HTS System Research Articles

Abstract 5322: High throughput chemotherapeutic drug screening system for gastric cancer: cure-GA

Abstract To discover clinically applicable anticancer drugs and predict therapeutic response for advanced gastric cancer, we developed a high-throughput drug (HTD) screening system that could rapidly evaluate drug reactivity using 3D cultured primary cells derived from gastric cancer (GC) patients. Primary cancer cells were isolated from fresh surgical specimens that resected from 143 GC patients using Gentle Max tissue dissociation system. Primary cells were mixed with Matrigel, and placed on a micropillar for three-dimensional (3D) culture. After the primary cells were stabilized in the complete culture medium (CCM) than added various chemotherapeutic drugs containing 5-FU, Oxaliplatin, and Paclitaxel in CCM and incubated for 7 days. Cell viability was determined through calcein staining and quantified scanned images. The IC50 for each drug was calculated by a sigmoidal dose-response curve, using the GraphPad Prism 9 program. The average weight of gastric cancer tissue used in the experiment was 300 mg (75 mg ~ 1930 mg), and the average number of dissociated viable cells for each tissue was 3.9 × 10^6 cells/case. About 2.4 × 10^5 live cell was required per drug, we were able to obtain an average of 6.4 (Min.2, Max 14) drug reactivity data per tissue using the HTD screening system. GC tissues obtained from the operating room were dissociated within 16 hours and then loaded into the HTS system within 3 hours. Cells were stabilized for 1 day in 3D culture plate and exposed to the drug for 7 days, and then data reports were made within 3 days. As a result, it was possible to obtain within 14 days from fresh surgical GC tissue to drug response data. Additionally, we confirmed that 3D cultured primary cells derived from GC tissues consistently preserved primary characters using IHC. Similar to their parental cancer tissue, GC 3D cultured primary cells derived from adenocarcinoma large glandular patterns and retain the expression of some marker proteins. In this study, we evaluated the drug response data for 101 cases (success rate 71%; 101/143) to 5-FU, Oxaliplatin, and Palitaxel, etc. using the HTD screening system and it was confirmed that individual patient had a difference response to each drug. Here we established the HTD screening system using 3D cultured GC patient derived primary cells. The advantages of this system were that it is the first model system that directly used patient-derived primary cells for drug screening, and it can rapidly evaluate drug reactivity to various anticancer drugs within 10 days. The HTD screening system based on patient-derived primary cells can provide that information to predict drug response and allow for finding more appropriate therapy for each patient. Citation Format: Jieun Lee, Jung Eun Kim, Sanjun Lee, Tae-Kyeong Lee, In Hee Kim, So Hee Yoon, Mira Yoo, Eunju Lee, Doo-Young Hwang, So Hyun Kang, Bo Sung Ku, Dong Woo Lee, Young Suk Park, Ji-Won K, Jin Won Kim, Sang-Hoon Ahn, Keun-Wook Lee, Hyung-Ho Kim, Hyun Jung Oh, Yun-Suhk Suh. High throughput chemotherapeutic drug screening system for gastric cancer: cure-GA. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5322.

Secretion of miRNA-326-3p by senescent adipose exacerbates myocardial metabolism in diabetic mice

BackgroundAdipose tissue homeostasis is at the heart of many metabolic syndromes such as diabetes. Previously it has been demonstrated that adipose tissues from diabetic patients are senescent but whether this contributes to diabetic cardiomyopathy (DCM) remains to be elucidated.MethodsThe streptozotocin (STZ) type 1 diabetic mice were established as animal model, and adult mouse ventricular myocytes (AMVMs) isolated by langendorff perfusion as well as neonatal mouse ventricular myocytes (NMVMs) were used as cell models. Senescent associated β galactosidase (SA-β-gal) staining and RT-qPCR were used to identify the presence of adipose senescence in diabetic adipose tissue. Senescent adipose were removed either by surgery or by senolytic treatment. Large extracellular vesicles (LEVs) derived from adipose tissue and circulation were separated by ultracentrifugation. Cardiac systolic and diastolic function was evaluated through cardiac ultrasound. Cardiomyocytes contraction function was evaluated by the Ionoptix HTS system and live cell imaging, mitochondrial morphology and functions were evaluated by transmission electron microscope, live cell fluorescent probe and seahorse analysis. RNA-seq for AMVMs and miRNA-seq for LEVs were performed, and bioinformatic analysis combined with RT-qPCR and Western blot were used to elucidate underlying mechanism that senescent adipose derives LEVs exacerbates myocardial metabolism.ResultsSA-β-gal staining and RT-qPCR identified the presence of adipose tissue senescence in STZ mice. Through surgical as well as pharmacological means we show that senescent adipose tissue participates in the pathogenesis of DCM in STZ mice by exacerbates myocardial metabolism through secretion of LEVs. Specifically, expression of miRNA-326-3p was up-regulated in LEVs isolated from senescent adipose tissue, circulation, and cardiomyocytes of STZ mice. Up-regulation of miRNA-326-3p coincided with myocardial transcriptomic changes in metabolism. Functionally, we demonstrate that miRNA-326-3p inhibited the expression of Rictor and resulted in impaired mitochondrial and contractile function in cardiomyocytes.ConclusionWe demonstrate for the first time that senescent adipose derived LEVs exacerbates myocardial metabolism through up-regulated miRNA-326-3p which inhibits Rictor in cardiomyocytes. Furthermore, reducing senescence burden in adipose tissue is capable of relieving myocardial metabolism disorder in diabetes mellitus.

Abstract 6039: Application of triple negative breast cancer patient-derived organoid and xenograft model for drug screening

Abstract Background: Breast cancer Patient Derived Organoids (PDOs) and patient-derived xenografts (PDXs) which recapitulate the tumor heterogeneity and molecular signatures of original tumor tissue, are useful tools to enable greater precision of both approved and experimental medicines through personalized therapeutic approaches. We focus on use of ex vivo PDOs, PDX-derived organoids (PDXOs), and vivo PDXs models for drug testing and personalized therapy. In this study, we investigated the oncogenic role and therapeutic potency of targeting WEE1 in breast cancer. Methods: Fresh surgical specimens of primary TNBC breast tumor (N=3) were used for PDOs. PDOs and PDX-derived organoids (PDXOs) were cultured in previously reported media compositions based on ECM hydrogel. PODs and PDXOs were subjected to drug screening for 22 anticancer drugs including chemoreagents and targeted drugs in the 3D HTS system. Drug sensitivity was tested in triplicate in different concentration ranges for 7 days. The IC50 for each drug was calculated by a sigmoidal dose-response curve, using the GraphPad Prism 9 program. For drug screening in PDXs models, PDOs were injected into the fourth mammary fat pads of NOD.Cg-Prkdcscid Il2rgtm1wjl/SzJ mice. WEE1 inhibitor (AZD1775) was administered by oral gavage (AZD1775, 30 mg/kg) for 3 weeks. Immunostaining of ER, PR, Her2, CK5 and Ki67 was performed in PODs, PODXs and PDX tumor tissues. Results: All PDOs, PDXs, and PDXOs were ER-, PR- and HER2- negative. PDOs and PDXOs (172T, 185T, 207T) showed differential response to 22 anticancer drugs. In PDOs and PDXOs models, 172T and 207T PDOs and PDXOs are highly sensitive to AZD1775, but 185T PDOs and PDXOs less sensitive or never respond to treatment with AZD1775 (10 µM). In PDX models, treatment with AZ1775 decreased tumor growth (P<0.0079) as well as distance metastasis in 207T-PDX mice, whereas in 172T- and 185T-PDX mice, the tumor growth and metastasis were not reduced compared with the vehicle group. Conclusion: Consequently, PDOs, PDXOs and PDXs recapitulated immunohistological signatures of original tumor tissues. The results of WEE1 inhibitor response between PDOs, PDXOs and PDXs were consistent. This study may provide a preclinical tool to screen drug responses to standard-of-care and newly identified drugs. Citation Format: Seungyeon Ryu, Hoe Suk Kim, Jung Eun KIM, So-Hyun Yoon, Sangeun Lee, Moonjou Baek, Han-Byoel Lee, Dong Woo Lee, Bosung Ku, Wonshik Han. Application of triple negative breast cancer patient-derived organoid and xenograft model for drug screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6039.

Abstract P5-02-02: Development of automated 3D high-throughput drug screening platform for patient-derived breast cancer organoids

Abstract Background Patient-derived cancer organoids, which reliably conserve original features of tumors, are emerging as an excellent model for predicting therapy response and drug screening. Developing optimized 3D high-throughput drug screening platform to establish patient-derived cancer organoids and simultaneously perform drug screening is essential for personalized medicine. Methods We established normal breast organoids (n=4) and breast cancer organoids (n=10) from 20 fresh surgical specimen (normal 7, tumor 13 cases). A number (500-2000) of normal and cancer cells were automatically dispensed with the ASFA™ Spotter ST and organoids were generated by hydrogel hanging-drop culture on Cellvitro™ Pillar platform (Medical & Bio Decision, South Korea). Organoids were subjected to drug screening for 17 anticancer drugs including chemoreagents and targeted drugs in the 3D HTS system. Drug sensitivity was tested in triplicate in different concentration ranges for 5 days. Drug cytotoxic effect was assessed by calcein AM staining. Acquisition and analysis of high-content 3D organoid images were peformed using ASFA™ SCANNER (Medical & Bio Decision, South Korea). The IC50 for each drug was calculated by a sigmoidal dose-response curve, using the GraphPad Prism 9 program. We analyzed a drug response index (DRI) using a prediction alogorithm to evalute drug sensitivity (DRI<-0.5) and resistance (DRI>0.5). Results We summarized the DRI value of patient-derived breast organoids of 7 drugs (Table 1). 6 tumor organoids (2T, 6T, 8T, 10T, 13T, 15T) showed high sensitivity to Docetaxel, Doxorubicin, Paclitaxel, and Gemcitabin while 4 tumor organoids (1T, 9T, 14T, 16T) were less sensitive and resistant. 2 tumor organoids (10T, 16T) were sensitive to Tamoxifen and 2 tumor organoids (6T, 8T) show high sensitivity to palbociclib and erlotinib. Normal organoids show less sensitivity and resistance to chemotherapeutic drugs. Drug response index >0.5 : resistancy top 30%, Drug response index <-0.5 : sensitivity top 30%. Conclusions Herein, we developed the hydrogel hanging-drop culture on Cellvitro™ Pillar platform for easily and rapidly high-throughput drug screening in patient-derived organoids using a small number of cells by testing clinically actionable drugs at different concentrations. There were different drug response indeces for each individual organoids to chemoreagents and targeted drugs. We anticipate that 3D high-throughput drug screenings platform based on patient-derived organoids can provide the information to predict drug response and allow for finding more appropriate therapy for individual patients. Table 1.DRI values of patient-derived breast organoids of 7 drugs.Patient No.Tumor/NormalDocetaxelPaclitaxelDoxorubicinTamoxifenGemcitabinePalbociclibErlotinib1TTumor1.051.261.080.060.590.26-2TTumor-0.64-0.72-0.60-0.08-0.79--6TTumor-0.98-0.99-2.330.00-1.44-2.31-1.608TTumor-0.32-0.50-0.250.64-1.07-1.47-0.669TTumor1.050.980.610.870.430.610.4410TTumor-0.84-0.77-0.78-1.680.790.610.0913TTumor-0.87-0.430.560.70-0.510.611.1714TTumor0.861.261.180.481.81--15TTumor-1.59-1.45-1.41--0.71-0.13-16TTumor1.051.261.18-1.70-1.22-2NNormal1.051.260.650.741.810.610.7810NNormal1.05-0.910.810.870.880.610.084NNormal-0.31-0.25-0.530.25-0.190.61-1.575NNormal1.051.130.380.75-0.630.610.10 Citation Format: Jungeun Kim, Hoe Suk Kim, Ga Yeon Kim, Kyung hyeun Park, Seung yeon Ryu, Sangeun Lee, Dong Woo Lee, Bosung Ku, Han-Byoel Lee, Wonshik Han. Development of automated 3D high-throughput drug screening platform for patient-derived breast cancer organoids [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-02-02.

Thrifty, Rapid Intestinal Monolayers (TRIM) Using Caco-2 Epithelial Cells for Oral Drug Delivery Experiments.

Caco-2 monolayers are the most common model of the intestinal epithelium and are critical to the development of oral drug delivery strategies and gastrointestinal disease treatments. However, current monolayer systems are cost- and/or time-intensive, hampering progress. This study evaluates two separate methods to reduce resource input: FB Essence as a fetal bovine serum (FBS) alternative and a new, 3-day Caco-2 system deemed "thrifty, rapid intestinal monolayers" (TRIM). Caco-2 cells were cultured with FB Essence and compared to cells in 10% FBS for proliferation and monolayer formation. TRIM were compared to commonly-used 21-day and Corning® HTS monolayer systems, as well as mouse intestines, for permeability behavior, epithelial gene expression, and tight junction arrangement. No amount of FB Essence maintained Caco-2 cells beyond 10 passages. In contrast, TRIM compared favorably in permeability and gene expression to intestinal tissues. Furthermore, TRIM cost $109 and required 1.3h of time per 24-well plate, compared to $164 and 3.7h for 21-day monolayers, and $340 plus 1.0h for the HTS system. TRIM offer a new approach to generating Caco-2 monolayers that resemble the intestinal epithelium. They are anticipated to accelerate the pace of in vitro intestinal experiments while easing financial burden.

Abstract 846: A novel high-throughput 3D screening system for EMT inhibitors

Abstract EMT (epithelial-mesenchymal transition) is a significant event in tumor metastasis and malignancy. Therefore, inhibition of EMT is considered to enable controlling of malignant transformation. In this study, we introduce an innovative three-dimensional (3D) high-throughput (HTS) system that leads to an identification of EMT inhibitors. At first, we established 3D-HTS EMT model with NanoCulture Plate (NCP) that provided a gel-free micro-patterned scaffold for cells forming spheroids naturally. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs. Citation Format: Kazuya Arai, M Mamunur Rahman, Manabu Itoh, Norio Masuda, Takanori Eguchi, Tetsuya Nakatsura, Stuart K. Calderwood. A novel high-throughput 3D screening system for EMT inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 846. doi:10.1158/1538-7445.AM2017-846

HTS System and Component Targets for a Distributed Aircraft Propulsion System

Distributed propulsion for aircraft is a radical concept in which the generation of thrust is decentralized from the main power sources. Various implementations of this concept are being considered as ways of reducing aircraft fuel burn and noise impact in the future, many relying on electrical transmission. However, the power required for aircraft propulsion is orders greater than the present electrical systems on aircraft. Efficiency and weight of the electrical components will be crucial for the feasibility of an aircraft with distributed propulsion and thus, HTS technology is of strong interest. This paper explains the interest in the technology and possible means of implementation in an aircraft, including considerations of the means of cryogenic cooling. Technology targets are presented on the basis of an aircraft that uses distributed propulsion to enable boundary layer ingestion and produces 50% of its thrust electrically.

2-31.ソフトコンピューティングと高速スクリーニングによる触媒開発(8) : 96ウェルマイクロプレートシステムとラジアル基底関数ネットワークによるメタノール合成用Cu-Zn-Al-Ga酸化物触媒の組成最適化((10)転換利用III,Session 2 天然ガス・メタンハイドレート等)

2-31.ソフトコンピューティングと高速スクリーニングによる触媒開発(8) : 96ウェルマイクロプレートシステムとラジアル基底関数ネットワークによるメタノール合成用Cu-Zn-Al-Ga酸化物触媒の組成最適化((10)転換利用III,Session 2 天然ガス・メタンハイドレート等)

Development of an HTS system to identify natural chemicals that specifically inhibit Escherichia coli O157:H7 adhesion to host cells

Adhesion to intestinal epithelium is critical for virulence and infection of Escherichia coli O157:H7, which is a major cause of foodborne illnesses. Targeting adhesion but not growth of the pathogen is an effective approach to control bacterial infections. In this study, a high-throughput screening system was developed to identify anti-adhesive natural chemicals using a reporter strain whose activity entirely depends on Ler since genes encoding the locus of enterocyte effacement (LEE) which forms attaching and effacing lesions are known to be activated by Ler. A selected hit, yomogin, suppressed the expression of virulent genes encoded within the LEE without affecting bacterial growth itself. Yomogin significantly reduced the adherence of E. coli O157:H7 to Caco-2 epithelial cells and cytotoxicity. These results indicated that the developed system allows for rapid and cost-effective discovery of novel anti-adhesive agents and yomogin can prevent early stage of E. coli O157:H7 pathogenesis.

Hidden Markov Model based Speech Synthesis: A Review

production of human system. This paper reviews recent research advances in field of speech synthesis with related to statistical parametric approach to speech synthesis based on HMM. In this approach, Hidden Markov Model based Text to speech synthesis (HTS) is reviewed in brief. The HTS is based on the generation of an optimal parameter sequence from subword HMMs. The quality of HTS framework relies on the accurate description of the phoneset. The most attractive part of HTS system is the prosodic characteristics of the voice can be modified by simply varying the HMM parameters, thus reducing the large storage requirement.

Modeling of Speaking Rate Influences on Mandarin Speech Prosody and Its Application to Speaking Rate-controlled TTS

A new data-driven approach to building a speaking rate-dependent hierarchical prosodic model (SR-HPM), directly from a large prosody-unlabeled speech database containing utterances of various speaking rates, to describe the influences of speaking rate on Mandarin speech prosody is proposed. It is an extended version of the existing HPM model which contains 12 sub-models to describe various relationships of prosodic-acoustic features of speech signal, linguistic features of the associated text, and prosodic tags representing the prosodic structure of speech. Two main modifications are suggested. One is designing proper normalization functions from the statistics of the whole database to compensate the influences of speaking rate on all prosodic-acoustic features. Another is modifying the HPM training to let its parameters be speaking-rate dependent. Experimental results on a large Mandarin read speech corpus showed that the parameters of the SR-HPM together with these feature normalization functions interpreted the effects of speaking rate on Mandarin speech prosody very well. An application of the SR-HPM to design and implement a speaking rate-controlled Mandarin TTS system is demonstrated. The system can generate natural synthetic speech for any given speaking rate in a wide range of 3.4-6.8 syllables/sec. Two subjective tests, MOS and preference test, were conducted to compare the proposed system with the popular HTS system. The MOS scores of the proposed system were in the range of 3.58-3.83 for eight different speaking rates, while they were in 3.09-3.43 for HTS. Besides, the proposed system had higher preference scores (49.8%-79.6%) than those (9.8%-30.7%) of HTS. This confirmed the effectiveness of the speaking rate control method of the proposed TTS system.

Novel Multiplexed Assay for Identifying SH2 Domain Antagonists of STAT Family Proteins

Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z’ values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.

A thienopyrimidine derivative induces growth inhibition and apoptosis in human cancer cell lines via inhibiting Aurora B kinase activity

Aurora kinases play a key role in the regulation of mitosis and have been regarded as promising targets of cancer therapy. In this paper we describe a thienopyrimidine derivative (S7), a novel potent ATP-competitive hit inhibitor of Aurora B kinase screened through a HTS system, with the IC50 141.12 nM in the biochemical kinase activity assay. Human tumor cells treated with S7 showed dose-dependent inhibition of auto-phosphorylation of Aurora B on Thr232 and another widely-used marker specific for Aurora B kinase, the phosphorylation of Histone H3 (Ser 10), demonstrating endogenous Aurora B kinase activity were inhibited at cellular level. Moreover, S7 treatment induced proliferation inhibition, colony formation inhibition and apoptosis of human tumor cell lines in a dose- and time-dependent manner.

Exploiting the mechanism of cellular glucose uptake to develop an image-based high-throughput screening system in living cells

Biophysical understanding of cellular glucose uptake led us to the development of an image-based high-throughput screening system by using a fluorescent glucose bioprobe, GB2. The accuracy, robustness, and practicality of our image-based HTS system were demonstrated through the pilot screening and the subsequent in vitro confirmation.

Quantum Dot‐Based Screening System for Discovery of G Protein‐Coupled Receptor Agonists

Cellular imaging has emerged as an important tool to unravel biological complexity and to accelerate the drug-discovery process, including cell-based screening, target identification, and mechanism of action studies. Recently, semiconductor nanoparticles known as quantum dots (QDs) have attracted great interest in cellular imaging applications due to their unique photophysical properties such as size, tunable optical property, multiplexing capability, and photostability. Herein, we show that QDs can also be applied to assay development and eventually to high-throughput/content screening (HTS/HCS) for drug discovery. We have synthesized QDs modified with PEG and primary antibodies to be used as fluorescent probes for a cell-based HTS system. The G protein-coupled receptor (GPCR) family is known to be involved in most major diseases. We therefore constructed human osteosarcoma (U2OS) cells that specifically overexpress two types of differently tagged GPCRs: influenza hemagglutinin (HA) peptide-tagged κ-opioid receptors (κ-ORs) and GFP-tagged A3 adenosine receptors (A3AR). In this study, we have demonstrated that 1) anti-HA antibody-conjugated QDs could specifically label HA-tagged κ-ORs, 2) subsequent treatment of QD-tagged GPCR agonists allowed agonist-induced translocation to be monitored in real time, 3) excellent emission spectral properties of QD permitted the simultaneous detection of two GPCRs in one cell, and 4) the robust imaging capabilities of the QD-antibody conjugates could lead to reproducible quantitative data from high-content cellular images. These results suggest that the present QD-based GPCR inhibitor screening system can be a promising platform for further drug screening applications.

Low Field MRI Detection With Tuned HTS SQUID Magnetometer

We set up a low field (LF) magnetic resonance imaging (MRI) system with a tuned high- <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">T</i> <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">c</sub> (HTS) superconducting quantum interference device (SQUID) consisting of a SQUID magnetometer and a liquid-nitrogen-cooled <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">LC</i> resonant pickup circuit, which are inductively coupled. Around 9 kHz, the configuration enhances the sensitivity of the system to 6-7 fT/√Hz. Meanwhile, the relatively large diameter of the coil <i xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">L</i> compensates the magnetometer's drawback of a small pickup area. We performed our experiments with a prepolarization field of 10 mT in a magnetically shielded room. The measurement field was 212.2 μT, corresponding to the Larmor frequency of 9.038 kHz. The compromise between the signal-to-noise ratio and the spatial resolution for our system was studied by recording one-dimensional MRI images of carrots under different gradient fields. For the sample of two carrot slices with center distance of 2.8 cm, a gradient field of 20 Hz/cm was suitable. Two-dimensional images were finally acquired. A HTS system for biological LF MRI detection by a tuned SQUID is demonstrated to be feasible.

High Temperature Superconducting Degaussing From Feasibility Study to Fleet Adoption

The need for increased magnetic signature control on Navy ships has lead to the development and adoption of a three axis advanced degaussing system. While this system is effective in reducing the ship magnetic signature, it requires significantly more copper cable than the legacy two axis systems. Degaussing only requires DC currents for field manipulation. Since DC applications is where HTS use excels, a feasibility study was conducted in FY04 to determine the benefits of HTS when used in an advanced degaussing system. Results showed reduced system size and weight, while remaining cost neutral. A series of lab based demonstrations were conducted proving out key aspects of an HTS DG system, most notably cooling a long length of flexible cryostat with gaseous helium. This led to an at sea demonstration of a single HTS DG loop aboard the USS Higgins. This was the first HTS system installed on an active combatant, and it made a successful magnetic range run in April 2009 demonstrating its capability to perform in a naval environment. This paper details the development of the HTS DG system from the initial feasibility study through the successful demonstration onboard the USS HIGGINS.

In Situ Evaluation of Kinetic Resolution Catalysts for Nitroaldol by Rationally Designed Fluorescence Probe

Development of effective chemical catalysts is a key concern in organic chemistry. Therefore, convenient screening systems for chemical catalysts are required, and although some fluorescence-based HTS systems have been developed, little attempt has been made to apply them to asymmetric catalysts. Therefore, we tried to develop a chiral fluorescence probe which can evaluate the reactivity and enantioselectivity of asymmetric catalysts. We focused on kinetic resolution catalysts as a target of our novel fluorescence probe, employing β-elimination following acylation of nitroaldol. Once the hydroxyl group of nitroaldol is acylated, β-elimination occurs immediately, affording nitro olefin. Therefore, we designed and synthesized a fluorescence probe with an asymmetric nitroaldol moiety. Its fluorescence intensity decreases dramatically upon β-elimination, so the fluorescence decrease is an indicator of the reaction yield. Thus, the enantioselectivity of kinetic resolution catalysts can be assessed simply by measuring the fluorescence intensities of the reaction mixtures of the two enantiomers; it is not necessary to purify the product. This fluorescence probe revealed that benzotetramisole is a superior catalyst for kinetic resolution of nitroaldol. Furthermore, we established an HTS system for asymmetric catalysts, using a fluorescence probe and benzotetramisole. To our knowledge, this is the first fluorescence-based HTS system for asymmetric catalysts.

Reduction of symptoms in binocular anomalies using computerized home therapy—HTS™

Background Asthenopic symptoms often are associated with various accommodative/vergence disorders. Recent studies have found that symptoms associated with convergence insufficiency are reduced by in-office vision therapy with supplemental home therapy. No studies have used standardized symptom questionnaires to evaluate the effectiveness of either in-office or home-based vision therapy in binocular anomalies other than convergence insufficiency. This retrospective study was designed to evaluate the changes in symptoms using an automated, home computer vision therapy program (HTS™) in accommodative/vergence disorders. Methods A retrospective study of 43 prepresbyopic patients who completed the HTS was performed. Before and immediately after treatment all patients in this study completed a 15-question symptom questionnaire (Convergence Insufficiency Symptom Survey). Treatment consisted of various accommodative and vergence activities. Results Initial symptoms scores on the scaled questionnaire were 32.8 (SD = 8.1); after therapy they were 20.6 (SD = 11.5). These changes were both clinically and statistically significant. Forty percent were “normalized” and 55% improved. Convergence amplitude improved from 22Δ to 53Δ after treatment, and divergence amplitudes improved from 15Δ to 25Δ. These findings were clinically significant. Lastly, more than 75% of the patients finished the program by 40 sessions (equivalent to 8 weeks). Conclusion Automated vision therapy delivered by the HTS system improved convergence and divergence amplitudes with a concomitant reduction in symptoms. The HTS system should be used on those patients with symptoms associated with an accommodative/vergence anomaly when in-office vision therapy supplemented with home therapy is not practical.

Novel high-throughput screening system for identifying STAT3–SH2 antagonists

Constitutive activation of the oncogenic transcription factor STAT3 frequently occurs in various human malignancies. STAT3 activation involves dimerization via intermolecular pTyr–SH2 interaction. Thus, antagonizing this interaction is a feasible approach to inhibit STAT3 activation for cancer therapy. In order to identify selective STAT3 inhibitors, we developed a biochemical HTS system based on AlphaScreen technology, which measures the abilities of test compounds to antagonize pTyr–SH2 interactions. We screened our chemical libraries using this system and identified 5,15-diphenylporphyrin (5,15-DPP) as a selective STAT3–SH2 antagonist. Selective inhibition of STAT3 nuclear translocation and DNA biding activity was observed in cells treated with 5,15-DPP. IL-6-dependent dimerization of STAT3, c-myc promoter binding and c-myc protein expression were all suppressed by 5,15-DPP, whereas no decrement in either expression or phosphorylation level of STAT3 was observed. Thus, the HTS assay system represented herein may be useful for identifying novel STAT3–SH2 antagonists.