Endogenous Interaction Research Articles

Managing piracy: Dual-channel strategy for digital contents

<p style='text-indent:20px;'>The Internet offers digital content disc producers the opportunities to design dual channels by introducing an online-direct store alongside traditional retail stores, but also leads related firms to suffer significant piracy problems. Using a game-theoretic framework, we explore dual-channel marketing optimality as a piracy-mitigating strategy for digital content sold in the physical disc format. We construct a price-setting game between a digital content producer and its independent retailer(s) in a pirated market by endogenizing the producer's copyright protection investments. We show that dual-channel marketing, a complement or a substitute for conventional copyright protection, can strategically mitigate the piracy level by increasing the equal-size retail sales volume. We also investigate how firms' pricing strategies and profits are influenced by the endogenous interaction of dual-channel marketing and copyright protection. We unexpectedly find that in a pirated market with insufficient copyright protection, dual-channel marketing can simultaneously raise firm pricing and sales volumes when the producer sells through a monopolistic retailer. We also identify the conditions under which dual-channel marketing can mitigate profit losses caused by piracy for the producer and the retailer(s). Unlike previous research which shows that dual-channel marketing benefits the producer and the monopolistic retailer because it mitigates double marginalization, in the pirated market, this win-win outcome occurs even if accompanied by aggravated double marginalization. Moreover, dual-channel marketing can mitigate all the firms' profit losses caused by piracy only when it can complement conventional copyright protection, i.e., when the producer sells through a monopolistic retailer or duopolistic retailers. In each situation, counter-intuitively, as copyright protection becomes increasingly costly, although the retailer(s) is (are) more willing to accept dual-channel marketing, the producer has a decreased incentive to design such sales channels.</p>

An Approach for the Real-Time Quantification of Cytosolic Protein-Protein Interactions in Living Cells.

In recent years, cell-based assays have been frequently used in molecular interaction analysis. Cell-based assays complement traditional biochemical and biophysical methods, as they allow for molecular interaction analysis, mode of action studies, and even drug screening processes to be performed under physiologically relevant conditions. In most cellular assays, biomolecules are usually labeled to achieve specificity. In order to overcome some of the drawbacks associated with label-based assays, we have recently introduced "cell-based molography" as a biosensor for the analysis of specific molecular interactions involving native membrane receptors in living cells. Here, we expand this assay to cytosolic protein-protein interactions. First, we created a biomimetic membrane receptor by tethering one cytosolic interaction partner to the plasma membrane. The artificial construct is then coherently arranged into a two-dimensional pattern within the cytosol of living cells. Thanks to the molographic sensor, the specific interactions between the coherently arranged protein and its endogenous interaction partners become visible in real time without the use of a fluorescent label. This method turns out to be an important extension of cell-based molography because it expands the range of interactions that can be analyzed by molography to those in the cytosol of living cells.

Knockdown of TUG1 rescues cardiomyocyte hypertrophy through targeting the miR-497/MEF2C axis.

The aim of this study was to investigate the detailed role and molecular mechanism of long noncoding RNA (lncRNA) taurine upregulated gene 1 (TUG1) in cardiac hypertrophy. Cardiac hypertrophy was established by transverse abdominal aortic constriction (TAC) in vivo or angiotensin II (Ang II) treatment in vitro. Levels of lncRNA TUG1, miR-497 and myocyte enhancer factor 2C (MEF2C) mRNA were assessed by quantitative reverse transcriptase PCR (qRT-PCR). Western blot assay was performed to determine the expression of MEF2C protein. The endogenous interactions among TUG1, miR-497 and MEF2C were confirmed by dual-luciferase reporter and RNA immunoprecipitation assays. Our data indicated that TUG1 was upregulated and miR-497 was downregulated in the TAC rat model and Ang II-induced cardiomyocytes. TUG1 knockdown or miR-497 overexpression alleviated the hypertrophy induced by Ang II in cardiomyocytes. Moreover, TUG1 acted as a sponge of miR-497, and MEF2C was directly targeted and repressed by miR-497. miR-497 overexpression mediated the protective role of TUG1 knockdown in Ang II-induced cardiomyocyte hypertrophy. MEF2C was a functional target of miR-497 in regulating Ang II-induced cardiomyocyte hypertrophy. In addition, TUG1 regulated MEF2C expression through sponging miR-497. Knockdown of TUG1 rescued Ang II-induced hypertrophy in cardiomyocytes at least partly through targeting the miR-497/MEF2C axis, highlighting a novel promising therapeutic target for cardiac hypertrophy treatment.

Targeting androgen regulation of TMPRSS2 and ACE2 as a therapeutic strategy to combat COVID-19

SummaryEpidemiological data showing increased severity and mortality of COVID-19 in men suggests a potential role for androgen in SARS-CoV-2 infection. Here, we present evidence for the transcriptional regulation of SARS-CoV-2 host cell receptor ACE2 and TMPRSS2 by androgen in mouse and human cells. Additionally, we demonstrate the endogenous interaction between TMPRSS2 and ACE2 in human cells and validate ACE2 as a TMPRSS2 substrate. Furthermore, camostat—a TMPRSS2 inhibitor—blocked the cleavage of pseudotype SARS-CoV-2 surface Spike without disrupting TMPRSS2-ACE2 interaction, thus providing evidence for the first time of a direct role of TMPRSS2 in priming the SARS-CoV-2 Spike, required for viral fusion to the host cell. Importantly, androgen-deprivation, anti-androgens, or camostat attenuated the SARS-CoV-2 S-mediated cellular entry. Together, our data provide a strong rationale for clinical evaluations of TMPRSS2 inhibitors and androgen-deprivation therapy/androgen receptor antagonists alone or in combination with antiviral drugs as early as clinically possible to prevent COVID-19 progression.

Cyclicality in non-state violence: theory and evidence

ABSTRACT What explains recurrent fluctuations in violence within militant groups? Temporal swings in the number of attacks perpetrated are common, but lack a general explanation. I provide evidence of cycles in attacks, and provide a theory of why organizational levels of violence vary serially. First, due to the contradictions of non-state violence, there exist, simultaneously, upward and downward pressures on its use. By “contradictions,” I mean that violence is both beneficial and detrimental to an organization. It can secure public support and government concessions, but can also tarnish a group’s reputation and invite retaliatory violence. Second, since the relative strength of each type of pressure (upward or downward) varies inversely with how much it is accommodated, organizations face disincentives to pause at high or low levels of violence. Groups that shift tactics to accommodate one pressure find they have activated another. For example, a group that curtails violent attacks to avoid government repression finds it lacks media attention. These pressures incentivize a reprise in violence, causing the up-and-down pattern to repeat. Finally, the cyclical pattern is reinforced by endogenous interactions between militants’ political environments and their decision-making processes. I provide suggestive support for the arguments by examining the Basque group ETA.

Ligand recognition and allosteric regulation of DRD1-Gs signaling complexes

Dopamine receptors, including D1- and D2-like receptors, are important therapeutic targets in a variety of neurological syndromes, as well as cardiovascular and kidney diseases. Here, we present five cryoelectron microscopy (cryo-EM) structures of the dopamine D1 receptor (DRD1) coupled to Gs heterotrimer in complex with three catechol-based agonists, a non-catechol agonist, and a positive allosteric modulator for endogenous dopamine. These structures revealed that a polar interaction network is essential for catecholamine-like agonist recognition, whereas specific motifs in the extended binding pocket were responsible for discriminating D1- from D2-like receptors. Moreover, allosteric binding at a distinct inner surface pocket improved the activity of DRD1 by stabilizing endogenous dopamine interaction at the orthosteric site. DRD1-Gs interface revealed key features that serve as determinants for G protein coupling. Together, our study provides a structural understanding of the ligand recognition, allosteric regulation, and G protein coupling mechanisms of DRD1.

Robust point-process Granger causality analysis in presence of exogenous temporal modulations and trial-by-trial variability in spike trains.

Assessing directional influences between neurons is instrumental to understand how brain circuits process information. To this end, Granger causality, a technique originally developed for time-continuous signals, has been extended to discrete spike trains. A fundamental assumption of this technique is that the temporal evolution of neuronal responses must be due only to endogenous interactions between recorded units, including self-interactions. This assumption is however rarely met in neurophysiological studies, where the response of each neuron is modulated by other exogenous causes such as, for example, other unobserved units or slow adaptation processes. Here, we propose a novel point-process Granger causality technique that is robust with respect to the two most common exogenous modulations observed in real neuronal responses: within-trial temporal variations in spiking rate and between-trial variability in their magnitudes. This novel method works by explicitly including both types of modulations into the generalized linear model of the neuronal conditional intensity function (CIF). We then assess the causal influence of neuron i onto neuron j by measuring the relative reduction of neuron j's point process likelihood obtained considering or removing neuron i. CIF's hyper-parameters are set on a per-neuron basis by minimizing Akaike's information criterion. In synthetic data sets, generated by means of random processes or networks of integrate-and-fire units, the proposed method recovered with high accuracy, sensitivity and robustness the underlying ground-truth connectivity pattern. Application of presently available point-process Granger causality techniques produced instead a significant number of false positive connections. In real spiking responses recorded from neurons in the monkey pre-motor cortex (area F5), our method revealed many causal relationships between neurons as well as the temporal structure of their interactions. Given its robustness our method can be effectively applied to real neuronal data. Furthermore, its explicit estimate of the effects of unobserved causes on the recorded neuronal firing patterns can help decomposing their temporal variations into endogenous and exogenous components.

Deciphering molecular interactions by proximity labeling.

Many biological processes are executed and regulated through the molecular interactions of proteins and nucleic acids. Proximity labeling (PL) is a technology for tagging the endogenous interaction partners of specific protein 'baits', via genetic fusion to promiscuous enzymes that catalyze the generation of diffusible reactive species in living cells. Tagged molecules that interact with baits can then be enriched and identified by mass spectrometry or nucleic acid sequencing. Here we review the development of PL technologies and highlight studies that have applied PL to the discovery and analysis of molecular interactions. In particular, we focus on the use of PL for mapping protein-protein, protein-RNA and protein-DNA interactions in living cells and organisms.

SPONGEdb: a pan-cancer resource for competing endogenous RNA interactions.

microRNAs (miRNAs) are post-transcriptional regulators involved in many biological processes and human diseases, including cancer. The majority of transcripts compete over a limited pool of miRNAs, giving rise to a complex network of competing endogenous RNA (ceRNA) interactions. Currently, gene-regulatory networks focus mostly on transcription factor-mediated regulation, and dedicated efforts for charting ceRNA regulatory networks are scarce. Recently, it became possible to infer ceRNA interactions genome-wide from matched gene and miRNA expression data. Here, we inferred ceRNA regulatory networks for 22 cancer types and a pan-cancer ceRNA network based on data from The Cancer Genome Atlas. To make these networks accessible to the biomedical community, we present SPONGEdb, a database offering a user-friendly web interface to browse and visualize ceRNA interactions and an application programming interface accessible by accompanying R and Python packages. SPONGEdb allows researchers to identify potent ceRNA regulators via network centrality measures and to assess their potential as cancer biomarkers through survival, cancer hallmark and gene set enrichment analysis. In summary, SPONGEdb is a feature-rich web resource supporting the community in studying ceRNA regulation within and across cancer types.

Intracellular IRF5 Dimerization Assay.

The intracellular interferon regulatory factor 5 (IRF5) dimerization assay is a technique designed to measure molecular interaction(s) with endogenous IRF5. Here, we present two methods that detect endogenous IRF5 homodimerization and interaction of endogenous IR5 with cell penetrating peptide (CPP) inhibitors. Briefly, to detect endogenous IRF5 dimers, THP-1 cells are incubated in the presence or absence of the IRF5-targeted CPP (IRF5-CPP) inhibitor for 30 min then the cells are stimulated with R848 for 1 h. Cell lysates are separated by native-polyacrylamide gel electrophoresis (PAGE) and IRF5 dimers are detected by immunoblotting with IRF5 antibodies. To detect endogenous interactions between IRF5 and FITC-labeled IRF5-CPP, an in-cell fluorescence resonance energy transfer (FRET) assay is used. In this assay, THP-1 cells are left untreated or treated with FITC-IRF5-CPP conjugated inhibitors for 1 h. Next, cells are fixed, permeabilized, and stained with anti-IRF5 and TRITC-conjugated secondary antibodies. Transfer of fluorescence can be measured and calculated as FRET units. These methods provide rapid and accurate assays to detect IRF5 molecular interactions.

Detection of Endogenous RASSF1A Interacting Proteins.

RASSF1A is a Ras effector that promotes the anti-proliferative properties of Ras. It acts as a scaffold protein that regulates several pro-apoptotic signaling pathways, thereby linking Ras to their regulation. However, accumulating evidence suggests that RASSF1A functions as a regulator of other additional biological processes, such as DNA repair and transcription, thereby implicating Ras in the modulation of these biological processes. The mechanisms by which RASSF1A modulates these processes is not fully understood but likely involves interacting with other effectors associated with these functions and coordinating their activity. Thus, to fully understand how RASSF1A manifests its activity, it is critical to identify RASSF1A interacting partners.Unfortunately, the reagents available for the detection of RASSF1A are of poor quality and also exhibit low sensitivity. Here we describe an immunoprecipitation protocol, taking into consideration the limitations of currently available reagents, that can reliably detect the endogenous interaction between RASSF1A and its binding partners.

USP13 interacts with cohesin and regulates its ubiquitination in human cells

Cohesin is a multiprotein ring complex that regulates 3D genome organization, sister chromatid cohesion, gene expression, and DNA repair. Cohesin is known to be ubiquitinated, although the mechanism, regulation, and effects of cohesin ubiquitination remain poorly defined. We previously used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in human HCT116 cells. Here we report that mass spectrometry analysis of dual-affinity purifications identified the USP13 deubiquitinase as a novel cohesin-interacting protein. Subsequent immunoprecipitation/Western blots confirmed the endogenous interaction in HCT116, 293T, HeLa, and RPE-hTERT cells; demonstrated that the interaction occurs specifically in the soluble nuclear fraction (not in the chromatin); requires the ubiquitin-binding domains (UBA1/2) of USP13; and occurs preferentially during DNA replication. Reciprocal dual-affinity purification of endogenous USP13 followed by mass spectrometry demonstrated that cohesin is its primary interactor in the nucleus. Ectopic expression and CRISPR knockout of USP13 showed that USP13 is paradoxically required for both deubiquitination and ubiquitination of cohesin subunits in human cells. USP13 was dispensable for sister chromatid cohesion in HCT116 and HeLa cells, whereas it was required for the dissociation of cohesin from chromatin as cells transit through mitosis. Together these results identify USP13 as a new cohesin-interacting protein that regulates the ubiquitination of cohesin and its cell cycle regulated interaction with chromatin.

Impaired endoplasmic reticulum-mitochondrial signaling in ataxia-telangiectasia.

SummaryThere is evidence that ATM mutated in ataxia-telangiectasia (A-T) plays a key role in protecting against mitochondrial dysfunction, the mechanism for which remains unresolved. We demonstrate here that ATM-deficient cells are exquisitely sensitive to nutrient deprivation, which can be explained by defective cross talk between the endoplasmic reticulum (ER) and the mitochondrion. Tethering between these two organelles in response to stress was reduced in cells lacking ATM, and consistent with this, Ca2+ release and transfer between ER and mitochondria was reduced dramatically when compared with control cells. The impact of this on mitochondrial function was evident from an increase in oxygen consumption rates and a defect in mitophagy in ATM-deficient cells. Our findings reveal that ER-mitochondrial connectivity through IP3R1-GRP75-VDAC1, to maintain Ca2+ homeostasis, as well as an abnormality in mitochondrial fusion defective in response to nutrient stress, can account for at least part of the mitochondrial dysfunction observed in A-T cells.

A case study of the pyramid scheme in China based on communication network

This paper discusses the structure and endogenous process of a pyramid scheme organization. While most previous studies focus on the operation mode of pyramid scheme and its destruction to social economy, we investigate the non-trivial microstructure and endogenous interaction within the pyramid scheme. By using the motif analysis and the exponential random graph model (ERGM), we conducted an in-depth study on the communication network of a specific pyramid scheme organizations. Through integration of the communication networks with the organizational hierarchical structure, we explore what is the typical structure of a pyramid scheme organization and how do people interact with each other in the organization. The results show that the pyramid scheme communication network is a sparse network and obeys a core–peripheral structure. In terms of personnel interactions, pyramid scheme communication network presents significant homogeneity, with minimal cross-community and cross-hierarchy interactions.

Targeting Androgen Regulation of TMPRSS2 and ACE2 as a Therapeutic Strategy to Combat COVID-19

The COVID-19 pandemic is expected to have an adverse effect on the progression of multiple cancers, including prostate cancer, due to the ensuing cytokine storm and associated oncogenic signaling. Epidemiological data showing increased severity and mortality of COVID-19 in men suggests a potential role for androgen in SARS-CoV-2 infection. Here, we present evidence for the transcriptional regulation of SARS-CoV-2 host cell receptor ACE2 and co-receptor TMPRSS2 by androgen in mouse tissues and human prostate and lung cell lines. Additionally, we demonstrate the endogenous interaction between TMPRSS2 and ACE2 in human cells and validate ACE2 as a TMPRSS2 substrate. In an overexpression model, and the prostate and lung cells, Camostat – a TMPRSS2 inhibitor, blocked the cleavage of pseudotype SARS-CoV-2 surface Spike without disrupting TMPRSS2-ACE2 interaction. Thus providing evidence for the first time a direct role of TMPRSS2 in priming the SARS-CoV-2 Spike protein, required for viral fusion to the host cell. Importantly, androgen-deprivation, anti-androgens such as enzalutamide/AR-PROTAC, or Camostat treatment attenuated the SARS-CoV-2 S-mediated entry in lung and prostate cells. Together, our preclinical data provide a strong rationale for clinical evaluations of the TMPRSS2 inhibitors, androgen-deprivation therapy and androgen receptor antagonists alone or in combination with anti-viral drugs as early as clinically possible to prevent inflammation driven COVID-19 progression.Funding: Research in I.A.A laboratory is supported by NIH (1-R01 CA249210-0), Department of Defense Idea Development Award (W81XWH-17-0404), and Sarcoma Foundation of America Research Award to I.A.A.Ethical Approval: In vivo mouse studies were performed following protocols approved by the Institutional Animal Care and Use Committee at the University of Pennsylvania and in compliance with all regulatory standards. C57BL/6J mice were procured from Jackson Laboratory (000664) and breed in house.Declaration of Interest: None to declare

ESR1 Mutation Status as a Biomarker for Radiation Response in ER-Positive Breast Cancer

Acquired mutations in oncogenic drivers in response to systemic therapies is a common mechanism of treatment resistance. How such mutations alter response to radiation therapy is poorly understood. Acquired mutations in the gene encoding for Estrogen Receptor (ESR1) are one of the most common mechanisms of endocrine therapy resistance in breast cancer. The goal of this study is to define the role of ESR1 mutations in altering response to therapeutic radiation in breast cancer and to develop therapeutic approaches to overcome such resistance. To test if ESR1 mutations influence response to radiation therapy, we employed ER-positive MCF7 and T47D cell lines which have been genome edited to knock in two of the most common ESR1 mutations found in breast cancer patients (Y537S and D538G). Clonogenic survival assays were carried out to characterize differences in radiation sensitivity of these cells. γ-H2AX and Comet assays were employed to study the differences in residual unrepaired DNA damage following radiation in these cell lines. MCF7 cell line-derived xenografts harboring the wild type or mutant ESR1 were employed to study differences in radiation resistance of these tumors in vivo. ER-positive breast cancer cell lines harboring ESR1 mutations exhibit resistance to radiation compared to the corresponding isogenic cell lines with the wild type ESR1. Following exposure to radiation, the ESR1 mutant cells have significantly less residual, unrepaired DNA damage. We also show an endogenous interaction between mutant ER proteins and BRD4, a bromodomain and extraterminal domain (BET) family protein. Recent studies have established a key role for BRD4 in repairing DNA double strand breaks via non-homologous end joining pathway. Pharmacologic BET inhibition with OTX015 overcomes ESR1 mutation-induced resistance to therapeutic radiation in breast cancer, both in vitro and in vivo. ESR1 mutations, which confer resistance to endocrine therapies, also confer resistance to radiation therapy. Recent studies show that ESR1 mutations are also enriched in non-metastatic, ER-positive breast cancer patients receiving neoadjuvant endocrine therapy. Our findings suggest that patients who develop ESR1 mutations under selective pressure of neoadjuvant endocrine therapy may respond poorly to subsequent radiation therapy and may experience increased risk of locoregional failure. In fact, a recent study reported enrichment of ESR1 mutations in locoregionally recurrent breast cancers. Our findings support prospective clinical studies for personalization of radiation treatments in breast cancer based on ESR1 mutation status.

Abstract S01-01: Therapeutic targeting of TMPRSS2 and ACE2 as a potential strategy to combat COVID-19

Abstract The novel SARS-CoV-2 infection responsible for the COVID-19 pandemic is expected to have an adverse effect on the progression of multiple cancers, including prostate cancer, due to the ensuing cytokine storm associated oncogenic signaling. A better understanding of the host cell factors and their regulators will help identify potential therapies to block SARS-CoV-2 infection at an early stage and thereby prevent cancer progression. Host cell infection by SARS-CoV-2 requires the binding of the viral spike S protein to ACE2 receptor and priming by the serine protease TMPRSS2—encoded by a well-known androgen response gene and highly expressed in patients diagnosed with prostate cancer. Epidemiologic data showing increased severity and mortality of SARS-CoV-2 disease in men suggest a possible role for androgen in the transcriptional activation of ACE2 and TMPRSS2 in the lungs and other primary infection sites. Here, by performing in vivo castration in mice, RT-PCR, immunoblotting, Co-IP, and pseudovirus infection assays in multiple cell lines, we present evidence for the transcriptional regulation of TMPRSS2 and ACE2 by androgen, their endogenous interaction, as well as a novel combination of drugs in blocking viral infection. In adult male mice, castration led to a significant loss in the expression of ACE2 and TMPRSS2 at the transcript and protein levels in the lung, heart, and small intestine. Intriguingly, castrated mice displayed a substantial increase in ACE2 in the kidney, which could potentially be due to the low blood pressure resulting from androgen deprivation. Endogenous TMPRSS2 and ACE2 were found to be physically interacting, as observed by reciprocal immunoprecipitation followed by immunoblotting. Importantly, along with full-length zymogen form, a prominent novel small isoform of TMPRSS2 was found to be associated with ACE2 in lung and prostate cells. In an overexpression system, camostat—a serine protease inhibitor specific to TMPRSS2—inhibited the cleavage of spike S, suggesting a direct role of this serine protease in priming the viral spike S protein, a prerequisite for an active infection. Furthermore, a combination of camostat, antiandrogen, and epigenetic drugs at sublethal concentrations blocked SARS-CoV-2 pseudovirus infection in multiple cell types. Together, our preclinical data provide a strong rationale for clinical evaluation of TMRPSS2 inhibitors, antiandrogens, and epigenetic drug combinations along with antiviral drug remdesivir as early as clinically possible to prevent progression to pneumonia and multiorgan failure as a result of hyperinflammatory responses in COVID-19 patients. Citation Format: Qu Deng, Reyaz Ur Rasool, Ramakrishnan Natesan, Irfan A. Asangani. Therapeutic targeting of TMPRSS2 and ACE2 as a potential strategy to combat COVID-19 [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr S01-01.

Abstract 6529: GI101, A novel CD80-IgG4-IL2 variant bispecific protein, inhibits tumor growth and induces anti-tumor immune response in multiple preclinical models

Abstract Introduction: Immune checkpoint blockades (ICBs) have revolutionized cancer treatment and broadened clinical applicability. However, the majority of patients still fail to respond to standard ICBs. To overcome such unmet needs in a clinical study, we designed GI-101, combining the extracellular domain of CD80 serve as a CTLA-4 blockade and an IL-2 variant that preferentially binds the IL-2 receptor β subunit (IL-2Rβ) together. The harmonizing mechanisms of action are projected to translate into improved clinical benefits for this first-in-class immune checkpoint inhibitor fusion protein, even in non-inflamed “cold” tumors. Methods: Binding affinity of GI101 to IL2Rs, CTLA4, and CD28 was determined by SPR. Immune cell proliferation was analyzed by CFSE assay. In vivo anti-tumor efficacy was tested by single or combination treatment on CT26, MC38 and B16F10 syngeneic tumor models. To elucidate the involvement of GI101 on tumor microenvironment (TME), immune cell population was analyzed by flow cytometry from tumor. Tumor specific T cells (surrogate marker, gp70) were measured by splenocyte proliferation assay and IFN-γ ELISPOT assay. RNA sequencing was performed to elucidate immune mechanism of GI-101. Results: GI101 highly binds to CTLA-4 (Kd, 2.9 nM) which leads to the reinforcement of endogenous CD80 and CD28 interaction resulting in the activation of T cells. Bivalent IL-2 variant of GI101 triggers both CD8+ T and NK cells proliferation in vitro and in vivo without Tregs proliferation. GI101 has no evidence for toxicity associated with IL-2 activity including vascular leakage syndrome and cytokine storm in non-GLP monkey studies whereas isolated mortality was observed in the anti-PD-1 and anti-CTLA4 combination treatment group. GI101 elicits restoration of immune functions in vitro settings using mouse splenocytes co-cultured with different PDL-1 and CTLA-4 expression level tumor cells. A dose-dependent (3 to 12 mg/kg) inhibition of tumor growth was observed in CT26 syngeneic models without toxicity. Immune profiling of tumor samples also revealed that a robust increment of M1 macrophages, CD8+ central memory T cells (Tcm) and Ki-67+ proliferating T cells but not Tregs in TME (p < 0.05). Tumor specific T cells were strongly proliferated when stimulated with CT26 neoantigens (gp70, RSPWFTTLI and MGPLIVLLL) in splenocyte. IFN-γ+ cells were significantly increased in draining lymph nodes from GI101 treated mice. Furthermore, drastic tumor regression was observed in MC38 tumor-bearing mice treated with GI101 and anti-PD-1 combination. Conclusion: GI101 facilitates the dual function of checkpoint blockade and IL2 activity that enhances the proliferation and activation of T and NK cells. This novel target drug is expected to be interpreted as superior clinical efficacy and safety as indicated even in ‘cold tumor' models. GI101 is the promising immune-oncology drug to replace the first-generation ICBs by single or combining with other immunotherapies. Our findings provide a rationale for further clinical investigations. Keywords: CD80, IL-2 variant, GI101, Bispecific fusion protein, immunotherapy Citation Format: Kyoung-Ho Pyo, Young Jun Koh, Chun-Bong Synn, Jae Chan Park, Jae-Hwan Kim, Yeongseon Byeon, Sung Eun Kim, Ji Min Lee, Ha Ni Jo, Wongeun Lee, Do Hee Kim, Sungwon Park, Yoo Jeong Song, Won Jae Lee, Ji Young Kim, Hyung Nam Ji, Sang Su Park, Kyung Wha Lee, Young Gyu Cho, Young Min Oh, Bo Gie Yang, Su Youn Nam, Myoung Ho Jang, Byoung Chul Cho. GI101, A novel CD80-IgG4-IL2 variant bispecific protein, inhibits tumor growth and induces anti-tumor immune response in multiple preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6529.

Revitalizing the race between technology and education: Investigating the growth strategy for the knowledge-based economy based on a CGE analysis

This study conducted quantitative policy experiments using a computable general equilibrium model to investigate how long-run economic growth can be achieved through the endogenous interactions between innovation and human capital accumulation. The analysis found that there are limits to driving productivity growth and enhancing growth potential when focusing solely on the expansion of technological innovation. However, our results suggest that promotion of the complementarity between technological innovation and human capital formation may alleviate the inequalities of wages and employment induced by skill-biased technological change, promoting balanced growth among industrial sectors with higher productivity improvement and scale effects. This study suggests that in order to spur long-run growth in knowledge-based economy, policymakers should establish educational infrastructure to support workers to move up the skill ladder and cope with rapid technological change.

Gas-Phase Protonation Thermodynamics of Biological Lipids: Experiment, Theory, and Implications.

Phospholipids are important to cellular function and are a vital structural component of plasma and organelle membranes. These membranes isolate the cell from its environment, allow regulation of the internal concentrations of ions and small molecules, and host diverse types of membrane proteins. It remains extremely challenging to identify specific membrane protein-lipid interactions and their relative strengths. Native mass spectrometry, an intrinsically gas-phase method, has recently been demonstrated as a promising tool for identifying endogenous protein-lipid interactions. However, to what extent the identified interactions reflect solution- versus gas-phase binding strengths is not known. Here, the "Extended" Kinetic Method and ab initio computations at three different levels of theory are used to experimentally and theoretically determine intrinsic gas-phase basicities (GB, ΔG for deprotonation of the protonated base) and proton affinities (PA, ΔH for deprotonation of the protonated base) of six lipids representing common phospholipid types. Gas-phase acidities (ΔG and ΔH for deprotonation) of neutral phospholipids are also evaluated computationally and ranked experimentally. Intriguingly, it is found that two of these phospholipids, sphingomyelin and phosphatidylcholine, have the highest GB of any small, monomeric biomolecules measured to date and are more basic than arginine. Phosphatidylethanolamine and phosphatidylserine are found to be similar in GB to basic amino acids lysine and histidine, and phosphatidic acid and phosphatidylglycerol are the least basic of the six lipid types studied, though still more basic than alanine. Kinetic Method experiments and theory show that the gas-phase acidities of these phospholipids are high but less extreme than their GB values, with phosphatidylserine and phosphatidylglycerol being the most acidic. These results indicate that sphingomyelin and phosphatidylcholine lipids can act as charge-reducing agents when dissociated from native membrane protein-lipid complexes in the gas phase and provide a straightforward model to explain the results of several recent native mass spectrometry studies of protein-lipid complexes.