Development of a Dual Factor Activatable Covalent Targeted Photoacoustic Imaging Probe for Tumor Imaging.

Photoacoustic imaging (PAI) is an emerging modality in biomedical imaging with superior imaging depth and specificity. However, PAI still has significant limitations, such as the background noise from endogenous chromophores. To overcome these limitations, we developed a covalent activity-based PAI probe, NOx-JS013, targeting NCEH1. NCEH1, a highly expressed and activated serine hydrolase in aggressive cancers, has the potential to be employed for the diagnosis of cancers. We show that NOx-JS013 labels active NCEH1 in live cells with high selectivity relative to other serine hydrolases. NOx-JS013 also presents its efficacy as a hypoxia-responsive imaging probe in live cells. Finally, NOx-JS013 successfully visualizes aggressive prostate cancer tumors in mouse models of PC3, while negligibly detected in tumors of non-aggressive LNCaP mouse models. These findings show that NOx-JS013 has the potential to be used to develop precision PAI reagents for detecting metastatic progression in various cancers.

Extended Kalman Filter-Based Vehicle Tracking Using Uniform Planar Array for Vehicle Platoon Systems.

We develop an extended Kalman filter-based vehicle tracking algorithm, specifically designed for uniform planar array layouts and vehicle platoon scenarios. We first propose an antenna placement strategy to design the optimal antenna array configuration for precise vehicle tracking in vehicle-to-infrastructure networks. Furthermore, a vehicle tracking algorithm is proposed to improve the position estimation performance by specifically considering the characteristics of the state evolution model for vehicles in the platoon. The proposed algorithm enables the sharing of corrected error transition vectors among platoon vehicles, for the purpose of enhancing the tracking performance for vehicles in unfavorable positions. Lastly, we propose an array partitioning algorithm that effectively divides the entire antenna array into sub-arrays for vehicles in the platoon, aiming to maximize the average tracking performance. Numerical studies verify that the proposed tracking and array partitioning algorithms improve the position estimation performance.

Simple method of measuring ocular rotation in supine position during small incision lenticule extraction.

AIM: To introduce a novel measurement method of static cyclotorsion in small incision lenticule extraction (SMILE) and to investigate the effect of preoperative parameters on cyclotorsion and the effect of cyclotorsion on postoperative outcomes. METHODS: The medical records of 242 patients and 484 eyes who underwent SMILE surgery were retrospectively reviewed. Preoperative intraocular pressure, refractive error, and corneal thickness were investigated. Refractive values and visual acuity were measured at 1d, 1, 3, and 6mo. Ocular cyclotorsion in the supine position was measured by calculating the location and angle of the incision site of the cornea in the anterior slit photograph taken after surgery. RESULTS: Of the total 484 eyes in 242 patients, preoperative mean spherical equivalent (SE) was -4.10±1.64 D, and the mean astigmatism was -0.82±0.74 D. Uncorrected distance visual acuity (UCVA) and SE improved significantly after the surgery. Moreover, 219 (45.2%) eyes had excyclotorsion, 235 (48.6%) eyes had incyclotorsion, and 30 (6.2%) eyes had no torsion. The right eyes tended to be excyclotorted, and the left eyes tended to be incyclotorted (P<0.01). The mean cyclotorsion was 1.18°±3.69°, and the mean absolute value of cyclotorsion was 3.14°±2.26°. The range of cyclotorsion was 0.5°-11.4°. It was found that the smaller the preoperative sphere, the higher the amount of cyclotorsion (r=0.11, P=0.016). There was no significant association between the amount of cyclotorsion and preoperative astigmatism. There was no correlation between sex, preoperative corneal thickness, preoperative intraocular pressure, amount of cyclotorsion, and direction of cyclotorsion. The ratio of right eye excyclotorsion and left eye incyclotorsion on 1d was higher than that at 1, 3, and 6mo (all P<0.01). There was no difference between the 1, 3, and 6mo results in the right and left eyes (P=0.15, P=0.16, respectively). CONCLUSION: The newly devised ocular cyclotorsion measurement method can be used to evaluate ocular cyclotorsion after SMILE. Preoperative SE is associated with the amount of cyclotorsion, however, cyclotorsion doesn't have a significant effect on the results of SMILE surgery.

Discovery of first-in-class inhibitors of ASH1L histone methyltransferase with anti-leukemic activity.

ASH1L histone methyltransferase plays a crucial role in the pathogenesis of different diseases, including acute leukemia. While ASH1L represents an attractive drug target, developing ASH1L inhibitors is challenging, as the catalytic SET domain adapts an inactive conformation with autoinhibitory loop blocking the access to the active site. Here, by applying fragment-based screening followed by medicinal chemistry and a structure-based design, we developed first-in-class small molecule inhibitors of the ASH1L SET domain. The crystal structures of ASH1L-inhibitor complexes reveal compound binding to the autoinhibitory loop region in the SET domain. When tested in MLL leukemia models, our lead compound, AS-99, blocks cell proliferation, induces apoptosis and differentiation, downregulates MLL fusion target genes, and reduces the leukemia burden in vivo. This work validates the ASH1L SET domain as a druggable target and provides a chemical probe to further study the biological functions of ASH1L as well as to develop therapeutic agents.

Full-Duplex Relay for Millimeter Wave Vehicular Platoon Communciations.

The safety and consistency of platoon-based driving are guaranteed via reliable communication between vehicles in a platoon. In this paper, we propose to exploit a full-duplex (FD) relay vehicle for millimeter-wave vehicular communications in platoon-based driving. Considering that a lead vehicle broadcasts information to all vehicles in a platoon, we consider two power allocation problems-maximizing broadcast information rates with a power constraint and minimizing power consumption with a quality-of-service constraint. In particular, for a four-vehicle platoon communication, we derive closed-form solutions for optimal power allocation and present numerical results to verify the performance of the proposed FD relay vehicles.

Synthesis and biological activity of 2-cyanoacrylamide derivatives tethered to imidazopyridine as TAK1 inhibitors.

The importance of transforming growth factor beta-activated kinase 1 (TAK1) to cell survival has been demonstrated in many studies. TAK1 regulates signalling cascades, the NF-κB pathway and the mitogen-activated protein kinase (MAPK) pathway. TAK1 inhibitors can induce the apoptosis of cancerous cells, and irreversible inhibitors such as (5Z)-7-oxozeaenol are highly potent. However, they can react non-specifically with cysteine residues in proteins, which may have serious adverse effects. Reversible covalent inhibitors have been suggested as alternatives. We synthesised imidazopyridine derivatives as novel TAK1 inhibitors, which have 2-cyanoacrylamide moiety that can form reversible covalent bonding. A derivative with 2-cyano-3-(6-methylpyridin-2-yl)acrylamide (13h) exhibited potent TAK1 inhibitory activity with an IC50 of 27 nM. It showed a reversible reaction with ß-mercaptoethanol, which supports its potential as a reversible covalent inhibitor.

Structure-activity relationship of 7-aryl-2-anilino-pyrrolopyrimidines as Mer and Axl tyrosine kinase inhibitors.

The TAM (Axl, Mer, and Tyro3) family is implicated in the survival and chemoresistance of tumours and has emerged as a potential therapeutic target. A novel series of 7-aryl-2-anilino-pyrrolopyrimidines were identified as potent Axl/Mer tyrosine kinase inhibitors without significant inhibition of Tyro3. A representative compound 27 exhibited IC50 values of 2 nM and 16 nM for Mer and Axl, respectively, and considerable inhibition for Mer phosphorylation in cells. Docking studies suggested that the formation of a salt bridge between the nitrogen of the aniline moiety with ASP678 of the Mer kinase domain as well as an interaction with the hinge region that most kinase inhibitors have in common would be essential to retain activity. These results could provide useful information for finding promising inhibitors of Axl/Mer for the treatment of cancer.

Visual Prognostic Factors of Epiretinal Membrane Surgery in Patients with Pseudophakia.

PURPOSE: To evaluate the visual prognostic factors in patients with pseudophakic epiretinal membrane (ERM) after vitrectomy using spectral domain optical coherence tomography (SD-OCT). METHOD: A retrospective review of patients with pseudophakic ERM having undergone vitrectomy was conducted. Best corrected visual acuity (BCVA) and SD-OCT were conducted before and 1, 3, and 6 months after vitrectomy. Known visual prognostic factors, such as inner-retina irregularity index, central foveal thickness (CFT), central inner retinal layer thickness (CIRLT), cone outer segment tip defect length, and photoreceptor outer segment length, were reviewed and their correlation with BCVA was analyzed. RESULTS: Forty-three patients (mean age: 64.88 ± 10.46 years) with pseudophakic ERM were included. BCVA significantly improved after vitrectomy (logMAR 0.30 ± 0.24 vs. 0.11 ± 0.14, p < 0.001). The preoperative high inner-retina irregularity index significantly correlated with poor postoperative BCVA in patients with pseudophakic ERM (correlation coefficient 0.583, p < 0.001). Postoperative improvements of inner retinal SD-OCT findings, such as inner-retina irregularity index, CFT, and CIRLT, were significantly associated with the amount of BCVA improvement after ERM surgery (correlation coefficients were as follows: inner-retina irregularity index - 0.711, p < 0.001; CFT - 0.462, p = 0.002; CIRLT - 0.596, p < 0.001). However, preoperative outer retinal SD-OCT findings were not associated with postoperative visual prognosis. CONCLUSION: From this study, we determined the visual prognostic factors of ERM surgery without confounding factors, such as visual acuity improvement following combined cataract surgery, and inner retinal SD-OCT findings more significantly associated with the visual prognosis of ERM surgery compared to outer retinal SD-OCT findings.

Exogenous Addition of 25-Hydroxycholesterol Reduces Level of Very Long-Chain Fatty Acids in X-Linked Adrenoleukodystrophy.

X-Linked adrenoleukodystrophy (X-ALD) is a severe metabolic disorder characterized by the accumulation of very long-chain fatty acids (VLCFAs). Recently, we demonstrated that levels of 25-hydroxycholesterol (25-HC) and cholesterol 25-hydroxylase (CH25H) were found to be elevated in X-ALD. Herein, we report that the exogenous addition of 25-HC significantly reduces C26:0 levels in X-ALD patient-derived fibroblasts and oligodendrocytes differentiated from induced pluripotent stem cells (iPSCs) derived from X-ALD patients. Moreover, 25-HC treatment was found to down-regulate the expression of ELOVL1, a key enzyme for the synthesis of C26. In addition, activation of liver X receptor (LXR), a molecular target of endogenous 25-HC, also reduced C26:0 level. The reduction of C26:0 levels by 25-HC treatment might result, at least partially, from the decrease of ELOVL1 expression as well as the activation of LXR. Our findings could provide a better understanding of the role of 25-HC in X-ALD and useful information to find therapeutic agents to treat X-ALD.

Design, synthesis and cytotoxicity of chimeric erlotinib-alkylphospholipid hybrids.

Two series of erlotinib-alkylphospholipid hybrids were prepared and evaluated for their antiproliferative activities against a panel of four cell lines representing lung, breast, liver and skin cancers using erlotinib and miltefosine as reference standards. Amide analogs elicited more enhanced cytotoxic activity than analogous esters. Amide derivatives 8d and 8e exhibited promising broad-spectrum antiproliferative activity and higher efficacy than reference erlotinib and miltefosine. Their cellular GI50 values was in the ranges of 24.7-46.9 µM and 26.8-43.1 µM for 8e and 8d respectively. Assay results of the inhibitory activity of the prepared compounds on EGFR kinase reaction and Akt phosphorylation in conjugation with statistical correlation analysis indicated that other mechanisms might contribute to their elicited cytotoxicities. In addition, statistical correlation analysis revealed that mechanisms of elicited cytotoxicities for amide series might be different from ester series. In addition, correlation analysis indicated variations in the mechanisms according to the types of cell line.

Click chemistry for improvement in selectivity of quinazoline-based kinase inhibitors for mutant epidermal growth factor receptors.

Discovery of mutant-selective kinase inhibitors is one of the challenges in medicinal chemistry and is a main issue for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. We tried to improve the selectivity of pan-HER inhibitors for mutant EGFRs. Utilizing click chemistry, triazole-tethered quinazoline derivatives were synthesized, based on a quinazoline scaffold showing pan-HER inhibition. The representative compound 5j exhibited 17- and 52-fold improved selectivity for EGFR L858R/T790M over wild-type EGFR and HER2, respectively, and demonstrated 6.7-fold more potent antiproliferative activity against PC9 cells harboring EGFR-activating mutation than gefitinib. Although the described quinazolines did not surpass pyrimidines as 3rd generation EGFR inhibitors in terms of selectivity for mutant EGFRs, our approach might provide information that would help in the identification of mutant-selective compounds among pan-HER inhibitors using the quinazoline scaffold.

A 3D microvascular network model to study the impact of hypoxia on the extravasation potential of breast cell lines.

Hypoxia is a common feature of the tumor microenvironment. Accumulating evidence has demonstrated hypoxia to be an important trigger of tumor cell invasion or metastasizes via hypoxia-signaling cascades, including hypoxia-inducible factors (HIFs). Microfluidic model can be a reliable in vitro tool for systematically interrogating individual factors and their accompanying downstream effects, which may otherwise be difficult to study in complex tumor tissues. Here, we used an in vitro model of microvascular networks in a microfluidic chip to measure the extravasation potential of breast cell lines subjected to different oxygen conditions. Through the use of HIF-1α knock-down cell lines, we also validated the importance of HIF-1α in the transmigration ability of human breast cell lines. Three human breast cell lines derived from human breast tissues (MCF10A, MCF-7 and MDA-MB-231) were used in this study to evaluate the role of hypoxia in promoting metastasis at different stages of cancer progression. Under hypoxic conditions, HIF-1α protein level was increased, and coincided with changes in cell morphology, viability and an elevated metastatic potential. These changes were accompanied by an increase in the rate of extravasation compared to normoxia (21% O2). siRNA knockdown of HIF-1α in hypoxic tumors significantly decreased the extravasation rates of all the cell lines tested and may have an effect on the function of metastatic and apoptotic-related cellular processes.

Effect of Gravity Acceleration on Choroidal and Retinal Nerve Fiber Layer Thickness: A Swept-Source Optical Coherence Tomography Study.

Purpose: The purpose of this study was to evaluate the effect of gravity acceleration on choroidal and retinal nerve fiber layer (RNFL) thickness using swept-source optical coherence tomography (SS-OCT). Methods: Thirteen healthy volunteers who planned to participate in human centrifuge training as part of the flight surgeon selection process enrolled this study. During centrifuge training, gravity was gradually increased up to six times that of sea level. All subjects underwent complete ophthalmologic examination and three-dimensional wide-scanning SS-OCT imaging (DRI OCT-1 Atlantis; Topcon, Tokyo, Japan). Imaging was performed before (baseline), immediately after, and 15, 30, and 60 minutes after centrifuge training. Changes in choroidal thickness, choroidal volume, retinal thickness, and RNFL thickness after centrifuge training were analyzed. Results: Mean choroidal thickness significantly and transiently decreased immediately (258.19 ± 73.54 µm, P < 0.001), 15 minutes (258.54 ± 75.12 µm, P = 0.001), and 30 minutes (254.31 ± 66.92, P = 0.001) after human centrifuge training, relative to baseline (273.35 ± 80.80 µm). However, the decreased choroidal thickness returned to baseline levels 1 hour after centrifuge training (270.12 ± 71.69 µm, P = 0.437). Mean retinal thickness and RNFL thickness were not significantly affected by human centrifuge training. In participants who suffered from gravity-force induced loss of consciousness (G-LOC) during training, the amount of the choroidal thickness decrease was larger than in participants who did not experience G-LOC. However, because of the small sample size, the difference, although large, was not statistically significant. Conclusions: Choroidal thickness and volume significantly and transiently decreased after human centrifuge training, which might reflect that choroidal perfusion was transiently decreased during human centrifuge training. Considering choroidal thickness decreased after human centrifuge training, long-term exposure to a high gravity environment may lead to ischemic injury to ocular structures.

Endogenous TRPV4 Expression of a Hybrid Neuronal Cell Line N18D3 and Its Utilization to Find a Novel Synthetic Ligand.

Primary sensory afferent neurons detect environmental and painful stimuli at their peripheral termini. A group of transient receptor potential ion channels (TRPs) are expressed in these neurons and constitute sensor molecules for the stimuli such as thermal, mechanical, and chemical insults. We examined whether a mouse sensory neuronal line, N18D3, shows the sensory TRP expressions and their functionality. In Ca2+ imaging and electrophysiology with these cells, putative TRPV4-mediated responses were observed. TRPV4-specific sensory modalities including sensitivity to a specific agonist, hypotonicity, or an elevated temperature were reproduced in N18D3 cells. Electrophysiological and pharmacological profiles conformed to those from native TRPV4 of primarily cultured neurons. The TRPV4 expression in N18D3 was also confirmed by RT-PCR and Western blot analyses. Thus, N18D3 cells may represent TRPV4-expressing sensory neurons. Further, using this cell lines, we discovered a novel synthetic TRPV4-specific agonist, MLV-0901. These results suggest that N18D3 is a reliable cell line for functional and pharmacological TRPV4 assays. The chemical information from the novel agonist will contribute to TRPV4-targeting drug design.

Identification of aminosulfonylarylisoxazole as microRNA-31 regulators.

The discovery of small-molecule regulators of microRNAs remains challenging, but a few have been reported. Herein, we describe small-molecule inhibitors of miR-31, a tumor-associated microRNA (miRNA), identified by high-throughput screening using a cell-based reporter assay. Aminosulfonylarylisoxazole compounds exhibited higher specificity for miR-31 than for six other miRNAs, i.e., miR-15a, miR-16, miR-21, miR-92a-1, miR-146a, and miR-155, and increased the expression of miR-31 target genes. The down-regulation of mature miR-31 was observed, while its precursor form increased following treatment with the compounds. Thus, the compounds may target the processing of pre-miR-31 into mature miR-31 and thereby inhibit the production of mature miR-31.

Transetherification of 2,4-dimethoxynitrobenzene by aromatic nucleophilic substitution.

In view of the few reports concerning aromatic nucleophilic substitution reactions featuring an alkoxy group as a leaving group, the aromatic nucleophilic substitution of 2,4-dimethoxynitrobenzene was investigated with a bulky t-butoxide nucleophile under microwave irradiation. The transetherification of 2,4-dimethoxynitrobenezene with sodium t-butoxide under specific conditions, namely for 20 min at 110°C in 10% dimethoxyethane in toluene, afforded the desired product in 87% yield with exclusive ortho-selectivity. A variety of reaction conditions were screened to obtain the maximum yield. The aromatic nucleophilic substitution of 2,4-dimethoxynitrobenzene with t-butoxide should be carried out under controlled conditions in order to avoid the formation of byproducts, unlike that of dihalogenated activated benzenes. Among the formed byproducts, a major compound was elucidated as 2,4-dimethoxy-N-(5-methoxy-2-nitrophenyl)aniline by X-ray crystallography.

Structure-Activity Relationship of Indole-Tethered Pyrimidine Derivatives that Concurrently Inhibit Epidermal Growth Factor Receptor and Other Angiokinases.

Antiangiogenic agents have been widely investigated in combination with standard chemotherapy or targeted cancer agents for better management of advanced cancers. Therapeutic agents that concurrently inhibit epidermal growth factor receptor and other angiokinases could be useful alternatives to combination therapies for epidermal growth factor receptor-dependent cancers. Here, we report the synthesis of an indole derivative of pazopanib using a bioisosteric replacement strategy, which was designated MKP101. MKP101 inhibited not only the epidermal growth factor receptor with an IC50 value of 43 nM but also inhibited angiokinases as potently as pazopanib. In addition, MKP101 effectively inhibited vascular endothelial growth factor-induced endothelial proliferation, tube formation, migration of human umbilical vein endothelial cells and proliferation of HCC827, an epidermal growth factor receptor-addicted cancer cell line. A docking model of MKP101 and the kinase domain of the epidermal growth factor receptor was generated to predict its binding mode, and validated by synthesizing and evaluating MKP101 derivatives. Additionally, a study of structure-activity relationships of indolylamino or indolyloxy pyrimidine analogues derived from MKP101 demonstrated that selectivity for epidermal growth factor receptor and other angiokinases, especially vascular endothelial growth factor receptor 2 depends on the position of substituents on pyrimidine and the type of link between pyrimidine and the indole moiety. We believe that this study could provide a basis for developing angiokinase inhibitors having high affinity for the epidermal growth factor receptor, from the pyrimidine scaffold.

3D bio-etching of a complex composite-like embryonic tissue.

Morphogenesis involves a complex series of cell signaling, migration and differentiation events that are coordinated as tissues self-assemble during embryonic development. Collective cell movements such as those that occur during morphogenesis have typically been studied in 2D with single layers of cultured cells adhering to rigid substrates such as glass or plastic. In vivo, the intricacies of the 3D microenvironment and complex 3D responses are pivotal in the formation of functional tissues. To study such processes as collective cell movements within 3D multilayered tissues, we developed a microfluidic technique capable of producing complex 3D laminar multicellular structures. We call this technique "3D tissue-etching" because it is analogous to techniques used in the microelectromechanics (MEMS) field where complex 3D structures are built by successively removing material from a monolithic solid through subtractive manufacturing. We use a custom-designed microfluidic control system to deliver a range of tissue etching reagents (detergents, chelators, proteases, etc.) to specific regions of multilayered tissues. These tissues were previously isolated by microsurgical excision from embryos of the African claw-toed frog, Xenopus laevis. The ability to shape the 3D form of multicellular tissues and to control 3D stimulation will have a high impact on tissue engineering and regeneration applications in bioengineering and medicine as well as provide significant improvements in the synthesis of highly complex 3D integrated multicellular biosystems.

Controlled surface topography regulates collective 3D migration by epithelial-mesenchymal composite embryonic tissues.

Cells in tissues encounter a range of physical cues as they migrate. Probing single cell and collective migratory responses to physically defined three-dimensional (3D) microenvironments and the factors that modulate those responses are critical to understanding how tissue migration is regulated during development, regeneration, and cancer. One key physical factor that regulates cell migration is topography. Most studies on surface topography and cell mechanics have been carried out with single migratory cells, yet little is known about the spreading and motility response of 3D complex multi-cellular tissues to topographical cues. Here, we examine the response to complex topographical cues of microsurgically isolated tissue explants composed of epithelial and mesenchymal cell layers from naturally 3D organized embryos of the aquatic frog Xenopus laevis. We control topography using fabricated micropost arrays (MPAs) and investigate the collective 3D migration of these multi-cellular systems in these MPAs. We find that the topography regulates both collective and individual cell migration and that dense MPAs reduce but do not eliminate tissue spreading. By modulating cell size through the cell cycle inhibitor Mitomycin C or the spacing of the MPAs we uncover how 3D topographical cues disrupt collective cell migration. We find surface topography can direct both single cell motility and tissue spreading, altering tissue-scale processes that enable efficient conversion of single cell motility into collective movement.

Mechanochemical actuators of embryonic epithelial contractility.

Spatiotemporal regulation of cell contractility coordinates cell shape change to construct tissue architecture and ultimately directs the morphology and function of the organism. Here we show that contractility responses to spatially and temporally controlled chemical stimuli depend much more strongly on intercellular mechanical connections than on biochemical cues in both stimulated tissues and adjacent cells. We investigate how the cell contractility is triggered within an embryonic epithelial sheet by local ligand stimulation and coordinates a long-range contraction response. Our custom microfluidic control system allows spatiotemporally controlled stimulation with extracellular ATP, which results in locally distinct contractility followed by mechanical strain pattern formation. The stimulation-response circuit exposed here provides a better understanding of how morphogenetic processes integrate responses to stimulation and how intercellular responses are transmitted across multiple cells. These findings may enable one to create a biological actuator that actively drives morphogenesis.