scholarly journals Using RNA Editing and ADARs to Increase Cell Sensitivity to Interferon Chemotherapies

2018 ◽  
Vol 1 (1) ◽  
Author(s):  
Eimile Oakes ◽  
Obi Nwosu ◽  
Pranathi Vadlamni ◽  
Heather Hundley
Keyword(s):  
Rna Editing ◽  
P Value ◽  
Protein Kinase R ◽  
Cellular Receptors ◽  
Tumor Cell Apoptosis ◽  
Interferon Stimulated Genes ◽  
Cellular Events ◽  
Ifn Therapy ◽  
Mixed Efficacy ◽  
Activate Protein Kinase

Background and Hypothesis: During viral infection, viral double stranded RNAs (dsRNAs) are bound by and activate pattern recognition receptors (PRRs). Activated PRRs signal several downstream cellular events, including activating transcription of interferon (IFN). IFN-β can bind to cellular receptors and increase transcription of PRRs, interferon stimulated genes (ISGs), activate Protein Kinase R (PKR) and trigger apoptosis. The ability of IFN to trigger apoptosis makes it a potential chemotherapeutic. However, IFN therapy has had mixed efficacy in inducing tumor cell apoptosis.  One mechanism for this failure may be intrinsic mechanisms that prevent dsRNA from binding to and activating PRRs. ADAR1 binds to dsRNA and catalyzes deamination of adenosine to inosine, a process known as RNA editing. Normally, ADAR1 inhibits cellular dsRNAs from initiating the IFN response. Recent studies have shown ADAR1 is overexpressed in several cancers and contributes to oncogenic phenotypes, suggesting inhibition of ADAR1 may increase sensitivity to IFN-β. The Hundley lab recently identified an inhibitor of ADAR1 called ADAR3. ADAR3 has no deaminase activity and is highly expressed in glioblastoma (GBM) tumors. We hypothesize that ADAR3 expressing GBM cells should exhibit a greater antiproliferative effect in response to IFN-b treatment compared to control.  Experimental Design or Project Methods: An MTT assay was conducted in which U87 ADAR3 +/- cell lines were exposed to IFN-β after 24 hours. Cell viability was measured for 3 days post-treatment.  Results: U87 ADAR3 + cells are more sensitive to IFN-β treatment (n=2 biological replicates, p-value= 0.04).  Conclusion and Potential Impact: The results suggest a mechanism to enhance IFN therapy for patients with ADAR3 expressing GBM.

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 ADAR1 RNA editing regulates endothelial cell functions via the MDA-5 RNA sensing signaling pathway

2021 ◽  
Vol 5 (3) ◽  
pp. e202101191
Author(s):  
Xinfeng Guo ◽  
Silvia Liu ◽  
Rose Yan ◽  
Vy Nguyen ◽  
Mazen Zenati ◽  
...  
Keyword(s):  
Gene Expression ◽  
Signaling Pathway ◽  
Rna Editing ◽  
Sequencing Analysis ◽  
Newborn Mice ◽  
Interferon Stimulated Genes ◽  
Cell Functions ◽  
Multiple Organs ◽  
Elevated Expression ◽  
Editing Enzyme

The RNA-sensing signaling pathway has been well studied as an essential antiviral mechanism of innate immunity. However, its role in non-infected cells is yet to be thoroughly characterized. Here, we demonstrated that the RNA sensing signaling pathway also reacts to the endogenous cellular RNAs in endothelial cells (ECs), and this reaction is regulated by the RNA-editing enzyme ADAR1. Cellular RNA sequencing analysis showed that EC RNAs endure extensive RNA editing, especially in the RNA transcripts of short interspersed nuclear elements. The EC-specific deletion of ADAR1 dramatically reduced the editing level on short interspersed nuclear element RNAs, resulting in newborn death in mice with damage evident in multiple organs. Genome-wide gene expression analysis revealed a prominent innate immune activation with a dramatically elevated expression of interferon-stimulated genes. However, blocking the RNA sensing signaling pathway by deletion of the cellular RNA receptor MDA-5 prevented interferon-stimulated gene expression and rescued the newborn mice from death. This evidence demonstrated that the RNA-editing/RNA-sensing signaling pathway dramatically modulates EC function, representing a novel molecular mechanism for the regulation of EC functions.

scholarly journals Interferon-Stimulated Genes—Mediators of the Innate Immune Response during Canine Distemper Virus Infection

2019 ◽  
Vol 20 (7) ◽  
pp. 1620 ◽  
Author(s):  
Daniela Klotz ◽  
Ingo Gerhauser
Keyword(s):  
Canine Distemper Virus ◽  
Effector Proteins ◽  
Type I ◽  
Canine Distemper ◽  
Oligoadenylate Synthetase ◽  
Protein Kinase R ◽  
Interferon Stimulated Genes ◽  
Cellular Origin ◽  
Mx Protein ◽  
Cerebellar Lesions

The demyelinating canine distemper virus (CDV)-leukoencephalitis represents a translational animal model for multiple sclerosis. The present study investigated the expression of type I interferon (IFN-I) pathway members in CDV-induced cerebellar lesions to gain an insight into their role in lesion development. Gene expression of 110 manually selected genes in acute, subacute and chronic lesions was analyzed using pre-existing microarray data. Interferon regulatory factor (IRF) 3, IRF7, signal transducer and activator of transcription (STAT) 1, STAT2, MX protein, protein kinase R (PKR), 2′-5′-oligoadenylate synthetase (OAS) 1 and interferon-stimulated gene (ISG) 15 expression were also evaluated using immunohistochemistry. Cellular origin of STAT1, STAT2, MX and PKR were determined using immunofluorescence. CDV infection caused an increased expression of the antiviral effector proteins MX, PKR, OAS1 and ISG15, which probably contributed to a restricted viral replication, particularly in neurons and oligodendrocytes. This increase might be partly mediated by IRF-dependent pathways due to the lack of changes in IFN-I levels and absence of STAT2 in astrocytes. Nevertheless, activated microglia/macrophages showed a strong expression of STAT1, STAT2 and MX proteins in later stages of the disease, indicating a strong activation of the IFN-I signaling cascade, which might be involved in the aggravation of bystander demyelination.

scholarly journals Identification and Characterization of Interferon-Induced Proteins That Inhibit Alphavirus Replication

2007 ◽  
Vol 81 (20) ◽  
pp. 11246-11255 ◽  
Author(s):  
Yugen Zhang ◽  
Crystal W. Burke ◽  
Kate D. Ryman ◽  
William B. Klimstra
Keyword(s):  
Sindbis Virus ◽  
Virus Genome ◽  
Gene Products ◽  
Antiviral Protein ◽  
Protein Kinase R ◽  
Interferon Stimulated Genes ◽  
Virulence Attenuation ◽  
Murine Fibroblast

ABSTRACT Alpha/beta interferon (IFN-α/β) produces antiviral effects through upregulation of many interferon-stimulated genes (ISGs) whose protein products are effectors of the antiviral state. Previous data from our laboratory have shown that IFN-α/β can limit Sindbis virus (SB) replication through protein kinase R (PKR)-dependent and PKR-independent mechanisms and that one PKR-independent mechanism inhibits translation of the infecting virus genome (K. D. Ryman et al., J. Virol. 79:1487-1499, 2005). Further, using Affymetrix microarray technology, we identified 44 genes as candidates for PKR/RNase L-independent IFN-induced antiviral activities. In the current studies, we have begun analyzing these gene products for antialphavirus activity using three techniques: (i) overexpression of the protein from SB vectors and assessment of virulence attenuation in mice; (ii) overexpression of the proteins in a stable tetracycline-inducible murine fibroblast culture system and assessment of effects upon SB replication; and (iii) small interfering RNA-mediated knockdown of gene mRNA in fibroblast cultures followed by SB replication assessment as above. Tested proteins included those we hypothesized had potential to affect virus genome translation and included murine ISG20, ISG15, the zinc finger antiviral protein (ZAP), viperin, p56, p54, and p49. Interestingly, the pattern of antiviral activity for some gene products was different between in vitro and in vivo assays. Viperin and ZAP attenuated virulence most profoundly in mice. However, ISG20 and ZAP potently inhibited SB replication in vitro, whereas and viperin, p56, and ISG15 exhibited modest replication inhibition in vitro. In contrast, p54 and p49 had little to no effect in any assay.

scholarly journals Dynamic Changes in the Expression of Interferon-Stimulated Genes in Joints of SPF Chickens Infected With Avian Reovirus

2021 ◽  
Vol 8 ◽  
Author(s):  
Sheng Wang ◽  
Liji Xie ◽  
Zhixun Xie ◽  
Lijun Wan ◽  
Jiaoling Huang ◽  
...  
Keyword(s):  
Viral Load ◽  
Zinc Finger Protein ◽  
Transmembrane Protein ◽  
Expression Patterns ◽  
Avian Reovirus ◽  
Protein Kinase R ◽  
Inhibitory Protein ◽  
Viral Challenge ◽  
Interferon Stimulated Genes ◽  
Real Time Quantitative Pcr

Avian reovirus (ARV) can induce many diseases as well as immunosuppression in chickens, severely endangering the poultry industry. Interferons (IFNs) play an antiviral role by inducing the expression of interferon-stimulated genes (ISGs). The effect of ARV infection on the expression of host ISGs is unclear. Specific-pathogen-free (SPF) chickens were infected with ARV strain S1133 in this study, and real time quantitative PCR was used to detect changes in the dynamic expression of IFNs and common ISGs in joints of SPF chickens. The results showed that the transcription levels of IFNA, IFNB, and several ISGs, including myxovirus resistance (MX), interferon-induced transmembrane protein 3 (IFITM3), protein kinase R (PKR), oligoadenylate synthase (OAS), interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), interferon-stimulated gene 12 (ISG12), virus inhibitory protein (VIPERIN), interferon-alpha-inducible protein 6 (IFI6), and integrin-associated protein (CD47), were upregulated in joints on days 1–7 of infection (the levels of increase of MX, IFIT5, OAS, VIPERIN, ISG12, and IFI6 were the most significant, at hundreds-fold). In addition, the expression levels of the ISGs encoding zinc finger protein 313 (ZFP313), and DNA damage–inducible transcript 4 (DDIT4) increased suddenly on the 1st or 2nd day, then decreased to control levels. The ARV viral load in chicken joints rapidly increased after 1 day of viral challenge, and the viral load remained high within 6 days of viral challenge. The ARV viral load sharply decreased starting on day 7. These results indicate that in SPF chicken joints, many ISGs have mRNA expression patterns that are basically consistent with the viral load in joints. IFNA, IFNB, and the ISGs MX, IFITM3, PKR, OAS, IFIT5, ISG12, VIPERIN, IFI6, and CD47 play important roles in defending against ARV invasion, inhibiting ARV replication and proliferation, and promoting virus clearance. These results enrich our understanding of the innate immune response mechanisms of hosts against ARV infection and provide a theoretical basis for prevention and control of ARV infection.

scholarly journals Lung Atelectasis Promotes Immune and Barrier Dysfunction as Revealed by Transcriptome Sequencing in Female Sheep

2020 ◽  
Vol 133 (5) ◽  
pp. 1060-1076
Author(s):  
Congli Zeng ◽  
Gabriel C. Motta-Ribeiro ◽  
Takuga Hinoshita ◽  
Marcos Adriano Lessa ◽  
Tilo Winkler ◽  
...  
Keyword(s):  
Immune Response ◽  
Differentially Expressed Genes ◽  
Barrier Function ◽  
P Value ◽  
Capillary Barrier ◽  
Gene Set Enrichment ◽  
Gene Set ◽  
Interferon Stimulated Genes ◽  
Female Sheep ◽  
Alveolar Capillary

Background Pulmonary atelectasis is frequent in clinical settings. Yet there is limited mechanistic understanding and substantial clinical and biologic controversy on its consequences. The authors hypothesize that atelectasis produces local transcriptomic changes related to immunity and alveolar–capillary barrier function conducive to lung injury and further exacerbated by systemic inflammation. Methods Female sheep underwent unilateral lung atelectasis using a left bronchial blocker and thoracotomy while the right lung was ventilated, with (n = 6) or without (n = 6) systemic lipopolysaccharide infusion. Computed tomography guided samples were harvested for NextGen RNA sequencing from atelectatic and aerated lung regions. The Wald test was used to detect differential gene expression as an absolute fold change greater than 1.5 and adjusted P value (Benjamini–Hochberg) less than 0.05. Functional analysis was performed by gene set enrichment analysis. Results Lipopolysaccharide-unexposed atelectatic versus aerated regions presented 2,363 differentially expressed genes. Lipopolysaccharide exposure induced 3,767 differentially expressed genes in atelectatic lungs but only 1,197 genes in aerated lungs relative to the corresponding lipopolysaccharide-unexposed tissues. Gene set enrichment for immune response in atelectasis versus aerated tissues yielded negative normalized enrichment scores without lipopolysaccharide (less than –1.23, adjusted P value less than 0.05) but positive scores with lipopolysaccharide (greater than 1.33, adjusted P value less than 0.05). Leukocyte-related processes (e.g., leukocyte migration, activation, and mediated immunity) were enhanced in lipopolysaccharide-exposed atelectasis partly through interferon-stimulated genes. Furthermore, atelectasis was associated with negatively enriched gene sets involving alveolar–capillary barrier function irrespective of lipopolysaccharide (normalized enrichment scores less than –1.35, adjusted P value less than 0.05). Yes-associated protein signaling was dysregulated with lower nuclear distribution in atelectatic versus aerated lung (lipopolysaccharide-unexposed: 10.0 ± 4.2 versus 13.4 ± 4.2 arbitrary units, lipopolysaccharide-exposed: 8.1 ± 2.0 versus 11.3 ± 2.4 arbitrary units, effect of lung aeration, P = 0.003). Conclusions Atelectasis dysregulates the local pulmonary transcriptome with negatively enriched immune response and alveolar–capillary barrier function. Systemic lipopolysaccharide converts the transcriptomic immune response into positive enrichment but does not affect local barrier function transcriptomics. Interferon-stimulated genes and Yes-associated protein might be novel candidate targets for atelectasis-associated injury. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

Two-Dimensional Crystallization of Tetanus Toxin

1985 ◽  
Vol 43 ◽  
pp. 528-529
Author(s):  
Jeffry A. Reidler ◽  
John P. Robinson
Keyword(s):  
Electron Microscopy ◽  
Tetanus Toxin ◽  
Structural Information ◽  
Three Dimensional ◽  
Two Dimensional ◽  
Membrane Interface ◽  
3D Reconstructions ◽  
Cellular Receptors ◽  
Computer Reconstruction ◽  
2D Crystals

We have prepared two-dimensional (2D) crystals of tetanus toxin using procedures developed by Uzgiris and Kornberg for the directed production of 2D crystals of monoclonal antibodies at an antigen-phospholipid monolayer interface. The tetanus toxin crystals were formed using a small mole fraction of the natural receptor, GT1, incorporated into phosphatidyl choline monolayers. The crystals formed at low concentration overnight. Two dimensional crystals of this type are particularly useful for structure determination using electron microscopy and computer image refinement. Three dimensional (3D) structural information can be derived from these crystals by computer reconstruction of photographs of toxin crystals taken at different tilt angles. Such 3D reconstructions may help elucidate the mechanism of entry of the enzymatic subunit of toxins into cells, particularly since these crystals form directly on a membrane interface at similar concentrations of ganglioside GT1 to the natural cellular receptors.

Total Hospital Costs and Readmission Rate of Patient-Specific Instrument in Total Knee Arthroplasty Patients

2020 ◽  
Author(s):  
Stephen Thomas ◽  
Ankur Patel ◽  
Corey Patrick ◽  
Gary Delhougne
Keyword(s):  
Total Knee Arthroplasty ◽  
Knee Arthroplasty ◽  
Hospital Cost ◽  
Readmission Rate ◽  
Hospital Costs ◽  
Patient Specific ◽  
P Value ◽  
Total Hospital Cost ◽  
Total Hospital ◽  
Total Knee

AbstractDespite advancements in surgical technique and component design, implant loosening, stiffness, and instability remain leading causes of total knee arthroplasty (TKA) failure. Patient-specific instruments (PSI) aid in surgical precision and in implant positioning and ultimately reduce readmissions and revisions in TKA. The objective of the study was to evaluate total hospital cost and readmission rate at 30, 60, 90, and 365 days in PSI-guided TKA patients. We retrospectively reviewed patients who underwent a primary TKA for osteoarthritis from the Premier Perspective Database between 2014 and 2017 Q2. TKA with PSI patients were identified using appropriate keywords from billing records and compared against patients without PSI. Patients were excluded if they were < 21 years of age; outpatient hospital discharges; evidence of revision TKA; bilateral TKA in same discharge or different discharges. 1:1 propensity score matching was used to control patients, hospital, and clinical characteristics. Generalized Estimating Equation model with appropriate distribution and link function were used to estimate hospital related cost while logistic regression models were used to estimate 30, 60, and 90 days and 1-year readmission rate. The study matched 3,358 TKAs with PSI with TKA without PSI patients. Mean total hospital costs were statistically significantly (p < 0.0001) lower for TKA with PSI ($14,910; 95% confidence interval [CI]: $14,735–$15,087) than TKA without PSI patients ($16,018; 95% CI: $15,826–$16,212). TKA with PSI patients were 31% (odds ratio [OR]: 0.69; 95% CI: 0.51–0.95; p-value = 0.0218) less likely to be readmitted at 30 days; 35% (OR: 0.65; 95% CI: 0.50–0.86; p-value = 0.0022) less likely to be readmitted at 60 days; 32% (OR: 0.68; 95% CI: 0.53–0.88; p-value = 0.0031) less likely to be readmitted at 90 days; 28% (OR: 0.72; 95% CI: 0.60–0.86; p-value = 0.0004) less likely to be readmitted at 365 days than TKA without PSI patients. Hospitals and health care professionals can use retrospective real-world data to make informed decisions on using PSI to reduce hospital cost and readmission rate, and improve outcomes in TKA patients.

Isolated Liner Revision for Total Knee Arthroplasty Instability: A Road That Should Remain Less Taken

2020 ◽  
Author(s):  
Jason D. Tegethoff ◽  
Rafael Walker-Santiago ◽  
William M. Ralston ◽  
James A. Keeney
Keyword(s):  
Total Knee Arthroplasty ◽  
Femoral Component ◽  
Knee Arthroplasty ◽  
Primary Total Knee Arthroplasty ◽  
P Value ◽  
Surgical Morbidity ◽  
Exact Test ◽  
Total Knee ◽  
Component Revision

AbstractIsolated polyethylene liner exchange (IPLE) is infrequently selected as a treatment approach for patients with primary total knee arthroplasty (TKA) prosthetic joint instability. Potential advantages of less immediate surgical morbidity, faster recovery, and lower procedural cost need to be measured against reoperation and re-revision risk. Few published studies have directly compared IPLE with combined tibial and femoral component revision to treat patients with primary TKA instability. After obtaining institutional review board (IRB) approval, we performed a retrospective comparison of 20 patients treated with IPLE and 126 patients treated with tibial and femoral component revisions at a single institution between 2011 and 2018. Patient demographic characteristics, medical comorbidities, time to initial revision TKA, and reoperation (90 days, <2 years, and >2 years) were assessed using paired Student's t-test or Fisher's exact test with a p-value <0.01 used to determine significance. Patients undergoing IPLE were more likely to undergo reoperation (60.0 vs. 17.5%, p = 0.001), component revision surgery (45.0 vs. 8.7%, p = 0.002), and component revision within 2 years (30.0 vs. 1.6%, p < 0.0001). Differences in 90-day reoperation (p = 0.14) and revision >2 years (p = 0.19) were not significant. Reoperation for instability (30.0 vs. 4.0%, p < 0.001) and infection (20.0 vs. 1.6%, p < 0.01) were both higher in the IPLE group. IPLE does not provide consistent benefits for patients undergoing TKA revision for instability. Considerations for lower immediate postoperative morbidity and cost need to be carefully measured against long-term consequences of reoperation, delayed component revision, and increased long-term costs of multiple surgical procedures. This is a level III, case–control study.

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