Search

Cholesterol is ubiquitous in cells; it plays a critical role in membrane structure and transport as well as in intracellular trafficking processes. There are suggestions that cholesterol metabolism is linked to innate immunity with inhibitors of DHCR7, the last enzyme in the cholesterol pathway, suggested to have potential as viral therapeutics nearly a decade ago. In fact, there are a number of highly prescribed pharmaceuticals that are off-target inhibitors of DHCR7, causing increased cellular levels of 7-dehydrodesmosterol (7-DHD) and 7-dehydrocholesterol (7-DHC). We report here dose-response studies of six such inhibitors on late-stage cholesterol biosynthesis in Neuro2a cells as well as their effect on infection of vesicular stomatitis virus (VSV). Four of the test compounds are FDA-approved drugs (cariprazine, trazodone, metoprolol, and tamoxifen), one (ifenprodil) has been the object of a recent Phase 2b COVID trial, and one (AY9944) is an experimental compound that has seen extensive use as a DHCR7 inhibitor. The three FDA-approved drugs inhibit replication of a GFP-tagged VSV with efficacies that mirror their effect on DHCR7. Ifenprodil and AY9944 have complex inhibitory profiles, acting on both DHCR7 and DHCR14, while tamoxifen does not inhibit DHCR7 and is toxic to Neuro2a at concentrations where it inhibits the Δ7-Δ8 isomerase of the cholesterol pathway. VSV itself affects the sterol profile in Neuro2a cells, showing a dose-response increase of dehydrolathosterol and lathosterol, the substrates for DHCR7, with a corresponding decrease in desmosterol and cholesterol. 7-DHD and 7-DHC are orders of magnitude more vulnerable to free radical chain oxidation than other sterols as well as polyunsaturated fatty esters, and the effect of these sterols on viral infection is likely a reflection of this fact of Nature.

Chemotherapy can precondition for immunotherapy by creating an environment for homeostatic lymphoproliferation and eliminating some of the suppressive immune networks. We found that combination therapy with anti-4-1BB and cyclophosphamide (CTX) produced synergistic anticancer effects in the poorly immunogenic B16 melanoma model in mice. The antitumor effect of the combination therapy depended mainly on CD8+ T cells, the 4-1BB–dependent expansion and differentiation of which into IFN-γ–producing CD11c+CD8+ T cells was enhanced by CTX. Anti-4-1BB induced a rapid repopulation of T and B cells from CTX-mediated lymphopenia. Anti-4-1BB protected naïve T cells from CTX and promoted proliferation of memory/effector and memory T cells. The combination treatment produced ∼60- and 2.2-fold more CTLs per tumor-associated antigen compared with CTX or anti-4-1BB alone, respectively. This indicates that anti-4-1BB promoted a preferential expansion of tumor-specific CD8+ T cells among the repopulated lymphocytes following CTX-mediated lymphopenia. CTX treatment enhanced 4-1BB expression on CD4 and CD8 T cells, and CTX alone or in combination with anti-4-1BB effectively suppressed peripheral regulatory T cells. Our results indicate that anti-4-1BB and CTX can be practical partners in cancer therapy because CTX creates an environment in which anti-4-1BB actively promotes the differentiation and expansion of tumor-specific CTLs. [Mol Cancer Ther 2009;8(2):469–78

Supplementary Fig. from Mechanisms involved in synergistic anticancer effects of anti-4-1BB and cyclophosphamide therapy

Supplementary Figures 1-6 from Mechanisms Involved in Synergistic Anticancer Immunity of Anti-4-1BB and Anti-CD4 Therapy

Supplementary Figures 1-9 from Combination Therapy with Cisplatin and Anti–4-1BB: Synergistic Anticancer Effects and Amelioration of Cisplatin-Induced Nephrotoxicity

Anti-4-1BB–mediated anticancer effects were potentiated by depletion of CD4+ cells in B16F10 melanoma-bearing C57BL/6 mice. Anti-4-1BB induced the expansion and differentiation of polyclonal tumor-specific CD8+ T cells into IFN-γ–producing CD11c+CD8+ T cells. The CD4+ cell depletion was responsible for facilitating immune cell infiltration into tumor tissues and removing some regulatory barriers such as T regulatory and indoleamine-2,3-dioxygenase (IDO)+ dendritic cells. Both monoclonal antibodies (mAb) contributed to the efficient induction of MHC class I molecules on the tumor cells in vivo. The effectors that mediated the anti-4-1BB effect were NKG2D+KLRG1+CD11c+CD8+ T cells that accumulated preferentially in the tumor tissues. Blocking NKG2D reduced the therapeutic effect by 20% to 26%, which may indicate that NKG2D contributes partially to tumor killing by the differentiated CD8+ T cells. Our results indicate that the combination of the two mAbs, agonistic anti-4-1BB and depleting anti-CD4, results in enhanced production of efficient tumor-killing CTLs, facilitation of their infiltration, and production of a susceptible tumor microenvironment. [Cancer Res 2007;67(18):8891–9]

Supplementary Figures S1-S3 from A Gene Expression Signature of Genetic Instability in Colon Cancer

Steroid receptor coactivator-3 (SRC-3) is a histone acetyltransferase and nuclear hormone receptor coactivator, located on 20q12, which is amplified in several epithelial cancers and well studied in breast cancer. However, its possible role in lung cancer pathogenesis is unknown. We found SRC-3 to be overexpressed in 27% of non–small cell lung cancer (NSCLC) patients (n = 311) by immunohistochemistry, which correlated with poor disease-free (P = 0.0015) and overall (P = 0.0008) survival. Twenty-seven percent of NSCLCs exhibited SRC-3 gene amplification, and we found that lung cancer cell lines expressed higher levels of SRC-3 than did immortalized human bronchial epithelial cells (HBEC), which in turn expressed higher levels of SRC-3 than did cultured primary human HBECs. Small interfering RNA–mediated downregulation of SRC-3 in high-expressing, but not in low-expressing, lung cancer cells significantly inhibited tumor cell growth and induced apoptosis. Finally, we found that SRC-3 expression is inversely correlated with gefitinib sensitivity and that SRC-3 knockdown results in epidermal growth factor receptor tyrosine kinase inhibitor–resistant lung cancers becoming more sensitive to gefitinib. Taken together, these data suggest that SRC-3 may be an important oncogene and therapeutic target for lung cancer. Cancer Res; 70(16); 6477–85. ©2010 AACR.

Supplementary Figures 1-3, Tables 1-3 from Steroid Receptor Coactivator-3 Expression in Lung Cancer and Its Role in the Regulation of Cancer Cell Survival and Proliferation

Genetic instability plays a central role in the development and progression of human cancer. Two major classes of genetic instability, microsatellite instability (MSI) and chromosome instability (microsatellite stable; MSS), are best understood in the context of colon cancer, where MSI tumors represent ∼15% of cases, and compared with MSS tumors, more often arise in the proximal colon and display favorable clinical outcome. To further explore molecular differences, we profiled gene expression in a set of 18 colon cancer cell lines using cDNA microarrays representing ∼21,000 different genes. Supervised analysis identified a robust expression signature distinguishing MSI and MSS samples. As few as eight genes predicted with high accuracy the underlying genetic instability in the original and in three independent sample sets, comprising 13 colon cancer cell lines, 61 colorectal tumors, and 87 gastric tumors. Notably, the MSI signature was retained despite genetically correcting the underlying instability, suggesting the signature reflects a legacy of the tumor having arisen from MSI, rather than sensing the ongoing state of MSI. Our findings support a model in which MSI and MSS preferentially target different genes and pathways in cancer. Further, among the MSI signature genes, our findings implicate a role of elevated metallothionein expression in the clinical behavior of MSI cancers.

Supplementary Tables S1-S6 from A Gene Expression Signature of Genetic Instability in Colon Cancer

In order to estimate the crack depth in concrete using time-of-flight, finite element analysis and experiments were performed on non-cracked concrete blocks and 45 mm and 70 mm vertical cracks. As a result of measuring the time-of-flight change by changing the positions of the transmitter and receiver, it was confirmed that the finite element analysis results agreed with the experimental results, and high accuracy was confirmed by various formulas for calculating the depth of defects using the obtained experimental measurements for comparison. In addition to the verification of the simulation and experimental theory, research was conducted through actual field cases, and methodologies for crack detection and depth evaluation for concrete structures were presented, and furthermore, the expected effects of improving the soundness and safety of structures were shown.

Severe asthma is characterized by steroid insensitivity and poor symptom control and is responsible for most asthma-related hospital costs. Therapeutic options remain limited, in part due to limited understanding of mechanisms driving severe asthma. Increased arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is increased in human asthmatic lungs. In this study, we show that PRMT5 drives allergic airway inflammation in a mouse model reproducing multiple aspects of human severe asthma. We find that PRMT5 is required in CD4+ T cells for chronic steroid-insensitive severe lung inflammation, with selective T cell deletion of PRMT5 robustly suppressing eosinophilic and neutrophilic lung inflammation, pathology, airway remodeling, and hyperresponsiveness. Mechanistically, we observed high pulmonary sterol metabolic activity, retinoic acid-related orphan receptor γt (RORγt), and Th17 responses, with PRMT5-dependent increases in RORγt’s agonist desmosterol. Our work demonstrates that T cell PRMT5 drives severe allergic lung inflammation and has potential implications for the pathogenesis and therapeutic targeting of severe asthma.

Background Ozone (O3) exposure causes respiratory effects including lung function decrements, increased lung permeability, and airway inflammation. Additionally, baseline metabolic state can predispose individuals to adverse health effects from O3. For this reason, we conducted an exploratory study to examine the effect of O3 exposure on derivatives of cholesterol biosynthesis: sterols, oxysterols, and secosteroid (25-hydroxyvitamin D) not only in the lung, but also in circulation. Methods We obtained plasma and induced sputum samples from non-asthmatic (n = 12) and asthmatic (n = 12) adult volunteers 6 hours following exposure to 0.4ppm O3 for 2 hours. We quantified the concentrations of 24 cholesterol precursors and derivatives by UPLC-MS and 30 cytokines by ELISA. We use computational analyses including machine learning to determine whether baseline plasma sterols are predictive of O3 responsiveness. Results We observed an overall decrease in the concentration of cholesterol precursors and derivatives (e.g. 27-hydroxycholesterol) and an increase in concentration of autooxidation products (e.g. secosterol-B) in sputum samples. In plasma, we saw a significant increase in the concentration of secosterol-B after O3 exposure. Machine learning algorithms showed that plasma cholesterol was a top predictor of O3 responder status based on decrease in FEV1 (>5%). Further, 25-hydroxyvitamin D was positively associated with lung function in non-asthmatic subjects and with sputum uteroglobin, whereas it was inversely associated with sputum myeloperoxidase and neutrophil counts. Conclusion This study highlights alterations in sterol metabolites in the airway and circulation as potential contributors to systemic health outcomes and predictors of pulmonary and inflammatory responsiveness following O3 exposure.

Coatings play a crucial role in protecting ships and marine structures from corrosion and extending their service life. The reliability of these coatings depends on their proper maintenance, which in turn, relies on the application of reliable diagnostic techniques. Non-destructive testing (NDT) techniques are useful in material diagnostics, such as detecting debonded zone in water. However, the challenging access environment in the ocean, and the high attenuation characteristics of the material itself add too many technical challenges. In this paper, we propose a guided wave-based technique for characterizing the bonded zone state of coatings, which uses FFT analysis in different bonded zone states. The proposed technique has been demonstrated to be effective in characterizing the bonded zone state of water coatings through numerical and experimental results.

Mounting evidence has shown that CETP has important physiological roles in adapting to chronic nutrient excess, specifically, to protect against diet-induced insulin resistance. However, the underlying mechanisms for the protective roles of CETP in metabolism are not yet clear. Mice naturally lack CETP expression. We used transgenic mice with a human CETP minigene (huCETP) controlled by its natural flanking region to further understand CETP-related physiology in response to obesity. Female huCETP mice and their wild-type littermates were fed a high-fat diet for 6 months. Blood lipid profile and liver lipid metabolism were studied. Insulin sensitivity was analyzed with euglycemic-hyperinsulinemic clamp studies combined with 3H-glucose tracer techniques. While high-fat diet feeding induced obesity for huCETP mice and their wild-type littermates lacking CETP expression, insulin sensitivity was higher for female huCETP mice than for their wild-type littermates. There was no difference in insulin sensitivity for male huCETP mice vs. littermates. The increased insulin sensitivity in females was largely caused by the better insulin-mediated suppression of hepatic glucose production. In huCETP females, CETP in the circulation decreased HDL-cholesterol content and increased liver cholesterol uptake and liver cholesterol and oxysterol contents, which was associated with the upregulation of LXR target genes in long-chain polyunsaturated fatty acid biosynthesis and PPARα target genes in fatty acid β-oxidation in the liver. The upregulated fatty acid β-oxidation may account for the improved fatty liver and liver insulin action in female huCETP mice. This study provides further evidence that CETP has beneficial physiological roles in the metabolic adaptation to nutrient excess by promoting liver fatty acid oxidation and hepatic insulin sensitivity, particularly for females.

Pelvic pain and vaginal bleeding are common symptoms in postpartum women presenting to the emergency room (ER). Pelvic ultrasonography plays a crucial role in evaluating symptomatic postpartum patients by allowing a rapid diagnosis and treatment initiation. The main goal of imaging is to distinguish between causes of pelvic pain and vaginal bleeding that may be managed conservatively and those requiring emergent intervention. This pictural essay focuses on the ultrasonographic features of common postpartum conditions for which patients may present to the ER with vaginal bleeding and pelvic pain, including retained products of conception, endometritis, uterine arteriovenous malformation, uterine artery pseudoaneurysm, ovarian vein thrombosis, bladder flap hematoma, and uterine dehiscence/rupture.

The heat exchanger (HE) is an important component of almost every energy generation system. Periodic inspection of the HEs is particularly important to keep high efficiency of the entire system. In this paper, a novel ultrasonic water immersion inspection method is presented based on circumferential wave (CW) propagation to detect defective HE. Thin patch-type piezoelectric elements with multiple resonance frequencies were adopted for the ultrasonic inspection of narrow-spaced HE in an immersion test. Water-filled HE was used to simulate defective HE because water is the most reliable indicator of the defect. The HE will leak water no matter what the defect pattern is. Furthermore, continuous wavelet transform (CWT) was used to investigate the received CW, and inverse CWT was applied to separate frequency bands corresponding to the thickness and lateral resonance modes of the piezoelectric element. Different arrangements of intact and leaky HE were tested with several pairs of thin piezoelectric patch probes in various instrumental setups. Also, direct waveforms in the water without HE were used as reference signals, to indicate instrumental gain and probe sensitivity. Moreover, all filtered CW corresponding to resonance modes together with the direct waveforms in the water were used to train the deep neural networks (DNNs). As a result, an automatic HE state classification method was obtained, and the accuracy of the applied DNN was estimated as 99.99%.

Inhaled ground level ozone (O(3)) has well described adverse health effects, which may be augmented in susceptible populations. While conditions, such as pre-existing respiratory disease, have been identified as factors enhancing susceptibility to O(3)-induced health effects, the potential for chemical interactions in the lung to sensitize populations to pollutant-induced responses has not yet been studied. In the airways, inhaled O(3) reacts with lipids, such as cholesterol, to generate reactive and electrophilic oxysterol species, capable of causing cellular dysfunction and inflammation. The enzyme regulating the final step of cholesterol biosynthesis, 7-dehydrocholesterol reductase (DHCR7), converts 7-dehydrocholesterol (7-DHC) to cholesterol. Inhibition of DHCR7 increases the levels of 7-DHC, which is much more susceptible to oxidation than cholesterol. Chemical analysis established the capacity for a variety of small molecule antipsychotic drugs, like Aripiprazole (APZ), to inhibit DHCR7 and elevate circulating 7-DHC. Our results show that APZ and the known DHCR7 inhibitor, AY9944, increase 7-DHC levels in airway epithelial cells and potentiate O(3)-induced IL-6 and IL-8 expression and cytokine release. Targeted immune-related gene array analysis demonstrates that APZ significantly modified O(3)-induced expression of 16 genes, causing dysregulation in expression of genes associated with leukocyte recruitment and inflammatory response. Additionally, we find that APZ increases O(3)-induced IL-6 and IL-8 expression in human nasal epithelial cells from male but not female donors. Overall, the evidence we provide describes a novel molecular mechanism by which chemicals, such as APZ, that perturb cholesterol biosynthesis affect O(3)-induced biological responses.

Inhalation of the ambient air pollutant ozone causes lung inflammation and can suppress host defense mechanisms, including impairing macrophage phagocytosis. Ozone reacts with cholesterol in the lung to form oxysterols, like secosterol A and secosterol B (SecoA and SecoB), which can form covalent adducts on cellular proteins. How oxysterol-protein adduction modifies the function of lung macrophages is unknown. Herein, we used a proteomic screen to identify lung macrophage proteins that form adducts with ozone-derived oxysterols. Functional ontology analysis of the adductome indicated that protein binding was a major function of adducted proteins. Further analysis of specific proteins forming adducts with SecoA identified the phagocytic receptors CD206 and CD64. Adduction of these receptors with ozone-derived oxysterols impaired ligand binding and corresponded with reduced macrophage phagocytosis. This work suggests a novel mechanism for the suppression of macrophage phagocytosis following ozone exposure through the generation of oxysterols and the formation of oxysterol-protein adducts on phagocytic receptors.

The coating is applied to prevent corrosion on the surface of ships or marine structures, and the thickness of the coating affects its anti-corrosion effect. As a result, non-destructive testing (NDT) is required to measure coating thickness, and ultrasonic NDT is a convenient and quick way to measure the thickness of underwater coatings. However, the offshore coating’s energy attenuation and absorption rates are high, the ultrasonic pulse echo test is difficult, and the testing environment is harsh. Because of the coating’s high attenuation, the distance of the optimal water delay line designed based on the reflection coefficient of the vertically incident wave is used. To accurately measure the thickness of the coating material, TOF of the reflected echo on the time-domain waveform was evaluated. The experimental results show that, when compared to caliper measurements, the coating thickness measured by the proposed method has a lower error and can be used for accurate measurement. The use of ultrasonic water immersion measurement is almost limitless in terms of size, location, and material of the object to be measured, and it is expected to be used to measure the thickness of the surface coating of ships or marine structures in the water.

The formation of lipid electrophile-protein adducts is associated with many disorders that involve perturbations of cellular redox status. The identities of adducted proteins and the effects of adduction on protein function are mostly unknown and an increased understanding of these factors may help to define the pathogenesis of various human disorders involving oxidative stress. 7-Dehydrocholesterol (7-DHC), the immediate biosynthetic precursor to cholesterol, is highly oxidizable and gives electrophilic oxysterols that adduct proteins readily, a sequence of events proposed to occur in Smith-Lemli-Opitz syndrome (SLOS), a human disorder resulting from an error in cholesterol biosynthesis. Alkynyl lanosterol (a-Lan) was synthesized and studied in Neuro2a cells, Dhcr7-deficient Neuro2a cells and human fibroblasts. When incubated in control Neuro2a cells and control human fibroblasts, a-Lan completed the sequence of steps involved in cholesterol biosynthesis and alkynyl-cholesterol (a-Chol) was the major product formed. In Dhcr7-deficient Neuro2a cells or fibroblasts from SLOS patients, the biosynthetic transformation was interrupted at the penultimate step and alkynyl-7-DHC (a-7-DHC) was the major product formed. When a-Lan was incubated in Dhcr7-deficient Neuro2a cells and the alkynyl tag was used to ligate a biotin group to alkyne-containing products, protein-sterol adducts were isolated and identified. In parallel experiments with a-Lan and a-7-DHC in Dhcr7-deficient Neuro2a cells, a-7-DHC was found to adduct to a larger set of proteins (799) than a-Lan (457) with most of the a-Lan protein adducts (423) being common to the larger a-7-DHC set. Of the 423 proteins found common to both experiments, those formed from a-7-DHC were more highly enriched compared to a DMSO control than were those derived from a-Lan. The 423 common proteins were ranked according to the enrichment determined for each protein in the a-Lan and a-7-DHC experiments and there was a very strong correlation of protein ranks for the adducts formed in the parallel experiments., Graphical abstract fx1

Background: Changes in cholesterol metabolism are common hallmarks of neurodevelopmental pathologies. A diverse array of genetic disorders of cholesterol metabolism support this claim as do multiple lines of research that demonstrate chemical inhibition of cholesterol biosynthesis compromises neurodevelopment. Recent work has revealed that a number of commonly used pharmaceuticals induce changes in cholesterol metabolism that are similar to changes induced by genetic disorders with devastating neurodevelopmental deficiencies. Objectives: We tested the hypothesis that common environmental toxicants may also impair cholesterol metabolism and thereby possibly contribute to neurodevelopmental toxicity. Methods: Using high-throughput screening with a targeted lipidomic analysis and the mouse neuroblastoma cell line, Neuro-2a, the ToxCast™ chemical library was screened for compounds that impact sterol metabolism. Validation of chemical effects was conducted by assessing cholesterol biosynthesis in human induced pluripotent stem cell (hiPSC)–derived neuroprogenitors using an isotopically labeled cholesterol precursor and by monitoring product formation with UPLC-MS/MS. Results: Twenty-nine compounds were identified as validated lead-hits, and four were prioritized for further study (endosulfan sulfate, tributyltin chloride, fenpropimorph, and spiroxamine). All four compounds were validated to cause hypocholesterolemia in Neuro-2a cells. The morpholine-like fungicides, fenpropimorph and spiroxamine, mirrored their Neuro-2a activity in four immortalized human cell lines and in a human neuroprogenitor model derived from hiPSCs, but endosulfan sulfate and tributyltin chloride did not. Conclusions: These data reveal the existence of environmental compounds that interrupt cholesterol biosynthesis and that methodologically hiPSC neuroprogenitor cells provide a particularly sensitive system to monitor the effect of small molecules on de novo cholesterol formation. https://doi.org/10.1289/EHP5053

When inhaled, ozone (O3) interacts with cholesterols of airway epithelial cell membranes or the lung-lining fluid, generating chemically reactive oxysterols. The mechanism by which O3-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O3 results in the formation of oxysterols, epoxycholesterol-α and -β and secosterol A and B (Seco A and Seco B), in cell lysates and apical washes. Similarly, bronchoalveolar lavage fluid obtained from human volunteers exposed to O3 contained elevated levels of these oxysterol species. As expected, O3-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly, expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1), which is regulated by activation of the liver X receptor (LXR), was suppressed in epithelial cells exposed to O3. Additionally, exposure of LXR knock-out mice to O3 enhanced pro-inflammatory cytokine production in the lung, suggesting LXR inhibits O3-induced inflammation. Using alkynyl surrogates of O3-derived oxysterols, our data demonstrate adduction of LXR with Seco A. Similarly, supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O3 exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared with stimulation with T0901317 alone, indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall, these data demonstrate that O3-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR, thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O3-derived formation of oxidized lipids in the airways and subsequent adverse health effects.

Ultrasonic wave can be widely used for water treatment; however, the efficiency is low compared to the input power used to generate the ultrasonic wave. To address this problem, the coiled-up space metamaterial, a zig-zag like spatial structure that can amplify the acoustic wave by creating the resonance, was adopted. The model structure of metamaterial with the optimized parameters found by using FEM simulation was fabricated with a 3D printer in order to test the advantage of this structure. The fabricated structure was submerged into the potassium iodide solution, and we compared the KI (aq) dosimetry result between the solution with and without the structure. As a result, we found that the coiled-up space metamaterial can increase the efficiency of the ultrasonic water treatment by 41% under the resonance condition of the container of the target solution. This result implies that the application of the coiled-up space into water treatment can increase the efficiency dramatically. The result can be widely implemented into ultrasonic cleansing and cell death induction as well.

Regulating blood cholesterol (Chol) levels by pharmacotherapy has successfully improved cardiovascular health. There is growing interest in the role of Chol precursors in the treatment of diseases. One sterol precursor, desmosterol (Des), is a potential pharmacological target for inflammatory and neurodegenerative disorders. However, elevating levels of the precursor 7-dehydrocholesterol (7-DHC) by inhibiting the enzyme 7-dehydrocholesterol reductase is linked to teratogenic outcomes. Thus, altering the sterol profile may either increase risk toward an adverse outcome or confer therapeutic benefit depending on the metabolite affected by the pharmacophore. In order to characterize any unknown activity of drugs on Chol biosynthesis, a chemical library of Food and Drug Administration-approved drugs was screened for the potential to modulate 7-DHC or Des levels in a neural cell line. Over 20% of the collection was shown to impact Chol biosynthesis, including 75 compounds that alter 7-DHC levels and 49 that modulate Des levels. Evidence is provided that three tyrosine kinase inhibitors, imatinib, ponatinib, and masitinib, elevate Des levels as well as other substrates of 24-dehydrocholesterol reductase, the enzyme responsible for converting Des to Chol. Additionally, the mechanism of action for ponatinib and masitinib was explored, demonstrating that protein levels are decreased as a result of treatment with these drugs.

This case study aims to verify the benefits of theme-based project learning for convergent science experiments. The study explores the possibilities of enhancing creative, integrated and collaborative teaching and learning abilities in science-gifted educa- tion. A convergent project-based science experiment program of physics, chemistry and biology with the theme of environment such as seawater and wetland was designed and applied to science-gifted secondary school students in an international science contest. The program was initiated with integration of physics, chemistry and biology, interrelating both field work and laboratory work. Besides, logical discussion and humanistic writing activities with environmental issues were followed. The participants were tasked to conduct hands-on multi-disciplinary projects for both in the fields and laboratories. The projects involve raising creative and critical thinking through interpreting col- lected data, predicting outcomes, drawing conclusions, and presenting results. The study shows a model of project-based convergent programs for integrated experimental composition to facilitate collaborative and creative learning as well as to improve students interests in related subjects. The study discusses ways to raise awareness of benefits from multi-disciplinary approaches through theme-based project learning in science-gifted education.

A quantitative analysis of violin sounds produced for different bowing positions over the full length of a violin string has been carried out. An automated bowing machine was employed in order to keep the bowing parameters constant. A 3-dimensional profile of the frequency spectrum was introduced in order to characterize the violin’s sound. We found that the fundamental frequency did not change for different bowing positions, whereas the frequencies of the higher harmonics were different. Bowing the string at 30 mm from the bridge produced musical sounds. The middle of the string was confirmed to be a dead zone, as reported in previous works. In addition, the quarter position was also found to be a dead zone. Bowing the string 90 mm from the bridge dominantly produces a fundamental frequency of 864 Hz and its harmonics.

Cholesterol undergoes ozonolysis to afford a variety of oxysterol products, including cholesterol-5,6-epoxide (CholEp) and the isomeric aldehydes secosterol A (seco A) and secosterol B (seco B). These oxysterols display numerous important biological activities, including protein adduction; however, much remains to be learned about the identity of the reactive species and the range of proteins modified by these oxysterols. Here, we synthesized alkynyl derivatives of cholesterol-derived oxysterols and employed a straightforward detection method to establish secosterols A and B as the most protein-reactive of the oxysterols tested. Model adduction studies with an amino acid, peptides, and proteins provide evidence for the potential role of secosterol dehydration products in protein adduction. Hydrophobic separation methods-Folch extraction and solid phase extraction (SPE)-were successfully applied to enrich oxysterol-adducted peptide species, and LC-MS/MS analysis of a model peptide-seco adduct revealed a unique fragmentation pattern (neutral loss of 390 Da) for that species. Coupling a hydrophobic enrichment method with proteomic analysis utilizing characteristic fragmentation patterns facilitates the identification of secosterol-modified peptides and proteins in an adducted protein. More broadly, these improved enrichment methods may give insight into the role of oxysterols and ozone exposure in the pathogenesis of a variety of diseases, including atherosclerosis, Alzheimer's disease, Parkinson's disease, and asthma.

AITR (activation-inducible TNFR family receptor, human GITR), an inducible costimulatory receptor of human T cells, is expressed constitutively on Treg and inducible on Teff upon antigen engagement. AITR co-stimulates T cell activation and recruits certain members of TRAF family upon interaction with its ligand. We examined whether the stimulation of distinct regions of extracellular domain of AITR can generate signals that promote different subsets of CD4+ T cells. We found that crosslinking AITR by agonistic anti-AITR Abs (A27, A41, and A35), that recognize three distinct regions of the extracellular domain, polarize CD4+ T cells into Th1, Th2 and Th17, respectively. The subset polarization evoked by these antibodies was the result of recruitment of specific TRAFs, phosphorylation of distinct STATs and expression of Th subset fate-determining master regulatory genes. Stimulation with A27 recruited TRAF-1 and -2, activated STAT-1, -4 and JNK1/2, and induced transcription factor T-bet. On the other hand, activation with A35 recruited TRAF-6, activated STAT-3 and p38 MAPK, and induced RORγt. In contrast, A41 recruited TRAF-3 and -5, activated STAT-5, -6 and ERK1/2, and produced GATA-3. Remarkably, A27 induced the conversion of CD25+Foxp3+ Treg cells to IFN-γ-producing Th1 cells with a concomitant suppression of Foxp3 and TGF-β expression both in healthy individuals as well as cancer patients. A35 also, but not A41, suppressed the expression of Foxp3 and induced IL-17 in Treg cells. A27 alone, but not others produced a strong anti-tumor effect in a human tumor xenograft model of humanized mice in an IFN-γ- and Treg conversion-dependent mechanism. Taken together, the data that AITR regulates Th subset polarization and eradicates solid tumors can be a target for broad-spectrum immune check point therapeutics against cancers and autoimmune diseases. Citation Format: Byoung S. Kwon, Seung J. Lee, Young H. Kim, Joong W. Lee, Sun H. Hwang, Dass S. Vinay. Activation-inducible TNFR family receptor (AITR) signaling mediates the polarization of CD4+ T cells into Th1, Th2, and Th17, converts Treg to Teff and eradicates solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3235.

In this study, focusing of ultrasonic Lamb wave by negative refraction with mode conversion from antisymmetric to symmetric mode was investigated. When a wave propagates backward by negative refraction, the energy flux is antiparallel to the phase velocity. Backward propagation of Lamb wave is quite well known, but the behavior of backward Lamb wave at an interface has rarely been investigated. A pin-type transducer is used to detect Lamb wave propagating on a steel plate with a step change in thickness. Conversion from forward to backward propagating mode leads to negative refraction and thus wave focusing. By comparing the amplitudes of received Lamb waves at a specific frequency measured at different distance between transmitter and interface, the focusing of Lamb wave due to negative refraction was confirmed.

To evaluate the suitability of the Weaponeering tool for the Surface to Surface Guided Missile at the AboveGround Buildings, we analyzed the relation among SSPD,   , CEP and the number of missiles, expected damage etc.. According to the analysis, if the target Length is 100m, and    is longer than the target length, the tool is suitable but shorter than it, the tool is not suitable. So Morris Driels's Weaponeering Tool needs new adaptive algorithm to Translate as the Target size and  .

A small library of pharmacologically active compounds (the NIH Clinical Collection) was assayed in Neuro2a cells to determine their effect on the last step in the biosynthesis of cholesterol, the transformation of 7-dehydrocholesterol (7-DHC) to cholesterol promoted by 7-dehydrocholesterol reductase, DHCR7. Of some 727 compounds in the NIH Clinical Collection, over 30 compounds significantly increased 7-DHC in Neuro2a cells when assayed at 1 μM. Active compounds that increased 7-DHC with a Z-score of +3 or greater generally gave rise to modest decreases in desmosterol and increases in lanosterol levels. Among the most active compounds identified in the library were the antipsychotic, antidepressant, and anxiolytic compounds that included perospirone, nefazodone, haloperidol, aripiprazole, trazodone, and buspirone. Fluoxetine and risperidone were also active at 1 μM, and another 10 compounds in this class of pharmaceuticals were identified in the screen at concentrations of 10 μM. Increased levels of 7-DHC are associated with Smith-Lemli-Opitz syndrome (SLOS), a human condition that results from a mutation in the gene that encodes DHCR7. The SLOS phenotype includes neurological deficits and congenital malformations, and it is linked to a higher incidence of autism spectrum disorder. The significance of the current study is that it identifies common pharmacological compounds that may induce a biochemical presentation similar to SLOS. Little is known about the side effects of elevated 7-DHC postdevelopmentally, and the elevated 7-DHC that results from exposure to these compounds may also be a confounder in the diagnosis of SLOS.

When inhaled, ozone (O3) interacts with cholesterols of airway epithelial cell membranes or the lung-lining fluid, generating chemically reactive oxysterols. The mechanism by which O3-derived oxysterols affect molecular function is unknown. Our data show that in vitro exposure of human bronchial epithelial cells to O3 results in the formation of oxysterols, epoxycholesterol-α and -β and secosterol A and B (Seco A and Seco B), in cell lysates and apical washes. Similarly, bronchoalveolar lavage fluid obtained from human volunteers exposed to O3 contained elevated levels of these oxysterol species. As expected, O3-derived oxysterols have a pro-inflammatory effect and increase NF-κB activity. Interestingly, expression of the cholesterol efflux pump ATP-binding cassette transporter 1 (ABCA1), which is regulated by activation of the liver X receptor (LXR), was suppressed in epithelial cells exposed to O3. Additionally, exposure of LXR knock-out mice to O3 enhanced pro-inflammatory cytokine production in the lung, suggesting LXR inhibits O3-induced inflammation. Using alkynyl surrogates of O3-derived oxysterols, our data demonstrate adduction of LXR with Seco A. Similarly, supplementation of epithelial cells with alkynyl-tagged cholesterol followed by O3 exposure causes observable lipid-LXR adduct formation. Experiments using Seco A and the LXR agonist T0901317 (T09) showed reduced expression of ABCA1 as compared with stimulation with T0901317 alone, indicating that Seco A-LXR protein adduct formation inhibits LXR activation by traditional agonists. Overall, these data demonstrate that O3-derived oxysterols have pro-inflammatory functions and form lipid-protein adducts with LXR, thus leading to suppressed cholesterol regulatory gene expression and providing a biochemical mechanism mediating O3-derived formation of oxidized lipids in the airways and subsequent adverse health effects.

Rishi Agarwal,1,2 Paramveer Saluja,1 Angela Pham,3 Karen Ledbetter,3 Sukhveer Bains,3 Seema Varghese,3 John Clements,1 Young H Kim41Synergy Medical Education Alliance, Michigan State University College of Human Medicine, Saginaw, Michigan, USA; 2MD Anderson Cancer Center, Houston, Texas, USA; 3Michigan State University, Saginaw, Michigan, USA; 4Seton Cancer Institute, Saginaw, Michigan, USAIntroduction: The current standard for treating operable early stage non-small cell lung cancer is surgical resection and for inoperable cases it is external beam radiotherapy. Lung functions are adversely affected with both the above treatments. CyberKnife treatment limits radiation damage by tracking targets moving with each breath. The effect of CyberKnife treatment on pulmonary function tests has not been well documented.Methods: Lung cancer patients who underwent CyberKnife treatment and had pre- and post-treatment pulmonary function tests were included. Paired t-tests were conducted. We also conducted subgroup analysis.Results : Thirty-seven patients were included. Median age was 73 years. No statistical difference between mean pre- and post-CyberKnife pulmonary function tests was found.Discussion: We observed that CyberKnife better preserves lung function status compared to current standards of care. It has shown to have very minimal side effects.Keywords: non-small cell lung cancer, radiation pneumonitis, radiotherapy, pulmonary function tests

(접수일자: 2011년 4월 11일; 수정일자: 2011년 6월 10일; 채택일자: 2011년 6월 30일)PC의 내장 사운드 카드를 이용하여 음향학 탐구학습을 수행하였다 . 사운드 카드는 함수발생기와 오실로스코프에 비해 가격이 저렴하고 학생들의 접근이 용이하다는 장점이 있다. 학생들은 다양한 음향 현상에서 발생하는 소리를 사운드 카드를 이용하여 녹음하고 주파수 스펙트럼을 분석 하였다. 소리굽쇠의 맥놀이, Rijke 관에서의 음의 발생, 물을 따를 때에 나는 소리 변화, 소리로 포도주잔 깨기 및 포도주 병마개를 딸 때에 발생하는 소리 등의 현상을 정량적으로 분석하면서 소리의 중첩, 공진 및 정상파에 대한 탐구를 수행하였다.핵심용어: 음향학 교육, 사운드 카드, 탐구활동투고분야: 일반 분야 (0.2)Inquiring activities on the acoustic phenomena have been carrie d out by using a sound card installed in a personal computer. A sound card is cheaper and more accessible to the st udents than the precision equipment such as a function generator or an oscilloscope. The students record the sounds fr om various acoustic phenomena to the sound card. Then they analyze the frequency spectrums of that sounds by usi ng a program. Inquired phenomena include beat by two tuning forks, sound from Rijke tube, pouring sound, brea king of a wine glass and pop-up sound of a wine bottle. Through these activities students perform quantitative analysis of various phenomena due to superposition, resonance and standing wave.Keywords: Education of acoustics, Sound cards, Inquiry activitiesASK subject classification: General Area (0.2)

Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogues of tyrosine gave rise to lipid peroxide-tyrosine (phenol) adducts in both aqueous micellar and organic solutions. The novel adducts were isolated and characterized by 1D and 2D NMR spectroscopy as well as by mass spectrometry (MS). The spectral data suggest that the polyunsaturated lipid peroxyl radicals give stable peroxide coupling products exclusively at the para position of the tyrosyl (phenoxy) radicals. These adducts have characteristic (13)C chemical shifts at 185 ppm due to the cross-conjugated carbonyl of the phenol-derived cyclohexadienone. The primary peroxide adducts subsequently undergo intramolecular Diels-Alder (IMDA) cyclization, affording a number of diastereomeric tricyclic adducts that have characteristic carbonyl (13)C chemical shifts at ~198 ppm. All of the NMR HMBC and HSQC correlations support the structure assignments of the primary and Diels-Alder adducts, as does MS collision-induced dissociation data. Kinetic rate constants and activation parameters for the IMDA reaction were determined, and the primary adducts were reduced with cuprous ion to give a phenol-derived 4-hydroxycyclohexa-2,5-dienone. No products from adduction of peroxyls at the phenolic ortho position were found in either the primary or cuprous reduction product mixtures. These studies provide a framework for understanding the nature of lipid-protein adducts formed by peroxyl-tyrosyl radical-radical termination processes. Coupling of lipid peroxyl radicals with tyrosyl radicals leads to cyclohexenone and cyclohexadienone adducts, which are of interest in and of themselves since, as electrophiles, they are likely targets for protein nucleophiles. One consequence of lipid peroxyl reactions with tyrosyls may therefore be protein-protein cross-links via interprotein Michael adducts.

Dendrogenin A (DDA) is a tumor suppressor metabolite identified in human tissues that arises from the conjugation of 5,6α-epoxycholesterol (5,6α-EC) with histamine (HA) by a yet unidentified enzyme. DDA is present in the normal breast but its levels were found drastically decreased in breast tumors, showing that a deregulation of DDA metabolism occurred during breast carcinogenesis. It was shown that DDA displayed chemopreventive and anticancer properties (de Medina et al, Nat Commun, 2013; Voisin et al, PNAS, 2017; Segala et al, Nat Commun, in press). In addition, DDA blocks the biosynthesis of a newly identified cholesterol tumor promoter named 6-oxo-cholestan-3β,5α-diol (OCDO) (Voisin et al, PNAS, 2017). DDA and OCDO arise from 5,6-EC. We showed the existence of a metabolic balance between these two 5,6-EC derivatives in normal breast and BC that controls or stimulates BC progression (Silvente-Poirot & Poirot, Science, 2014, Voisin et al, PNAS, 2017). We addressed here the question of the identification and characterization of the DDA synthase (DDAS) and we determined whether its expression could reflect DDA levels in patient breast tumor and normal tissue. We report that the recombinant human glutathione transferase A1-1 (GST A1-1) produced DDA from 5,6α-EC and histamine (HA). The chemical characterization of the DDA product was performed by chromatography and mass spectrometry fragmentation. DDAS activity was found to be a new and important activity of GST A1-1 in addition to known glutathione transferase and steroid isomerase activities. The measured Michaelis constants of GST A1-1 for its new substrates were: Km5,6α-EC=0.27±0.05 µM and KmHA=0.35±0.3 µM, and the maximum velocity for the transformation of each substrates Vm5,6α-EC=0.81±0.2 µmol.min-1.mg and VmHA=0.66±0.2 µmol.min-1.mg. Interestingly, we showed that OCDO and other ring-B oxysterols, as well as several natural substrates and product of the GST A1-1, were potent inhibitors of DDAS activity while xenobiotics substrates of GST, and side chain oxysterols were not. Patient BC samples (n=50) showed significant decreased DDA levels and lower GST A1-1 protein expression compared to normal matched tissues, indicating that the decreased production of DDA in tumors is due to decreased expression of its enzyme. The analyses of two human BC mRNA databases from the Barts Cancer Institute (London, UK) and the Curie Institute (Paris, France) showed that the expression of GST A1-1 was lost in ER(+) BC tumors compared to normal breast tissue. Interestingly, DDAS was selectively expressed in the cytoplasm of epithelial cells from lactating ducts and lobular terminal units. Since these cells are the origin of most BC, the loss of DDAS expression and DDA biosynthesis combine to OCDO production, which controls DDAS activity, may constitute a major oncogenic process leading to BC development in human. Citation Format: Marc Poirot, Emmanuel Noguer, Florence Dalenc, Regis Soules, Lisa Barrett, Arnaud Rives, Hye-Young Kim, Brigitta Sjödin, Camille Franchet, Pilippe Rochaix, Raphaelle Duprez-Paumier, Magali Lacroix-Triki, Thomas Filleron, Leonor Chaltiel, Louise Jones, Emanuala Gadaleta, Claude Chalala, Sergio Roman-Roman, Thierry Dubois, Ned A. Porter, Bengt Mannervik, Michel Record, Sandrine Silvente-Poirot. Characterization of the enzyme generating the cholesterol metabolite and tumor suppressor dendrogenin A in the breast and its deregulations in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5238.

Reactive electrophiles generated by lipid peroxidation are thought to contribute to cardiovascular disease and other oxidative stress-related pathologies by covalently modifying proteins and affecting critical protein functions. The difficulty of capturing and analyzing the relatively small fraction of modified proteins complicates identification of the protein targets of lipid electrophiles. We recently synthesized a biotin-modified linoleoylglycerylphosphatidycholine probe called PLPBSO (Tallmanet al.Chem. Res. Toxicol.2007, 7, 227−23417305406), which forms typical linoleate oxidation products and covalent adducts with model peptides and proteins. Supplementation of human plasma with PLPBSO followed by free radical oxidation resulted in covalent adduction of PLPBSO to plasma proteins, which were isolated with streptavidin and identified by liquid chromatography-tandem mass spectrometry (LC-MS−MS). Among the most highly modified proteins was apolipoprotein A1 (ApoA1), which is the core component of high density lipoprotein (HDL). ApoA1 phospholipid adduct sites were mapped by LC-MS−MS of tryptic peptides following mild base hydrolysis to release esterified phospholipid adducts. Several carboxylated adducts formed from phospholipid-esterified 9,12-dioxo-10(E)-dodecenoic acid (KODA), 9-hydroxy, 12-oxo-10(E)-dodecenoic acid (HODA), 7-oxoheptanoic acid, 8-oxooctanoic acid, and 9-oxononanoic acid were identified. Free radical oxidations of isolated HDL also generated adducts with 4-hydroxynonenal (HNE) and other noncarboxylated electrophiles, but these were only sporadically identified in the PLPBSO-adducted ApoA1, suggesting a low stoichiometry of modification in the phospholipid-adducted protein. Both phospholipid electrophiles and HNE adducted His162, which resides in an ApoA1 domain involved in the activation of Lecithin-cholesterol acyltransferase and maturation of the HDL particle. ApoA1 lipid electrophile adducts may affect protein functions and provide useful biomarkers for oxidative stress., We describe the use of PLPBSO as a probe to identify major protein targets of oxidized phospholipids in human plasma. This approach identified apolipoprotein A1 (ApoA1) as the principal target of PLPBSO oxidation products and led us to map adducts from lipid oxidation products on this protein. We report the identification of adducts from several phospholipid electrophiles, including carboxy-terminal adducts. We further evaluated the sites of ApoA1 adduction by the prototypical lipid electrophile HNE. ApoA1 lipid electrophile adducts may affect protein functions and provide useful biomarkers for oxidative stress.