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.

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.

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.

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.

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.

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.

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.

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.

TGF-ß sensitivity is determined by N-linked glycosylation of the type II TGF-ß receptor.

N-linked glycosylation is a critical determinant of protein structure and function, regulating processes such as protein folding, stability and localization, ligand-receptor binding and intracellular signalling. TßRII [type II TGF-ß (transforming growth factor ß) receptor] plays a crucial role in the TGF-ß signalling pathway. Although N-linked glycosylation of TßRII was first demonstrated over a decade ago, it was unclear how this modification influenced TßRII biology. In the present study, we show that inhibiting the N-linked glycosylation process successfully hinders binding of TGF-ß1 to TßRII and subsequently renders cells resistant to TGF-ß signalling. The lung cancer cell line A549, the gastric carcinoma cell line MKN1 and the immortal cell line HEK (human embryonic kidney)-293 exhibit reduced TGF-ß signalling when either treated with two inhibitors, including tunicamycin (a potent N-linked glycosylation inhibitor) and kifunensine [an inhibitor of ER (endoplasmic reticulum) and Golgi mannosidase I family members], or introduced with a non-glycosylated mutant version of TßRII. We demonstrate that defective N-linked glycosylation prevents TßRII proteins from being transported to the cell surface. Moreover, we clearly show that not only the complex type, but also a high-mannose type, of TßRII can be localized on the cell surface. Collectively, these findings demonstrate that N-linked glycosylation is essentially required for the successful cell surface transportation of TßRII, suggesting a novel mechanism by which the TGF-ß sensitivity can be regulated by N-linked glycosylation levels of TßRII.

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.

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.

Synaptic scaffolding molecule binds to and regulates vasoactive intestinal polypeptide type-1 receptor in epithelial cells.

BACKGROUND & AIMS: Vasoactive intestinal polypeptide (VIP) is a principal regulator of fluid and electrolyte secretion in the gastrointestinal system. The VIP type-1 receptor (VPAC1), a class II G-protein-coupled receptor, contains a putative C-terminal PDZ-binding motif. A yeast 2-hybrid screen indicated that the C-terminus of VPAC1 bound to the PDZ domain of synaptic scaffolding molecule (S-SCAM, also known as membrane-associated guanylate kinase inverted-2 [MAGI-2]). We analyzed the association between S-SCAM and VPAC1. METHODS: The biochemical properties and physiologic significance of the interaction between VPAC1 and S-SCAM were examined in heterologous expression systems, T84 colonic epithelial cells, and human pancreas and colon tissues using an integrated molecular and physiologic approach. RESULTS: The physical interaction between VPAC1 and S-SCAM was confirmed by immunoprecipitation in HEK 293 mammalian cells and human pancreatic and colonic tissues. Immunocytochemical analysis indicated that S-SCAM recruited VPAC1 to the junctional area near the apical end of the lateral membrane in T84 cells. Several lines of evidence revealed that S-SCAM inhibits VPAC1 activation. Overexpression of S-SCAM inhibited VPAC1-mediated cAMP production and agonist-induced VPAC1 internalization in HEK 293 and HeLa cells. In addition, S-SCAM decreased the VPAC1-mediated current through the cystic fibrosis transmembrane conductance regulator in Xenopus oocytes, especially at low concentrations of VIP. Importantly, loss of S-SCAM increased VIP-induced short-circuit currents in T84 monolayers, which endogenously express VPAC1 and S-SCAM. CONCLUSIONS: S-SCAM/MAGI-2 interacts with and regulates VPAC1 intracellular localization in epithelial cells and inhibits VPAC1 agonist-induced activation and internalization.

Defective Notch activation in microenvironment leads to myeloproliferative disease.

Despite the great importance of nonhematopoietic cells constituting the microenvironment for normal hematopoiesis, the cellular interactions between nonhematopoietic cells themselves are largely unknown. Using the Cre-loxP system in mice to inactivate Mind bomb-1 (Mib1), an essential component for Notch ligand endocytosis, here we show that the development of an MPD is dependent on defective Notch activation in the microenvironment. Our 2 independent Mib1 conditional knockout (CKO) mouse lines each developed a myeloproliferative disease (MPD), with gradual accumulations of immature granulocytes. The mutant mice showed hepatosplenomegaly, anemia, granulocytosis, and leukocyte infiltration in multiple organs and finally died at approximately 20 weeks of age. We were surprised to find that the transplantation of wild-type bone marrow cells into the Mib1-null microenvironment resulted in a de novo MPD. Moreover, by introducing the constitutively active intracellular domain of Notch1 in the Mib1-null background, we show that active Notch1 expression in the Mib1-null microenvironment significantly suppressed the disease progression, suggesting that the MPD development in the Mib1 CKO mice is due to defective Notch activation in the nonhematopoietic cells. These findings demonstrate that normal hematopoiesis absolutely requires Notch activation through the Notch ligand-receptor interaction between microenvironmental cells themselves and shed light on the microenvironment that fosters hematopoietic disorders.

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.

Receptor activator of NF-kappaB ligand regulates the proliferation of mammary epithelial cells via Id2.

Receptor activator of NF-kappaB ligand (RANKL) is a key regulator for mammary gland development during pregnancy. RANKL-deficient mice display impaired development of lobulo-alveolar mammary structures. Similar mammary gland defects have been reported in mice lacking Id2. Here we report that RANKL induces the proliferation of mammary epithelial cells via Id2. RANKL triggers marked nuclear translocation of Id2 in mammary epithelial cells. In vivo studies further demonstrated the defective nuclear translocation of Id2, but the normal expression of cyclin D1, in the mammary epithelial cells of rankl-/- mice. In vitro studies with nuclear localization sequence-tagged Id2 revealed that the nuclear localization of Id2 itself is critical for the downregulation of p21 promoter activity. Moreover, RANKL stimulation failed to induce cell growth and to downregulate p21 expression in Id2-/- mammary epithelial cells. Our results indicate that the inhibitor of helix-loop-helix protein, Id2, is critical to control the proliferation of mammary epithelial cells in response to RANKL stimulation.

Expression and subcellular localization of thymosin beta15 following kainic acid treatment in rat brain.

Thymosin beta15 (Tbeta15) is a pleiotropic factor which exerts multiple roles in the development of nervous system and brain diseases. In this study, we found that the expressions of Tbeta15 mRNA and protein were substantially increased in several brain regions including hippocampal formation and cerebral cortex, following kainic acid (KA)-evoked seizures in rat. Interestingly, a subset of cortex neurons exhibited nuclear Tbeta15 immunoreactivity upon KA treatment. Furthermore, translocation of Tbeta15 from cytosol to nuclei was observed in cultured neurons or HeLa cells during staurosporine (STS)-induced apoptosis, which was also verified by time-lapse imaging of YFP-tagged Tbeta15. It appeared that localization of Tbeta15 is restricted to the cytosol in normal condition by its G-actin-interacting domain, because site-directed mutagenesis of this region resulted in the nuclear localization of Tbeta15 in the absence of STS treatment. To explore the role of nuclear Tbeta15, we enforced Tbeta15 to localize in the nuclei by fusion of Tbeta15 with nuclear localization signal (NLS-Tbeta15). However, overexpression of NLS-Tbeta15 did not alter the viability of cells in response to STS treatment. Collectively, these results suggest that nuclear localization of Tbeta15 is a controlled process during KA or STS stimulation, although its functional significance is yet to be clarified.

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.