scholarly journals Robust RNA editing via recruitment of endogenous ADARs using circular guide RNAs

2021 ◽  
Author(s):  
Dhruva Katrekar ◽  
James Yen ◽  
Yichen Xiang ◽  
Anushka Saha ◽  
Dario Meluzzi ◽  
...  
Keyword(s):  
Rna Editing ◽  
Transcript Level ◽  
Rnase H ◽  
Type I ◽  
Rna Molecules ◽  
Guide Rnas ◽  
Cellular Machinery ◽  
Dna Vectors

ABSTRACTAkin to short-hairpin RNAs and antisense oligonucleotides which efficaciously recruit endogenous cellular machinery such as Argonaute and RNase H to enable targeted RNA knockdown, simple long antisense guide RNAs (1) can recruit endogenous adenosine deaminases acting on RNA (ADARs) to enable programmable A-to-I RNA editing, without requiring co-delivery of any exogenous proteins. This approach is highly specific, however the efficiency is typically lower than observed with enzyme overexpression. Conjecturing this was due in part to the short half-life and residence times of guide RNAs, here we engineer highly stable circular ADAR recruiting guide RNAs (cadRNAs), which can be delivered not only by genetically encoding on DNA vectors, but also via transfection of RNA molecules transcribed in vitro. Using these cadRNAs, we observed robust RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, vastly improved efficiency and durability of RNA editing, and high transcriptome-wide specificity. High transcript-level specificity was achieved by further engineering to reduce bystander editing. Additionally, in vivo delivery of cadRNAs via adeno-associated viruses (AAVs) enabled robust 38% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers, and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler (MPS I-H) syndrome. Taken together, cadRNAs enable efficacious programmable RNA editing with application across diverse protein modulation and gene therapeutic settings.

scholarly journals Effects of vascular endothelial growth factor on isolated fetal alveolar type II cells

2004 ◽  
Vol 286 (6) ◽  
pp. L1293-L1301 ◽  
Author(s):  
William Raoul ◽  
Bernadette Chailley-Heu ◽  
Anne-Marie Barlier-Mur ◽  
Christophe Delacourt ◽  
Bernard Maître ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Transcript Level ◽  
Alveolar Type ◽  
Vegf Receptor ◽  
Type I ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells

Previous investigations gained from in vivo or lung explant studies suggested that VEGF is an autocrine proliferation and maturation factor for developing alveolar type II cells. The objective of this work was to determine whether VEGF exerted its growth and maturation effects directly on isolated type II cells. These were isolated from 19-day fetal rat lung and cultured in defined medium. The presence of VEGF receptor-2 was assessed in cultured cells at the pre- and posttranslational levels. Recombinant VEGF165, formerly found to be active on lung explants, failed to enhance type II cell proliferation estimated by thymidine and 5-bromo-2′-deoxy-uridine incorporation. It increased choline incorporation in saturated phosphatidylcholine by 27% but did not increase phospholipid surfactant pool size. VEGF (100 ng/ml) left unchanged the transcript level of surfactant proteins (SP)-A, SP-C, and SP-D but increased SP-B transcripts to four times the control steady-state level. VEGF slightly retarded, but did not prevent, the in vitro transdifferentiation of type II into type I cells, as assessed by immunolabeling of the type I cell marker T1α. We conclude that, with the exception of SP-B expression, which appears to be controlled directly, the previously observed effects of this VEGF isoform on type II cells are likely to be exerted indirectly through reciprocal paracrine interactions involving other lung cell types.

scholarly journals A Novel Member of the RNase D Exoribonuclease Family Functions in Mitochondrial Guide RNA Metabolism in Trypanosoma brucei

2011 ◽  
Vol 286 (12) ◽  
pp. 10329-10340 ◽  
Author(s):  
Sara L. Zimmer ◽  
Sarah M. McEvoy ◽  
Jun Li ◽  
Jun Qu ◽  
Laurie K. Read
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Editing ◽  
Sequence Similarity ◽  
Mitochondrial Gene ◽  
Sequence Information ◽  
Rna Turnover ◽  
Guide Rna ◽  
Guide Rnas

RNA turnover and RNA editing are essential for regulation of mitochondrial gene expression in Trypanosoma brucei. RNA turnover is controlled in part by RNA 3′ adenylation and uridylation status, with trans-acting factors also impacting RNA homeostasis. However, little is known about the mitochondrial degradation machinery or its regulation in T. brucei. We have identified a mitochondrial exoribonuclease, TbRND, whose expression is highly up-regulated in the insect proliferative stage of the parasite. TbRND shares sequence similarity with RNase D family enzymes but differs from all reported members of this family in possessing a CCHC zinc finger domain. In vitro, TbRND exhibits 3′ to 5′ exoribonuclease activity, with specificity toward uridine homopolymers, including the 3′ oligo(U) tails of guide RNAs (gRNAs) that provide the sequence information for RNA editing. Several lines of evidence generated from RNAi-mediated knockdown and overexpression cell lines indicate that TbRND functions in gRNA metabolism in vivo. First, TbRND depletion results in gRNA tails extended by 2–3 nucleotides on average. Second, overexpression of wild type but not catalytically inactive TbRND results in a substantial decrease in the total gRNA population and a consequent inhibition of RNA editing. The observed effects on the gRNA population are specific as rRNAs, which are also 3′-uridylated, are unaffected by TbRND depletion or overexpression. Finally, we show that gRNA binding proteins co-purify with TbRND. In summary, TbRND is a novel 3′ to 5′ exoribonuclease that appears to have evolved a function highly specific to the mitochondrion of trypanosomes.

Peculiarities of the course of type I allergic reactions in patients with blood diseases

2020 ◽  
pp. 40-50
Author(s):  
A. Nikitina
Keyword(s):  
Search Engines ◽  
Anaphylactic Shock ◽  
Type I ◽  
Allergic Reactions ◽  
Common Cause ◽  
Blood Diseases ◽  
Treatment Regimens ◽  
Modern Methods

Analysis of literature data presented in search engines — Elibrary, PubMed, Cochrane — concerning the risk of developing type I allergic reactions in patients with blood diseases is presented. It is shown that the most common cause of type I allergic reactions is drugs included in the treatment regimens of this category of patients. The article presents statistics on the increase in the number of drug allergies leading to cases of anaphylactic shock in patients with blood diseases. Modern methods for the diagnosis of type I allergic reactions in vivo and in vitro are considered.

scholarly journals Collagen-Based Electrospun Materials for Tissue Engineering: A Systematic Review

2021 ◽  
Vol 8 (3) ◽  
pp. 39
Author(s):  
Britani N. Blackstone ◽  
Summer C. Gallentine ◽  
Heather M. Powell
Keyword(s):  
Systematic Review ◽  
Tissue Engineering ◽  
Collagen Type I ◽  
The Body ◽  
Pool Data ◽  
Type I ◽  
High Concentrations ◽  
Increased Strength

Collagen is a key component of the extracellular matrix (ECM) in organs and tissues throughout the body and is used for many tissue engineering applications. Electrospinning of collagen can produce scaffolds in a wide variety of shapes, fiber diameters and porosities to match that of the native ECM. This systematic review aims to pool data from available manuscripts on electrospun collagen and tissue engineering to provide insight into the connection between source material, solvent, crosslinking method and functional outcomes. D-banding was most often observed in electrospun collagen formed using collagen type I isolated from calfskin, often isolated within the laboratory, with short solution solubilization times. All physical and chemical methods of crosslinking utilized imparted resistance to degradation and increased strength. Cytotoxicity was observed at high concentrations of crosslinking agents and when abbreviated rinsing protocols were utilized. Collagen and collagen-based scaffolds were capable of forming engineered tissues in vitro and in vivo with high similarity to the native structures.

scholarly journals E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peng Gao ◽  
Xianwei Ma ◽  
Ming Yuan ◽  
Yulan Yi ◽  
Guoke Liu ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Type I Interferon ◽  
Zinc Fingers ◽  
E3 Ligase ◽  
Type I ◽  
E3 Ligases ◽  
Protein Posttranslational Modifications ◽  
Antiviral Innate Immunity

AbstractUbiquitination is one of the most prevalent protein posttranslational modifications. Here, we show that E3 ligase Nedd4l positively regulates antiviral immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3. Deficiency of Nedd4l significantly impairs type I interferon and proinflammatory cytokine production induced by virus infection both in vitro and in vivo. Nedd4l deficiency inhibits virus-induced ubiquitination of TRAF3, the binding between TRAF3 and TBK1, and subsequent phosphorylation of TBK1 and IRF3. Nedd4l directly interacts with TRAF3 and catalyzes K29-linked ubiquitination of Cys56 and Cys124, two cysteines that constitute zinc fingers, resulting in enhanced association between TRAF3 and E3 ligases, cIAP1/2 and HECTD3, and also increased K48/K63-linked ubiquitination of TRAF3. Mutation of Cys56 and Cys124 diminishes Nedd4l-catalyzed K29-linked ubiquitination, but enhances association between TRAF3 and the E3 ligases, supporting Nedd4l promotes type I interferon production in response to virus by catalyzing ubiquitination of the cysteines in TRAF3.

scholarly journals Effects of chitosan-collagen dressing on wound healing in vitro and in vivo assays

2021 ◽  
Vol 19 ◽  
pp. 228080002198969
Author(s):  
Min-Xia Zhang ◽  
Wan-Yi Zhao ◽  
Qing-Qing Fang ◽  
Xiao-Feng Wang ◽  
Chun-Ye Chen ◽  
...  
Keyword(s):  
Wound Healing ◽  
Wound Dressing ◽  
Type I Collagen ◽  
Inhibition Zone ◽  
Negative Control ◽  
Type I ◽  
Nih3t3 Cells ◽  
Post Operation

The present study was designed to fabricate a new chitosan-collagen sponge (CCS) for potential wound dressing applications. CCS was fabricated by a 3.0% chitosan mixture with a 1.0% type I collagen (7:3(w/w)) through freeze-drying. Then the dressing was prepared to evaluate its properties through a series of tests. The new-made dressing demonstrated its safety toward NIH3T3 cells. Furthermore, the CCS showed the significant surround inhibition zone than empty controls inoculated by E. coli and S. aureus. Moreover, the moisture rates of CCS were increased more rapidly than the collagen and blank sponge groups. The results revealed that the CCS had the characteristics of nontoxicity, biocompatibility, good antibacterial activity, and water retention. We used a full-thickness excisional wound healing model to evaluate the in vivo efficacy of the new dressing. The results showed remarkable healing at 14th day post-operation compared with injuries treated with collagen only as a negative control in addition to chitosan only. Our results suggest that the chitosan-collagen wound dressing were identified as a new promising candidate for further wound application.

scholarly journals mTOR inhibitors potentially reduce TGF-β2-induced fibrogenic changes in trabecular meshwork cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nozomi Igarashi ◽  
Megumi Honjo ◽  
Makoto Aihara
Keyword(s):  
Trabecular Meshwork ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Mtor Inhibitors ◽  
In Vivo Model ◽  
Type I ◽  
Fibrotic Response ◽  
Alpha Smooth Muscle Actin

AbstractWe examined the effects of mTOR inhibitors on the fibrotic response induced by transforming growth factor-beta2 (TGF-β2) in cultured human trabecular meshwork (hTM) cells. TGF-β2-induced expression of fibronectin, collagen type I, alpha 1 chain (COL1A1), and alpha-smooth muscle actin (αSMA) in hTM cells was examined in the presence or absence of mTOR inhibitors using quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The migration rates of hTM cells were examined in the presence of TGF-β2 with or without mTOR inhibitors. An in vitro study showed that the expression of fibronectin, COL1A1, and αSMA was upregulated by TGF-β2 treatment of hTM cells; such upregulation was significantly suppressed by mTOR inhibitors. The inhibitors significantly reduced the migration rate of TGF-β2-stimulated hTM cells. mTOR inhibitors may usefully reduce the fibrotic response of hTM cells and we may have to explore if it is also effective in in vivo model.

scholarly journals In Vitro Effects of Sulforaphane on Interferon-Driven Inflammation and Exploratory Evaluation in Two Healthy Volunteers

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3602
Author(s):  
Elena Genova ◽  
Maura Apollonio ◽  
Giuliana Decorti ◽  
Alessandra Tesser ◽  
Alberto Tommasini ◽  
...  
Keyword(s):  
Healthy Volunteers ◽  
Supportive Therapy ◽  
P Value ◽  
Type I ◽  
Interferon Signature ◽  
Interferon Stimulated Genes ◽  
Immune System Activation ◽  
In Vitro Effects

Interferonopathies are rare genetic conditions defined by systemic inflammatory episodes caused by innate immune system activation in the absence of pathogens. Currently, no targeted drugs are authorized for clinical use in these diseases. In this work, we studied the contribution of sulforaphane (SFN), a cruciferous-derived bioactive molecule, in the modulation of interferon-driven inflammation in an immortalized human hepatocytes (IHH) line and in two healthy volunteers, focusing on STING, a key-component player in interferon pathway, interferon signature modulation, and GSTM1 expression and genotype, which contributes to SFN metabolism and excretion. In vitro, SFN exposure reduced STING expression as well as interferon signature in the presence of the pro-inflammatory stimulus cGAMP (cGAMP 3 h vs. SFN+cGAMP 3 h p value < 0.0001; cGAMP 6 h vs. SFN+cGAMP 6 h p < 0.001, one way ANOVA), restoring STING expression to the level of unstimulated cells. In preliminary experiments on healthy volunteers, no appreciable variations in interferon signature were identified after SFN assumption, while only in one of them, presenting the GSTM1 wild type genotype related to reduced SFN excretion, could a downregulation of STING be recorded. This study confirmed that SFN inhibits STING-mediated inflammation and interferon-stimulated genes expression in vitro. However, only a trend towards the downregulation of STING could be reproduced in vivo. Results obtained have to be confirmed in a larger group of healthy individuals and in patients with type I interferonopathies to define if the assumption of SFN could be useful as supportive therapy.

scholarly journals Slow viral propagation during initial phase of infection leads to viral persistence in mice

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Haifeng C. Xu ◽  
Ruifeng Wang ◽  
Prashant V. Shinde ◽  
Lara Walotka ◽  
Anfei Huang ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Evasion ◽  
Protein Transport ◽  
Adaptive Immune System ◽  
Viral Persistence ◽  
Lymphocytic Choriomeningitis ◽  
Type I ◽  
Viral Propagation

AbstractImmune evasion of pathogens can modify the course of infection and impact viral persistence and pathology. Here, using different strains of the lymphocytic choriomeningitis virus (LCMV) model system, we show that slower propagation results in limited type I interferon (IFN-I) production and viral persistence. Specifically, cells infected with LCMV-Docile exhibited reduced viral replication when compared to LCMV-WE and as a consequence, infection with LCMV-Docile resulted in reduced activation of bone marrow derived dendritic cells (BMDCs) and IFN-I production in vitro in comparison with LCMV-WE. In vivo, we observed a reduction of IFN-I, T cell exhaustion and viral persistence following infection of LCMV-Docile but not LCMV-WE. Mechanistically, block of intracellular protein transport uncovered reduced propagation of LCMV-Docile when compared to LCMV-WE. This reduced propagation was critical in blunting the activation of the innate and adaptive immune system. When mice were simultaneously infected with LCMV-Docile and LCMV-WE, immune function was restored and IFN-I production, T cell effector functions as well as viral loads were similar to that of mice infected with LCMV-WE alone. Taken together, this study suggests that reduced viral propagation can result in immune evasion and viral persistence.

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