation of membrane phospholipids, containing the polyunsaturated fatty acid docosahexaenoic acid, results in the accumulation of an end product, 2-(ω-carboxyethyl)pyrrole (CEP), which was shown to have proangiogenic and proinflammatory functions. Although CEP is continuously accumulated during chronic processes, such as tumor progression and atherosclerosis, its level during wound healing return to normal when the wound is healed, suggesting the existence of a specific clearance mechanism. OBJECTIVE: To identify the cellular and molecular mechanism for CEP clearance. METHODS AND RESULTS: Here, we show that macrophages are able to bind, scavenge, and metabolize carboxyethylpyrrole derivatives of proteins but not structurally similar ethylpyrrole derivatives, demonstrating the high specificity of the process. F4/80(hi) and M2-skewed macrophages are much more efficient at CEP binding and scavenging compared with F4/80(lo) and M1-skewed macrophages. Depletion of macrophages leads to increased CEP accumulation in vivo. CEP binding and clearance are dependent on 2 receptors expressed by macrophages, CD36 and toll-like receptor 2. Although knockout of each individual receptor results in diminished CEP clearance, the lack of both receptors almost completely abrogates macrophages' ability to scavenge CEP derivatives of proteins. CONCLUSIONS: Our study demonstrates the mechanisms of recognition, scavenging, and clearance of pathophysiologically active products of lipid oxidation in vivo, thereby contributing to tissue protection against products of oxidative stress.

Management of recurrent paravalvular leakage in a very high-risk patient: a case report.

Interventional device closure has emerged as a less invasive alternative to surgery in the management of paravalvular leakage. However, this procedure involves various problems such as a high probability of residual leakage or hemolysis. Here, we report a case of residual paravalvular leakage despite two attempts at interventional closure in a patient with a history of four previous mitral valve replacements. The fifth operation for the primary repair of paravalvular leakage was performed successfully. Careful evaluation before the procedure and specially designed devices are essential for the interventional treatment of paravalvular leakage. Surgery can be performed adequately in the management of paravalvular leakage even in high-risk patients.

Oxidative stress in angiogenesis and vascular disease.

Despite the damaging effect on tissues at a high concentration, it has been gradually established that oxidative stress plays a positive role during angiogenesis. In adults, physiological or pathological angiogenesis is initiated by tissue demands for oxygen and nutrients, resulting in a hypoxia/reoxygenation cycle, which, in turn promotes the formation of reactive oxygen species (ROS). The ROS can be generated either endogenously, through mitochondrial electron transport chain reactions and nicotinamide adenine dinucleotide phosphate oxidase, or exogenously, resulting from exposure to environmental agents, such as ultraviolet or ionizing radiation. In many conditions, ROS promotes angiogenesis, either directly or via the generation of active oxidation products, including peroxidized lipids. The latter lipid metabolites are generated in excess during atherosclerosis, thereby linking atherogenic processes and pathological angiogenesis. Although the main mechanism of oxidative stress-induced angiogenesis involves hypoxia-inducible factor/vascular endothelial growth factor (VEGF) signaling, recent studies have identified several pathways that are VEGF-independent. This review aims to provide a summary of the past and present views on the role of oxidative stress as a mediator and modulator of angiogenesis, and to highlight newly identified mechanisms.

Glucosamine hydrochloride exerts a protective effect against unilateral ureteral obstruction-induced renal fibrosis by attenuating TGF-ß signaling.

UNLABELLED: Renal fibrosis is a common consequence of unilateral ureteral obstruction, which provides a useful model to investigate the pathogenesis of obstructive nephropathy and progressive renal fibrosis. Transforming growth factor (TGF-ß1) has been recognized as a key mediator in renal fibrosis by stimulating matrix-producing fibrogenic cells and promoting extracellular matrix deposition. Therefore, considerable efforts have been made to regulate TGF-ß signaling for antifibrotic therapy. Here, we investigated the mode of action of glucosamine hydrochloride (GS-HCl) on TGF-ß1-induced renal fibrosis. In the obstructed kidneys and TGF-ß1-treated renal cells, GS-HCl significantly decreased renal expression of α-smooth muscle actin, collagen I, and fibronectin. By investigating the inhibitory mechanism of GS-HCl on renal fibrosis, we found that GS-HCl suppressed TGF-ß signaling by inhibiting N-linked glycosylation of the type II TGF-ß receptor (TßRII), leading to an inefficient trafficking of TßRII to the membrane surface. Defective N-glycosylation of TßRII further suppressed the TGF-ß1-binding to TßRII, thereby decreasing TGF-ß signaling. Notably, GS-HCl treatment significantly reduced TGF-ß1-induced up-regulation of Smad2/3 phosphorylation and transcriptional activity in vivo and in vitro. Taken together, GS-HCl-mediated regulation of TGF-ß signaling exerted an antifibrotic effect, thereby ameliorating renal fibrosis. Our study suggests that GS-HCl would be a promising agent for therapeutic intervention for preventing TGF-ß1-induced renal fibrosis in kidney diseases. KEY MESSAGE: Glucosamine-mediated attenuation of TGF-ß signaling ameliorates renal fibrosis in vivo TGF-ß1-induced fibrogenic action is reduced by glucosamine in vitro N-glycosylation of the type II TGF-ß receptor is suppressed by glucosamine Glucosamine-induced defective N-glycosylation of TßRII decreases TGF-ß signaling.

Patterns of isotope uptake in sequential postoperative bone scan in undisplaced femoral-neck fractures.

PURPOSE: We aimed to investigate the changing pattern of isotope uptake in the sequential bone scan test for the prediction of osteonecrosis of the femoral head in patients with an undisplaced femoral-neck fracture. METHODS: Fifty-four cases of sequential bone scan for nondisplaced femoral-neck fracture treated by internal fixation with cannulated screws between 2000 and 2009 were retrospectively studied. The mean follow-up period was 4.2 years. The first postoperative bone scan was performed two weeks postoperatively in all patients. Second, third, and fourth follow-up bone scans were performed at one to six months, 12-18 months, and 18-24 months postoperatively. RESULTS: Mean femoral-head ratio (FHR) in the first postoperative bone scan was 0.99. Although it was under 1.0 in 38 patients (70.4 % of the 54 patients), only one patient developed osteonecrosis of the femoral head. The others showed hot uptake in their second follow-up bone scan. Mean FHRs in the second, third, and fourth postoperative bone scans were 1.69, 1.29, and 1.05, respectively, and there were significant statistical differences in each follow-up period (P = 0.035). In addition, there were unique patterns of isotope uptake with the passage of time, such as cold uptake in the early stage, hot uptake in a couple of months, and iso-uptake in the late stage. CONCLUSIONS: Early postoperative bone scan results should not be over interpreted when predicting osteonecrosis of the femoral head.