Abstract 15541: Novel Molecular Entity for Effective Cardiac Regeneration: 5’ppp Microrna Induces Reprogramming and Accelerates Cardiomyocyte Maturation

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Jiabiao Hu ◽  
Conrad Hodgkinson ◽  
Syeda S Baksh ◽  
Richard E Pratt ◽  
Victor J Dzau
Keyword(s):  
Pattern Recognition ◽  
Cardiac Regeneration ◽  
Molecular Entity ◽  
Rna Molecules ◽  
Novel Approach ◽  
Nfkb Activation ◽  
Recognition Receptor ◽  
Modest Effect

We have shown that miR combo directly reprograms fibroblasts into cardiomyocytes. However, both in vitro and in vivo, the generated cardiomyocytes are immature. Recently, we discovered that cardiomyocyte maturation is enhanced by NFkB activation. While miR combo initiates reprogramming, the effect on NFkB activity is modest. Therefore, miR combo only has a modest effect on maturation. We have now made an important discovery that 5’triphosphorylation (5’ppp) of RNA molecules markedly enhance NFkB activation; leading to increased and accelerated maturation of reprogramed cardiomyocytes. The addition of 5’ppp-RNA increased the number of mature cardiomyocytes by 4-fold (N=5, P<0.05). Moreover, 5’ppp-RNA also accelerated the appearance of mature cardiomyocyte mRNAs (1 day after transfection vs 4 days for unmodified RNA). Importantly, these effects were lost when the 5’ppp moiety was removed. Knockdown studies suggest that 5’ppp-RNA binds to the Pattern Recognition Receptor Rig1. MNase-seq and ChIP-seq analysis suggests that 5’ppp-RNA activates YY1 (N=4, P<0.001). We next asked if 5’ppp modification of the miRNAs within miR combo would also enhance cardiomyocyte maturation; thus, providing a single molecular entity that can initiate reprogramming and accelerate maturation of cardiomyocytes. Indeed, 5’ppp modification of the miR combo miR-1 increased the expression of the mature cardiomyocyte markers Actn2 (4-fold, N=3, P<0.05) and Myh6 (4-fold, N=3, P<0.05). In conclusion, 5’ppp-miR combo is a novel approach that has the advantage of both initiating reprogramming and accelerating cardiomyocyte maturation and should lead to more effective cardiac regeneration and restore cardiac function.

Natural pattern-recognition-receptor agonists as adjuvants for in situ vaccination lymphoma immunotherapy.

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. 123-123 ◽  
Author(s):  
Mark Aleynick ◽  
Paul Peng ◽  
Linda Hammerich ◽  
Ranjan Upadhyay ◽  
Netonia Marshall ◽  
...  
Keyword(s):  
Pattern Recognition ◽  
T Cell ◽  
Immune Activation ◽  
Pattern Recognition Receptor ◽  
Receptor Agonists ◽  
Prophylactic Vaccines ◽  
Recognition Receptor

123 Background: In patients with low-grade lymphoma, in situ vaccination has yielded both partial and complete remissions in clinical trials. Though clinical responses have been observed with multiple pattern recognition receptor agonists (PRRa), the optimal immune stimulant is unknown. We hypothesize that natural PRRa, such as the attenuated pathogens or subunits found in common prophylactic vaccines, could target multiple PRR in a physiologically relevant context and lead to a more robust activation of dendritic cells (DCs) versus synthetic PRRa. Methods: 20 vaccines, including BCG, Typhim Vi, MMR-II, etc. were screened in vitro, where DC phenotype and function were evaluated by flow cytometry. Flt3L-mobilized DC ability to phagocytose, process, present, and cross-present soluble protein or tumor derived antigen, were assessed using CRISPR gene-edited, β2m(-/-) GFP-lymphoma cells and a novel GFP-specific (‘JEDI’) CD8 T cell system. Vaccine mechanism of immune activation was elucidated using a library of PRR-null macrophage cell lines. Potent vaccines were also evaluated in vivo in a Flt3L-primed in situ vaccination using the A20 murine lymphoma model. Results: Several vaccines induced robust DC activation and several showed significant increases in subsequent T cell activation, proliferation, and tumor killing, suggesting increased antigen processing and cross-presentation by DCs. Some vaccines, either as single agents or in combination, were significantly more effective than synthetic PRRa in activating DCs and inducing a T cell response. In vivo, vaccine combination therapies induced tumor regression in a majority of animals, suggesting synergistic immune activation. Conclusions: This data suggests prophylactic vaccines are effective clinical-grade DC activators and can be repurposed for use in the in situ vaccination maneuver, with immediate translation into the clinic. Additionally, by extensive in vitro evaluation in parallel with in vivo studies, this work aims to identify a predictive in vitro molecular immune signature that correlates closely with adjuvant efficacy in vivo.

scholarly journals HSP90-dependent PUS7 overexpression facilitates the metastasis of colorectal cancer cells by regulating LASP1 abundance

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Dan Song ◽  
Ming Guo ◽  
Shuai Xu ◽  
Xiaotian Song ◽  
Bin Bai ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Intellectual Development ◽  
Molecular Mechanisms ◽  
Hsp90 Inhibitor ◽  
Pseudouridine Synthase ◽  
Novel Approach ◽  
Cell Metastasis ◽  
Underlying Mechanisms

Abstract Background Pseudouridine synthase (PUS) 7 is a member of the PUS family that catalyses pseudouridine formation. It has been shown to be involved in intellectual development and haematological malignancies. Nevertheless, the role and the underlying molecular mechanisms of PUS7 in solid tumours, such as colorectal cancer (CRC), remain unexplored. This study elucidated, for the first time, the role of PUS7 in CRC cell metastasis and the underlying mechanisms. Methods We conducted immunohistochemistry, qPCR, and western blotting to quantify the expression of PUS7 in CRC tissues as well as cell lines. Besides, diverse in vivo and in vitro functional tests were employed to establish the function of PUS7 in CRC. RNA-seq and proteome profiling analysis were also applied to identify the targets of PUS7. PUS7-interacting proteins were further uncovered using immunoprecipitation and mass spectrometry. Results Overexpression of PUS7 was observed in CRC tissues and was linked to advanced clinical stages and shorter overall survival. PUS7 silencing effectively repressed CRC cell metastasis, while its upregulation promoted metastasis, independently of the PUS7 catalytic activity. LASP1 was identified as a downstream effector of PUS7. Forced LASP1 expression abolished the metastasis suppression triggered by PUS7 silencing. Furthermore, HSP90 was identified as a client protein of PUS7, associated with the increased PUS7 abundance in CRC. NMS-E973, a specific HSP90 inhibitor, also showed higher anti-metastatic activity when combined with PUS7 repression. Importantly, in line with these results, in human CRC tissues, the expression of PUS7 was positively linked to the expression of HSP90 and LASP1, and patients co-expressing HSP90/PUS7/LASP1 showed a worse prognosis. Conclusions The HSP90-dependent PUS7 upregulation promotes CRC cell metastasis via the regulation of LASP1. Thus, targeting the HSP90/PUS7/LASP1 axis may be a novel approach for the treatment of CRC.

scholarly journals Plasma cells shape the mesenchymal identity of ovarian cancers through transfer of exosome-derived microRNAs

2021 ◽  
Vol 7 (9) ◽  
pp. eabb0737
Author(s):  
Zhengnan Yang ◽  
Wei Wang ◽  
Linjie Zhao ◽  
Xin Wang ◽  
Ryan C. Gimple ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Ovarian Cancer Cells ◽  
Mesenchymal Phenotype ◽  
Ovarian Cancers ◽  
Novel Approach

Ovarian cancer represents a highly lethal disease that poses a substantial burden for females, with four main molecular subtypes carrying distinct clinical outcomes. Here, we demonstrated that plasma cells, a subset of antibody-producing B cells, were enriched in the mesenchymal subtype of high-grade serous ovarian cancers (HGSCs). Plasma cell abundance correlated with the density of mesenchymal cells in clinical specimens of HGSCs. Coculture of nonmesenchymal ovarian cancer cells and plasma cells induced a mesenchymal phenotype of tumor cells in vitro and in vivo. Phenotypic switch was mediated by the transfer of plasma cell–derived exosomes containing miR-330-3p into nonmesenchymal ovarian cancer cells. Exosome-derived miR-330-3p increased expression of junctional adhesion molecule B in a noncanonical fashion. Depletion of plasma cells by bortezomib reversed the mesenchymal characteristics of ovarian cancer and inhibited in vivo tumor growth. Collectively, our work suggests targeting plasma cells may be a novel approach for ovarian cancer therapy.

scholarly journals THER-05. GENETICALLY STABLE POLIOVIRUS VECTOR CARRYING H3.3K27M ANTIGEN FOR TREATMENT OF DIFFUSE MIDLINE GLIOMA BY INTRAMUSCULAR INJECTION

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii472-iii472
Author(s):  
Mubeen Mosaheb ◽  
Daniel Landi ◽  
Elena Dobrikova ◽  
Michael Brown ◽  
Yuanfan Yang ◽  
...  
Keyword(s):  
T Cell ◽  
Antigen Presentation ◽  
Intramuscular Injection ◽  
Cd8 T Cell ◽  
Cytokine Profiles ◽  
Cell Immunity ◽  
Novel Approach ◽  
Diffuse Midline Glioma

Abstract BACKGROUND H3 K27M-mutant diffuse midline glioma (DMG) is invariably lethal. Viruses naturally engage innate immunity, induce antigen presentation, and mediate CD8 T cell priming against foreign antigens. Polioviruses, in particular, are uniquely tropic for dendritic cells (DC) and potently activate DC, inducing Th1-dominant cytokine profiles, CD8 T cell immunity, and enhanced epitope presentation. Thus, poliovirus is ideally suited for vectored delivery of signature tumor neoantigens, e.g. the H3 K27M feature of DMG. However, poliovirus vector design is inherently limited by genetic instability and the underlying neuropathogenicity of poliovirus. METHODS We created a genetically stable, polio:rhinovirus chimera vector devoid of neuropathogenicity and modified for stable expression of the HLA-A2 restricted H3.3 K27M antigen (RIPO (H3.3)). RESULTS RIPO(H3.3) infects, activates, and induces H3.3K27M antigen presentation in DCs in vitro. Given intramuscularly in vivo, RIPO(H3.3) recruits and activates DCs with Th1-dominant cytokine profiles, efficiently primes H3.3K27M-specific CD8 T cells, induces antigen-specific CD8 T cell migration to the tumor site, delays tumor growth, and enhances survival in murine tumor models. CONCLUSION This novel approach leverages the unique ability of polioviruses to activate DCs while simultaneously introducing the H3.3 K27M antigen. In this way, DCs are activated optimally in situ, while being simultaneously infected to express/present tumor antigen. RIPO(H3.3), given by intramuscular injection, will be evaluated in a clinical trial for children with H3 K27M-mutant diffuse midline glioma.

scholarly journals Hierarchy of evidence in interpretation of clinical significance of drug interactions

2012 ◽  
Vol 65 (1-2) ◽  
pp. 45-49
Author(s):  
Bozana Nikolic ◽  
Miroslav Savic
Keyword(s):  
Drug Metabolism ◽  
Drug Interactions ◽  
Clinical Significance ◽  
Health Professionals ◽  
Molecular Entity ◽  
Population Pharmacokinetic ◽  
Pharmacokinetic Studies ◽  
Potential Interactions

Introduction. Since drug interactions may result in serious adverse effects or failure of therapy, it is of huge importance that health professionals base their decisions about drug prescription, dispensing and administration on reliable research evidence, taking into account the hierarchy of data sources for evaluation. Clinical Significance of Potential Interactions - Information Sources. The sources of data regarding drug interactions are numerous, beginning with various drug reference books. However, they are far from uniformity in the way of choosing and presenting putative clinically relevant interactions. Clinical Significance of Potential Interactions - Interpretation of Information. The difficulties in interpretation of drug interactions are illustrated through the analysis of a published example involving assessment made by two different groups of health professionals. Systematic Evaluation of Drug-Drug Interaction. The potential for interactions is mainly investigated before marketing a drug. Generally, the in vitro, followed by in vivo studies are to be performed. The major metabolic pathways involved in the metabolism of a new molecular entity, as well as the potential of induction of human enzymes involved in drug metabolism are to be examined. In the field of interaction research it is possible to make use of the population pharmacokinetic studies as well as of the pharmacodynamic assessment, and also the postregistration monitoring of the reported adverse reactions and other literature data. Conclusion. In vitro and in vivo drug metabolism and transport studies should be conducted to elucidate the mechanisms and potential for drug-drug interactions. The assessment of their clinical significance should be based on well-defined and validated exposure-response data.

scholarly journals Automated analysis of filopodial length and spatially resolved protein concentration via adaptive shape tracking

2016 ◽  
Vol 27 (22) ◽  
pp. 3616-3626 ◽  
Author(s):  
Tanumoy Saha ◽  
Isabel Rathmann ◽  
Abhiyan Viplav ◽  
Sadhana Panzade ◽  
Isabell Begemann ◽  
...  
Keyword(s):  
Protein Concentration ◽  
Growth Dynamics ◽  
Automated Analysis ◽  
Cell Types ◽  
Control Mechanisms ◽  
Spatially Resolved ◽  
Novel Approach ◽  
Associated Proteins

Filopodia are dynamic, actin-rich structures that transiently form on a variety of cell types. To understand the underlying control mechanisms requires precise monitoring of localization and concentration of individual regulatory and structural proteins as filopodia elongate and subsequently retract. Although several methods exist that analyze changes in filopodial shape, a software solution to reliably correlate growth dynamics with spatially resolved protein concentration along the filopodium independent of bending, lateral shift, or tilting is missing. Here we introduce a novel approach based on the convex-hull algorithm for parallel analysis of growth dynamics and relative spatiotemporal protein concentration along flexible filopodial protrusions. Detailed in silico tests using various geometries confirm that our technique accurately tracks growth dynamics and relative protein concentration along the filopodial length for a broad range of signal distributions. To validate our technique in living cells, we measure filopodial dynamics and quantify spatiotemporal localization of filopodia-associated proteins during the filopodial extension–retraction cycle in a variety of cell types in vitro and in vivo. Together these results show that the technique is suitable for simultaneous analysis of growth dynamics and spatiotemporal protein enrichment along filopodia. To allow readily application by other laboratories, we share source code and instructions for software handling.

Abstract 129: Embryonic Stem Cell Derived Exosomes Revive Endogenous Repair Mechanisms In Failing Heart

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Mohsin Khan ◽  
Suresh K Verma ◽  
Alexander R Mackie ◽  
Erin Vaughan ◽  
Srikanth Garikipati ◽  
...  
Keyword(s):  
Stem Cells ◽  
Therapeutic Potential ◽  
Embryonic Stem ◽  
Cardiac Regeneration ◽  
Great Promise ◽  
Target Cells ◽  
Free System ◽  
Teratoma Formation

Rationale: Embryonic stem cells (ESCs) hold great promise for cardiac regeneration but are susceptible to ethical concerns, lack of autologous donors and teratoma formation. Recently, it has been observed that beneficial effects of stem cells are mediated by exosomes secreted out under various physiological conditions. ESCs have the ability to produce exosomes however their effect in the context of the heart is unknown. Objective: Determine the effect of ESC derived exosomes for cardiac repair and modulation of CPCs functions in the heart following myocardial infarction. Methods and Results: Exosomes were isolated from murine ESCs (mES Ex) or embryonic fibroblasts (MEFs) by ultracentrifugation and verified by Flotillin-1 immunoblot analysis. Induction of pluripotent markers, survival and in vitro tube formation was enhanced in target cells receiving ESC exosomes indicating therapeutic potential of mES Ex. mES Ex administration resulted in enhanced neovascularization, cardiomyocyte survival and reduced fibrosis post infarction consistent with resurgence of cardiac proliferative response. Importantly, mES Ex mediated considerable enhancement of cardiac progenitor cell (CPC) survival, proliferation and cardiac commitment concurrent with increased c-kit+ CPCs in vivo 4 weeks after mES Ex transfer. miRNA Array analysis of ESC and MEF exosomes revealed significantly high expression of miR290-295 cluster in the ESC exosomes compared to MEF exosomes. The underlying beneficial effect of mES Ex was tied to delivery of ESC miR-294 to the heart and in particular CPCs thereby promoting CPC survival and proliferation as analyzed by FACS based cell death analysis and CyQuant assay respectively. Interestingly, enhanced G1/S transition was observed in CPCs treated with miR-294 in conjunction with significant reduction of G1 phase. Conclusion: In conclusion, mES Ex provide a novel cell free system for cardiac regeneration with the ability to modulate both cardiomyocyte and CPC based repair programs in the heart thereby avoiding the risk of teratoma formation associated with ESCs.

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