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Marmosets have emerged as a valuable primate model in ophthalmic research due to their similarity to the human visual system and their potential for generating transgenic models to advance the development of therapies. In this study, we isolated and cultured primary retinal pigment epithelium (RPE) cells from marmosets to investigate the mechanisms underlying RPE dysfunction in aging and age-related macular degeneration (AMD). We confirmed that our culture conditions and materials supported the formation of RPE monolayers with functional tight junctions that closely resembled the in vivo RPE. Since serum has been shown to induce epithelial–mesenchymal transition (EMT) in RPE cells, we compared the effects of fetal bovine serum (FBS) with serum-free supplements B27 on transepithelial electrical resistance (TER), cell proliferation, and morphological characteristics. Additionally, we assessed the age-related morphological changes of in vivo and primary RPE cells. Our results indicate that primary marmoset RPE cells exhibit in vivo-like characteristics, while cells obtained from an older donor show evidence of aging, including a failure to form a polarized monolayer, low TER, and delayed cell cycle. In conclusion, our primary marmoset RPE cells provide a reliable in vitro model for developing novel therapeutics for visual-threatening disorders such as AMD, which can be used before animal experiments using marmosets.

Supplementary Data from The Reversible Fourth-Generation EGFR Tyrosine Kinase Inhibitor OBX02–011 Overcomes C797S-Mediated Resistance in Lung Cancer

Osimertinib is an irreversible third-generation EGFR tyrosine kinase inhibitor (TKI) that was initially developed to overcome the EGFR T790M mutation and is used as a standard therapy in patients with advanced non–small cell lung cancer (NSCLC) with EGFR-activating mutations. Despite the remarkable initial efficacy, osimertinib, like other EGFR-TKIs, is limited by the emergence of acquired resistance. As the EGFR mutation C797S has been identified as a key driver of acquired resistance to osimertinib, development of a drug that targets this clinically relevant mutation could help improve patient outcomes. Here, we report the discovery and preclinical efficacy of OBX02–011, a reversible fourth-generation EGFR-TKI that overcomes the EGFR C797S mutation. Compared with approved EGFR-TKIs, OBX02–011 showed potent anticancer effects and inhibited EGFR-related signaling in various models, including those harboring the EGFR C797S mutation. In addition, in transgenic mouse models (EGFRL858R/T790M/C797S), OBX02–011 treatment effectively inhibited tumor growth and EGFR activity, leading to enhanced survival. Collectively, these results suggest that OBX02–011 may be a promising new EGFR-TKI to overcome C797S-mediated resistance in NSCLC.Significance:OBX02–011 is designed to target EGFR C797S and can overcome EGFR double and triple mutations to effectively treat lung cancer.

This file contains Supplemental figures, tables, and legends that accompany the main figures, as indicated in each legend. There are 3 supplemental tables and 6 supplemental figures. Table S1. List of potential target of the GNS compounds that were profiled in Figure 1 of the main text. Table S2. Half-maximal inhibitor concentration of the EGFR inhibitors tested. Table S3. Blood-brain-barrier penetration of the GNS compounds as assessed by the indicated pharmacokinetic measurements. Figure S1. Structural modeling studies show the binding characteristics of the indicated EGFR inhibitors. Figure S2. The effects of each EGFR inhibitor in the cell lines expressing mutant or wild type EGFR. Figure S3. In vivo effects of the GNS compounds. Figure S4. In vivo anti-tumor effects of the GNS compounds. Figure S5. In vivo anti-tumor effects of the GNS compounds in intracranial EGFR-mutant lung cancer. Figure S6. In vivo anti-tumor effects of the indicated EGFR inhibitors in intracranial EGFR-mutant lung cancer.

The clinical utility of approved EGFR small-molecule kinase inhibitors is plagued both by toxicity against wild-type EGFR and by metastatic progression in the central nervous system, a disease sanctuary site. Here, we report the discovery and preclinical efficacy of GNS-1486 and GNS-1481, two novel small-molecule EGFR kinase inhibitors that are selective for T790M-mutant isoforms of EGFR. Both agents were effective in multiple mouse xenograft models of human lung adenocarcinoma (T790M-positive or -negative), exhibiting less activity against wild-type EGFR than existing approved EGFR kinase inhibitors (including osimertinib). In addition, GNS-1486 showed superior potency against intracranial metastasis of EGFR-mutant lung adenocarcinoma. Our results offer a preclinical proof of concept for new EGFR kinase inhibitors with the potential to improve therapeutic index and efficacy against brain metastases in patients. Cancer Res; 77(5); 1200–11. ©2017 AACR.

Supplementary Table from The Reversible Fourth-Generation EGFR Tyrosine Kinase Inhibitor OBX02–011 Overcomes C797S-Mediated Resistance in Lung Cancer

Supplementary Figure Legends 1-6 from Pierce1, a Novel p53 Target Gene Contributing to the Ultraviolet-Induced DNA Damage Response

Supplementary Table 1 from Pierce1, a Novel p53 Target Gene Contributing to the Ultraviolet-Induced DNA Damage Response

The placenta regulates maternal-fetal communication, and its defect leads to significant pregnancy complications. The maternal and embryonic circulations are primitively connected in early placentation, but the function of the placenta during this developmentally essential period is relatively unknown. We thus performed a comparative proteomic analysis of the placenta before and after primary placentation and found that the metabolism and transport of lipids were characteristically activated in this period. The placental fatty acid (FA) carriers in specific placental compartments were upregulated according to gestational age, and metabolomic analysis also showed that the placental transport of FAs increased in a time-dependent manner. Further analysis of two mutant mice models with embryonic lethality revealed that lipid-related signatures could reflect the functional state of the placenta. Our findings highlight the importance of the nutrient transport function of the primary placenta in the early gestational period and the role of lipids in embryonic development. Summary Sentence The placenta is activated characteristically in terms of lipid transport during primary placentation, and the lipid-related signatures closely reflect the functional state of the placenta.

Background Cytidine-5′-diphosphate choline (CDP-choline) has been suggested to exert neuroprotective and neuroreparative effects and may be beneficial for patients with stimulant dependence. This randomized, double-blind, placebo-controlled study in methamphetamine (MA) dependence investigated effects of CDP-choline on the brain structures and their associations with craving and MA use. Methods MA users (n = 44) were randomized to receive 2 g/day of CDP-choline (n = 22) or placebo (n = 22) for 8 weeks. Patients underwent brain magnetic resonance imaging (MRI) at baseline and 8-week follow-up. Healthy individuals (n = 27) were also examined using brain MRI at the same interval. Voxel-based morphometry analysis was conducted to examine changes in gray matter (GM) volumes and their associations with craving and MA use. Results Craving for MA was significantly reduced after the 8 week-treatment with CDP-choline (p = 0.01), but not with the placebo treatment (p = 0.10). There was no significant difference in the total number of MA-negative urine samples between the two groups (p = 0.19). With CDP-choline treatment, GM volumes in the left middle frontal gyrus (p = 0.001), right hippocampus (p = 0.009), and left precuneus (p = 0.001) were significantly increased compared to the placebo and control groups. Increased GM volumes in the left middle frontal gyrus with CDP-choline treatment were associated with reduced craving for MA (Spearman's ρ = −0.56, p = 0.03). In addition, the right hippocampal volume increases were positively associated with the total number of MA-negative urine results in the CDP-choline group (Spearman's ρ = 0.67, p = 0.006). Conclusion Our findings suggest that CDP-choline may increase GM volumes of MA-dependent patients, which may be related to decreases in MA use and craving.

C1q/tumor necrosis factor–related protein 9 (CTRP9) is an adipokine and has high potential as a therapeutic target. However, the role of CTRP9 in cardiovascular disease pathogenesis remains unclear. We found CTRP9 to induce HDAC7 and p38 MAPK phosphorylation via tight regulation of AMPK in vascular endothelial cells, leading to angiogenesis through increased MEF2 activity. The expression of CTRP9 and atheroprotective MEF2 was decreased in plaque tissue of atherosclerotic patients and the ventricle of post-infarction mice. CTRP9 treatment inhibited the formation of atherosclerotic plaques in ApoE KO and CTRP9 KO mice. In addition, CTRP9 induced significant ischemic injury prevention in the post-MI mice. Clinically, serum CTRP9 levels were reduced in patients with MI compared with healthy controls. In summary, CTRP9 induces a vasoprotective response via the AMPK/HDAC7/p38 MAPK pathway in vascular endothelial cells, whereas its absence can contribute to atherosclerosis and MI. Hence, CTRP9 may represent a valuable therapeutic target and biomarker in cardiovascular diseases.

Although epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have shown dramatic response and improvement in treating lung cancer with mutant EGFR, the emergence of drug resistance remains a major problem. In particular, some mutations including T790 M and C797S have been recognized as mechanisms of acquired resistance because they weaken binding affinity to drugs. To date, many attempts have been made to develop a new drug for overcoming acquired resistance to EGFR-TKIs, including secondary mutations. However, an appropriate animal model to evaluate in vivo efficacy during novel drug development remains lacking. In this study, we generated a novel transgenic mouse model that conditionally expresses human EGFR

Osimertinib is an irreversible third-generation EGFR tyrosine kinase inhibitor (TKI) that was initially developed to overcome the EGFR T790M mutation and is used as a standard therapy in patients with advanced non–small cell lung cancer (NSCLC) with EGFR-activating mutations. Despite the remarkable initial efficacy, osimertinib, like other EGFR-TKIs, is limited by the emergence of acquired resistance. As the EGFR mutation C797S has been identified as a key driver of acquired resistance to osimertinib, development of a drug that targets this clinically relevant mutation could help improve patient outcomes. Here, we report the discovery and preclinical efficacy of OBX02–011, a reversible fourth-generation EGFR-TKI that overcomes the EGFR C797S mutation. Compared with approved EGFR-TKIs, OBX02–011 showed potent anticancer effects and inhibited EGFR-related signaling in various models, including those harboring the EGFR C797S mutation. In addition, in transgenic mouse models (EGFRL858R/T790M/C797S), OBX02–011 treatment effectively inhibited tumor growth and EGFR activity, leading to enhanced survival. Collectively, these results suggest that OBX02–011 may be a promising new EGFR-TKI to overcome C797S-mediated resistance in NSCLC. Significance: OBX02–011 is designed to target EGFR C797S and can overcome EGFR double and triple mutations to effectively treat lung cancer.

CRISPR-Cas systems, including Cas9 and Cpf1 (Cas12a), are promising tools for generating gene knockout mouse models. Unlike Cas9, Cpf1 can generate multiple crRNAs from a single concatemeric crRNA precursor, which is favorable for multiplex gene editing. Recently, a hybrid guide RNA (hgRNA) system employing both Cas9 and Cpf1 was developed for multiplex gene editing. As the crRNA of Cpf1 was linked to the 3' end of the sgRNA for Cas9, it can be split into separate guide RNAs by Cpf1. To examine whether this Cas9-Cpf1 hybrid system is suitable for multiplex gene knockouts in the mouse embryo, we generated an hgRNA that simultaneously targets the mouse Il10ra gene by Cas9 and mouse Dr3 (or Tnfrsf25, death receptor3) gene by Cpf1. The expression of hgRNA from a single promoter induced significant indels at each gene in cultured mouse cells upon the co-expression of both Cas9 and Cpf1. Interestingly, the hgRNA exhibited comparable Cas9-mediated indel activity without Cpf1 expression. Similarly, when the hgRNA was co-microinjected with both Cas9 and Cpf1 mRNAs into mouse zygotes at the pronuclear stage, founder mice were generated harboring mutations in both the Il10ra and Dr3 genes. However, when Cas9 mRNA was used alone without Cpf1 mRNA, the mouse Il10ra gene targeting was significantly decreased. These results indicate that the hgRNA system is a possible tool for multiplex gene targeting in the mouse embryo.

With a plethora of technology available to support teaching and learning, preservice teachers are expected to become well-versed in technology literacy and competencies through their teacher educat...

KRAS-mutant non-small cell lung cancer (NSCLC) is a major lung cancer subtype that leads to many cancer-related deaths worldwide. Although numerous studies on KRAS-mutant type NSCLC have been conducted, new oncogenic or tumor suppressive genes need to be detected because a large proportion of NSCLC patients does not respond to currently used therapeutics. Here, we show the tumor-promoting function of a cell cycle-related protein, PIERCE1, in KRAS-mutant NSCLC. Mechanistically, PIERCE1 depletion inhibits cell growth and AKT phosphorylation (pAKT) at S473, which is particularly observed in KRAS-mutant lung cancers. Analyses of AKT-related genes using microarray, immunoblotting, and real-time quantitative PCR indicated that PIERCE1 negatively regulates the gene expression of the AKT suppressor, TRIB3, through the CHOP pathway, which is a key regulatory pathway for TRIB3 expression. Similarly, in vivo analyses of PIERCE1 depletion in the KRAS mutation-related lung cancer mouse models revealed the suppressive effect of PIERCE1 knockout in urethane- and KRASG12D-induced lung tumorigenesis with decreased pAKT levels observed in the tumors. Tissue microarrays of human lung cancers indicated the expression of PIERCE1 in 83% of lung cancers and its correlation with pAKT expression. Thus, we illustrate how PIERCE1 depletion may serve as a therapeutic strategy against KRAS-mutant NSCLC and propose the clinical benefit of PIERCE1.

A conical-shaped Si dioxide nano-pattern was employed to sapphire substrate in order to improve the light extraction efficiency of light-emitting diodes. The conical-shaped Si dioxide nano-patterns were fabricated on a 2-inch sapphire wafer using direct imprinting of hydrogen silsesquioxane material. A blue-LED structure was grown on conical-shaped silicon-dioxide nano-patterned sapphire substrates. Photoluminescence and electroluminescence measurements were used to confirm the effectiveness of the nanoscale Si oxide patterned sapphire. An improvement in the luminescence efficiency was observed when nanoscale Si oxide patterned sapphire substrate was used. 1.5 times higher PL intensity and 1.6 times higher EL intensity were observed for GaN LED structure grown on nanoscale Si oxide patterned sapphire, compared to LED structure grown on conventional flat sapphire wafer.

Tumor-derived exosomes (TEXs) contain enriched miRNAs that act as novel non-invasive biomarkers for cancer diagnosis and play a role in cancer progression. We investigated the exosomal miRNAs that affect cancer progression in non-small cell lung cancer (NSCLC) and identified the specific molecules involved. We identified that specific miRNAs in NSCLC cell-released exosomes can modulate angiogenesis, among which miR-619-5p was the most potent inducer. RCAN1.4 was identified as a target of miR-619-5p and its suppression promoted angiogenesis. Furthermore, the suppression of RCAN1.4 induced cell proliferation and metastasis in NSCLC cells. In patients with NSCLC, the level of RCAN1.4 expression was significantly lower, and that of miR-619-5p significantly higher, in tumor than normal lung tissues. miR-619-5p expression was higher than normal in exosomes isolated from the plasma of NSCLC patients. Finally, hypoxic conditions induced miR-619-5p upload into NSCLC cell-derived exosomes. Our findings indicate that exosomal miR-619-5p promotes the growth and metastasis of NSCLCs by regulating RCAN1.4 and can serve as a diagnostic indicator for these lung cancers.

In this study, two different conical-shape nano-patterns were fabricated on the 2-inch diameter sapphire wafer in order to improve the efficiency of light-emitting diodes. The conical-shape nano-patterns were fabricated on the sapphire wafer using a nanoimprint technique and dry etching method. A blue LED structure was grown on the nanoscale-patterned sapphire substrates. The photoluminescence and electroluminescence were measured to confirm the effectiveness of the nano-scale patterns. An improvement in the luminescence efficiency was observed in case that nano-patterned sapphire substrate was used; a 1.44 times-higher photoluminescence intensity and a 1.5 times-higher electroluminescence intensity were observed, compared to those of the light-emitting diodes structure grown on a conventional flat sapphire wafer.

Retinal progenitor cells (RPCs) divide in limited numbers to generate the cells comprising vertebrate retina. The molecular mechanism that leads RPC to the division limit, however, remains elusive. Here, we find that the hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) in an RPC subset by deletion of tuberous sclerosis complex 1 (Tsc1) makes the RPCs arrive at the division limit precociously and produce Müller glia (MG) that degenerate from senescence-associated cell death. We further show the hyperproliferation of Tsc1-deficient RPCs and the degeneration of MG in the mouse retina disappear by concomitant deletion of hypoxia-induced factor 1-alpha (Hif1a), which induces glycolytic gene expression to support mTORC1-induced RPC proliferation. Collectively, our results suggest that, by having mTORC1 constitutively active, an RPC divides and exhausts mitotic capacity faster than neighboring RPCs, and thus produces retinal cells that degenerate with aging-related changes.

The emergence of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) with activating EGFR mutations is a major hindrance to treatment. We investigated the effects of p53 in primary sensitivity and acquired resistance to EGFR-TKIs in NSCLC cells. Changes in sensitivity to EGFR-TKIs were determined using p53 overexpression or knockdown in cells with activating EGFR mutations. We investigated EMT-related molecules, morphologic changes, and AXL induction to elucidate mechanisms of acquired resistance to EGFR-TKIs according to p53 status. Changes in p53 status affected primary sensitivity as well as acquired resistance to EGFR-TKIs according to cell type. Firstly, p53 silencing did not affect primary and acquired resistance to EGFR-TKIs in PC-9 cells, but it led to primary resistance to EGFR-TKIs through AXL induction in HCC827 cells. Secondly, p53 silencing in H1975 cells enhanced the sensitivity to osimertinib through the emergence of mesenchymal-to-epithelial transition, and the emergence of acquired resistance to osimertinib in p53 knockout cells was much slower than in H1975 cells. Furthermore, two cell lines (H1975 and H1975/p53KO) demonstrated the different mechanisms of acquired resistance to osimertinib. Lastly, the introduction of mutant p53-R273H induced the epithelial-to-mesenchymal transition and exerted resistance to EGFR-TKIs in cells with activating EGFR mutations. These findings indicate that p53 mutations can be associated with primary or acquired resistance to EGFR-TKIs. Thus, the status or mutations of p53 may be considered as routes to improving the therapeutic effects of EGFR-TKIs in NSCLC.

Background: Tumor-derived exosomal microRNAs are key elements of the cell-cell communications response to lots of stimuli. However, various functions of the exosome in tumor suppression by radiotherapy (RT) are not clearly understood. Our study showed a previously unknown interaction of p53 and histone deacetylase 5 (HDAC5) by radiation exposure in hepatocellular carcinoma (HCC). Methods: Using serial ultracentrifugation methods, radiation and non-radiation exosomes were purified to investigate the radioresistance of miRNA151a-3p. Radiation doses were treated in 2gy and 4gy using radiation equipment X-RAD 320 to observe the expression of HDAC5 and p53 in hepatic cancer cells. Exosomal miRNA bioinformatics analysis was conducted to find a variation in the miRNA configuration inside Exosome after radiation exposure.Results: HDAC5 and p53 interacted by exposure to radiation, which increased exosome release and altered microRNAs' composition within exosomes. Also, we have described the intercommunication occurring between irradiated and untreated cells via exosomal microRNAs that affect tumor proliferation. In particular, the expression of exosomal microR151a-3p was markedly reduced by radiation treatment. We confirmed that inhibition of exosomal microR151a-3p promotes suppression of non-irradiated cancer cells, thereby increasing RT sensitivity. Conclusion: our present findings demonstrated HDAC5 is a key component of the p53-mediated release of exosomes resulting in tumor suppression through exosomal microRNA-151a-3p in response to radiation. Finally, we highlight the important role of exosomal microRNA 151a-3p as a biomarker in enhancing RT sensitivity.

Despite the strong influence of the gut microbiota on atherosclerosis, a causal relationship between atherosclerosis pathophysiology and gut microbiota is still unverified. This study was performed to determine the impact of the gut microbiota on the pathogenesis of atherosclerosis caused by genetic deficiency. To elucidate the influence of the gut microbiota on atherosclerosis pathogenesis, an atherosclerosis-prone mouse model (C1q/TNF-related protein 9-knockout (CTRP9-KO) mice) was generated. The gut microbial compositions of CTRP9-KO and WT control mice were compared. Fecal microbiota transplantation (FMT) was performed to confirm the association between gut microbial composition and the progression of atherosclerosis. FMT largely affected the gut microbiota in both CTRP9-KO and WT mice, and all transplanted mice acquired the gut microbiotas of the donor mice. Atherosclerotic lesions in the carotid arteries were decreased in transplanted CTRP9-KO mice compared to CTRP9-KO mice prior to transplantation. Conversely, WT mice transplanted with the gut microbiotas of CTRP9-KO mice showed the opposite effect as that of CTRP9-KO mice transplanted with the gut microbiotas of WT mice. Here, we show that CTRP9 gene deficiency is related to the distribution of the gut microbiota in subjects with atherosclerosis. Transplantation of WT microbiotas into CTRP9-KO mice protected against the progression of atherosclerosis. Conversely, the transplantation of CTRP9-KO microbiotas into WT mice promoted the progression of atherosclerosis. Treating atherosclerosis by restoring gut microbial homeostasis may be an effective therapeutic strategy.

Genetically engineered mouse (GEM) models have been revolutionizing the biomedical studies on deciphering the physiological roles of genes in vivo. In addition to deactivating a gene in mice, diverse strategies have been created to monitor gene expressions and molecular dynamics of specific proteins in vivo. Although gene targeting in mouse embryonic stem (ES) cells was essential for the precise engineering of the mouse genome over almost three decades, this process is a time-consuming, expensive, and laborious one. These days, new technologies that directly apply engineered endonucleases, such as zinc-finger nucleases (ZFNs), Transcription Activator-Like Effector (TALE) Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, into the mouse zygotes are enabling us to rapidly replace conventional gene targeting in mouse ES cells. In this chapter, we will describe the principles of reporter mouse strains and the recent advances in generating them using engineered endonucleases.

BiVO4 has a thickness limitation because of carrier diffusion length; thus, the light-absorption efficiency is limited. To resolve this issue, we propose coating Mo:BiVO4 on nanopatterned electrode...