scholarly journals Serum Amyloid A3 Promoter-Driven Luciferase Activity Enables Visualization of Diabetic Kidney Disease

2022 ◽  
Vol 23 (2) ◽  
pp. 899
Author(s):  
Tolulope Peter Saliu ◽  
Nao Yazawa ◽  
Kotaro Hashimoto ◽  
Kenshu Miyata ◽  
Ayane Kudo ◽  
...  
Keyword(s):  
Functional Foods ◽  
Inflammatory Responses ◽  
Early Stage ◽  
Serum Amyloid ◽  
Luciferase Reporter ◽  
Non Invasive ◽  
In Vivo Bioluminescence Imaging ◽  
Bioluminescence Signal ◽  
Direct Cytotoxicity

The early detection of diabetic nephropathy (DN) in mice is necessary for the development of drugs and functional foods. The purpose of this study was to identify genes that are significantly upregulated in the early stage of DN progression and develop a novel model to non-invasively monitor disease progression within living animals using in vivo imaging technology. Streptozotocin (STZ) treatment has been widely used as a DN model; however, it also exhibits direct cytotoxicity to the kidneys. As it is important to distinguish between DN-related and STZ-induced nephropathy, in this study, we compared renal responses induced by the diabetic milieu with two types of STZ models: multiple low-dose STZ injections with a high-fat diet and two moderate-dose STZ injections to induce DN. We found 221 genes whose expression was significantly altered during DN development in both models and identified serum amyloid A3 (Saa3) as a candidate gene. Next, we applied the Saa3 promoter-driven luciferase reporter (Saa3-promoter luc mice) to these two STZ models and performed in vivo bioluminescent imaging to monitor the progression of renal pathology. In this study, to further exclude the possibility that the in vivo bioluminescence signal is related to renal cytotoxicity by STZ treatment, we injected insulin into Saa3-promoter luc mice and showed that insulin treatment could downregulate renal inflammatory responses with a decreased signal intensity of in vivo bioluminescence imaging. These results strongly suggest that Saa3 promoter activity is a potent non-invasive indicator that can be used to monitor DN progression and explore therapeutic agents and functional foods.

scholarly journals NMR microsystem for label-free characterization of 3D nanoliter microtissues

2020 ◽  
Author(s):  
Marco Grisi ◽  
Gaurasundar M. Conley ◽  
Kyle J. Rodriguez ◽  
Erika Riva ◽  
Lukas Egli ◽  
...  
Keyword(s):  
Early Stage ◽  
Cmos Technology ◽  
Time Frame ◽  
Ease Of Use ◽  
Chemical Information ◽  
Spectroscopic Techniques ◽  
Label Free ◽  
Alcoholic Fatty Liver ◽  
Non Invasive

AbstractPerforming chemical analysis at the nanoliter (nL) scale is of paramount importance for medicine, drug development, toxicology, and research. Despite the numerous methodologies available, a tool for obtaining chemical information non-invasively is still missing at this scale. Observer effects, sample destruction and complex preparatory procedures remain a necessary compromise1. Among non-invasive spectroscopic techniques, one able to provide holistic and highly resolved chemical information in-vivo is nuclear magnetic resonance (NMR)2,3. For its renowned informative power and ability to foster discoveries and life-saving applications4,5, efficient NMR at microscopic scales is highly sought after6–10, but so far technical limitations could not match the stringent necessities of microbiology, such as biocompatible handling, ease of use, and high throughput. Here we introduce a novel microsystem, which combines CMOS technology with 3D microfabrication, enabling nL NMR as a platform tool for non-invasive spectroscopy of organoids, 3D cell cultures, and early stage embryos. In this study we show its application to microlivers models simulating non-alcoholic fatty liver disease (NAFLD), demonstrating detection of lipid metabolism dynamics in a time frame of 14 days based on 117 measurements of single 3D human liver microtissues.

scholarly journals In vivo non-invasive monitoring of dystrophin correction in a new Duchenne muscular dystrophy reporter mouse

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Leonela Amoasii ◽  
Hui Li ◽  
Yu Zhang ◽  
Yi-Li Min ◽  
Efrain Sanchez-Ortiz ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Genetic Disorder ◽  
Dystrophin Gene ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Gene Correction ◽  
Reading Frame ◽  
Non Invasive

Abstract Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by mutations in the dystrophin gene. To enable the non-invasive analysis of DMD gene correction strategies in vivo, we introduced a luciferase reporter in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. We evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of CRISPR/Cas9 gene editing machinery with adeno-associated virus. Bioluminescence monitoring revealed efficient and rapid restoration of dystrophin protein expression in affected skeletal muscles and the heart. Our results provide a sensitive non-invasive means of monitoring dystrophin correction in mouse models of DMD and offer a platform for testing different strategies for amelioration of DMD pathogenesis.

scholarly journals SAG/ROC2 E3 ligase regulates skin carcinogenesis by stage-dependent targeting of c-Jun/AP1 and IκB-α/NF-κB

2007 ◽  
Vol 178 (6) ◽  
pp. 1009-1023 ◽  
Author(s):  
Qingyang Gu ◽  
G. Tim Bowden ◽  
Daniel Normolle ◽  
Yi Sun
Keyword(s):  
Early Stage ◽  
Nuclear Factor Κb ◽  
Tumor Formation ◽  
Luciferase Reporter ◽  
Activator Protein 1 ◽  
Cellular Functions ◽  
Accelerated Proliferation ◽  
F Box Protein

Sensitive to apoptosis gene (SAG)/regulator of cullins-2–Skp1-cullin–F-box protein (SCF) E3 ubiquitin ligase regulates cellular functions through ubiquitination and degradation of protein substrates. We report that, when expressed in mouse epidermis driven by the K14 promoter, SAG inhibited TPA-induced c-Jun levels and activator protein-1 (AP-1) activity in both in vitro primary culture, in vivo transgenic mice, and an AP-1– luciferase reporter mouse model. After AP-1 inactivation, epidermal proliferation induced by 7,12-dimethylbenz(a)-anthracene/12-O-tetradecanoylphorbol-13-acetate at the early stage of carcinogenesis was substantially inhibited. Later stage tumor formation was also substantially inhibited with prolonged latency and reduced frequency of tumor formation. Interestingly, SAG expression increased tumor size, not because of accelerated proliferation, but caused by reduced apoptosis resulting, at least in part, from nuclear factor κB (NF-κB) activation. Thus, SAG, in a manner depending on the availability of F-box proteins, demonstrated early-stage suppression of tumor formation by promoting c-Jun degradation, thereby inhibiting AP-1, and later-stage enhancement of tumor growth, by promoting inhibitor of κBα degradation to activate NF-κB and inhibit apoptosis.

scholarly journals Comprehensive analysis of PPARγ agonist activities of stereo-, regio-, and enantio-isomers of hydroxyoctadecadienoic acids

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Aya Umeno ◽  
Mami Sakashita ◽  
Sakiko Sugino ◽  
Kazutoshi Murotomi ◽  
Tsugumi Okuzawa ◽  
...  
Keyword(s):  
Early Stage ◽  
Biological Activities ◽  
Docking Simulation ◽  
Luciferase Reporter ◽  
Water Ligand ◽  
Peroxisome Proliferator ◽  
Agonist Activity ◽  
Peroxisome Proliferator Activated Receptor ◽  
Pparγ Agonist

Abstract Hydroxyoctadecadienoic acids (HODEs) are produced by oxidation and reduction of linoleates. There are several regio- and stereo-isomers of HODE, and their concentrations in vivo are higher than those of other lipids. Although conformational isomers may have different biological activities, comparative analysis of intracellular function of HODE isomers has not yet been performed. We evaluated the transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ), a therapeutic target for diabetes, and analyzed PPARγ agonist activity of HODE isomers. The lowest scores for docking poses of 12 types of HODE isomers (9-, 10-, 12-, and 13-HODEs) were almost similar in docking simulation of HODEs into PPARγ ligand-binding domain (LBD). Direct binding of HODE isomers to PPARγ LBD was determined by water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR experiments. In contrast, there were differences in PPARγ agonist activities among 9- and 13-HODE stereo-isomers and 12- and 13-HODE enantio-isomers in a dual-luciferase reporter assay. Interestingly, the activity of 9-HODEs was less than that of other regio-isomers, and 9-(E,E)-HODE tended to decrease PPARγ-target gene expression during the maturation of 3T3-L1 cells. In addition, 10- and 12-(Z,E)-HODEs, which we previously proposed as biomarkers for early-stage diabetes, exerted PPARγ agonist activity. These results indicate that all HODE isomers have PPARγ-binding affinity; however, they have different PPARγ agonist activity. Our findings may help to understand the biological function of lipid peroxidation products.

scholarly journals RIPK1 Expression Associates with Inflammation in Early Atherosclerosis in Humans and Can be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice

Circulation ◽  
2020 ◽  
Author(s):  
Denuja Karunakaran ◽  
My-Anh Nguyen ◽  
Michele Geoffrion ◽  
Dianne Vreeken ◽  
Zachary Lister ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Activation ◽  
Inflammatory Responses ◽  
Early Stage ◽  
Oxidized Ldl ◽  
Loss Of Function ◽  
Aortic Sinus ◽  
Atherosclerotic Lesions

Background: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. Previously, we showed that macrophages in the atherogenic plaque undergo RIPK3-MLKL-dependent programmed necroptosis in response to sterile ligands such as oxidized LDL and damage-associated patterns (DAMPs) and necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1, which acts as a master switch that controls whether the cell undergoes NFκB-dependent inflammation, caspase-dependent apoptosis or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is largely driven by NFκB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NFκB-dependent inflammation in early atherogenic lesions and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. Methods: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and using loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 anti-sense oligonucleotides (ASO) to Apoe -/- mice fed a cholesterol-rich (Western) diet for 8 weeks. Results: We find RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 ASOs led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, p<0.01) and plasma inflammatory cytokines (IL-1α, IL-17A, p<0.05) compared to controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NFκB, TNFα, IL-1α) and in vivo LPS- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin and monocyte attachment. Conclusions: We have identified RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

scholarly journals NMR microsystem for label-free characterization of 3D nanoliter microtissues

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Marco Grisi ◽  
Gaurasundar M. Conley ◽  
Kyle J. Rodriguez ◽  
Erika Riva ◽  
Lukas Egli ◽  
...  
Keyword(s):  
Early Stage ◽  
Cmos Technology ◽  
Time Frame ◽  
Ease Of Use ◽  
Chemical Information ◽  
Spectroscopic Techniques ◽  
Label Free ◽  
Alcoholic Fatty Liver ◽  
Non Invasive

AbstractPerforming chemical analysis at the nanoliter (nL) scale is of paramount importance for medicine, drug development, toxicology, and research. Despite the numerous methodologies available, a tool for obtaining chemical information non-invasively is still missing at this scale. Observer effects, sample destruction and complex preparatory procedures remain a necessary compromise. Among non-invasive spectroscopic techniques, one able to provide holistic and highly resolved chemical information in-vivo is nuclear magnetic resonance (NMR). For its renowned informative power and ability to foster discoveries and life-saving applications, efficient NMR at microscopic scales is highly sought after, but so far technical limitations could not match the stringent necessities of microbiology, such as biocompatible handling, ease of use, and high throughput. Here we introduce a novel microsystem, which combines CMOS technology with 3D microfabrication, enabling nL NMR as a platform tool for non-invasive spectroscopy of organoids, 3D cell cultures, and early stage embryos. In this study we show its application to microlivers models simulating non-alcoholic fatty liver disease, demonstrating detection of lipid metabolism dynamics in a time frame of 14 days based on 117 measurements of single 3D human liver microtissues.

scholarly journals LOMIX, a Mixture of Flaxseed Linusorbs, Exerts Anti-Inflammatory Effects through Src and Syk in the NF-κB Pathway

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 859 ◽  
Author(s):  
Zubair Ahmed Ratan ◽  
Deok Jeong ◽  
Nak Yoon Sung ◽  
Youn Young Shim ◽  
Martin J. T. Reaney ◽  
...  
Keyword(s):  
Acute Toxicity ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Model Systems ◽  
Kinase Assay ◽  
Luciferase Reporter ◽  
No Production ◽  
Anti Inflammatory

Although flax (Linum usitatissimum L.) has long been used as Ayurvedic medicine, its anti-inflammatory role is still unclear. Therefore, we aimed to investigate the anti-inflammatory role of a linusorb mixture (LOMIX) recovered from flaxseed oil. Effects of LOMIX on inflammation and its mechanism of action were examined using several in vitro assays (i.e., NO production, real-time PCR analysis, luciferase-reporter assay, Western blot analysis, and kinase assay) and in vivo analysis with animal inflammation models as well as acute toxicity test. Results: LOMIX inhibited NO production, cell shape change, and inflammatory gene expression in stimulated RAW264.7 cells through direct targeting of Src and Syk in the NF-κB pathway. In vivo study further showed that LOMIX alleviated symptoms of gastritis, colitis, and hepatitis in murine model systems. In accordance with in vitro results, the in vivo anti-inflammatory effects were mediated by inhibition of Src and Syk. LOMIX was neither cytotoxic nor did it cause acute toxicity in mice. In addition, it was found that LOB3, LOB2, and LOA2 are active components included in LOMIX, as assessed by NO assay. These in vitro and in vivo results suggest that LOMIX exerts an anti-inflammatory effect by inhibiting the inflammatory responses of macrophages and ameliorating symptoms of inflammatory diseases without acute toxicity and is a promising anti-inflammatory medication for inflammatory diseases.

scholarly journals Fosl1 is vital to heart regeneration upon apex resection in adult Xenopus tropicalis

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Hai-Yan Wu ◽  
Yi-Min Zhou ◽  
Zhu-Qin Liao ◽  
Jia-Wen Zhong ◽  
You-Bin Liu ◽  
...  
Keyword(s):  
Early Stage ◽  
Dominant Negative ◽  
Luciferase Reporter ◽  
Heart Regeneration ◽  
Cell Cycle Regulators ◽  
Cardiomyocyte Proliferation ◽  
Neonatal Mice ◽  
Heart Injury

AbstractCardiovascular disease is the leading cause of death in the world due to losing regenerative capacity in the adult heart. Frogs possess remarkable capacities to regenerate multiple organs, including spinal cord, tail, and limb, but the response to heart injury and the underlying molecular mechanism remains largely unclear. Here we demonstrated that cardiomyocyte proliferation greatly contributes to heart regeneration in adult X. tropicalis upon apex resection. Using RNA-seq and qPCR, we found that the expression of Fos-like antigen 1 (Fosl1) was dramatically upregulated in early stage of heart injury. To study Fosl1 function in heart regeneration, its expression was modulated in vitro and in vivo. Overexpression of X. tropicalis Fosl1 significantly promoted the proliferation of cardiomyocyte cell line H9c2. Consistently, endogenous Fosl1 knockdown suppressed the proliferation of H9c2 cells and primary cardiomyocytes isolated from neonatal mice. Taking use of a cardiomyocyte-specific dominant-negative approach, we show that blocking Fosl1 function leads to defects in cardiomyocyte proliferation during X. tropicalis heart regeneration. We further show that knockdown of Fosl1 can suppress the capacity of heart regeneration in neonatal mice, but overexpression of Fosl1 can improve the cardiac function in adult mouse upon myocardium infarction. Co-immunoprecipitation, luciferase reporter, and ChIP analysis reveal that Fosl1 interacts with JunB and promotes the expression of Cyclin-T1 (Ccnt1) during heart regeneration. In conclusion, we demonstrated that Fosl1 plays an essential role in cardiomyocyte proliferation and heart regeneration in vertebrates, at least in part, through interaction with JunB, thereby promoting expression of cell cycle regulators including Ccnt1.

scholarly journals High-Density Lipoproteins and Serum Amyloid A (SAA)

2021 ◽  
Vol 23 (2) ◽  
Author(s):  
Nancy R. Webb
Keyword(s):  
Serum Amyloid A ◽  
Inflammatory Responses ◽  
Inflammatory Diseases ◽  
Density Lipoprotein ◽  
High Density ◽  
Serum Amyloid ◽  
High Density Lipoproteins ◽  
Amyloid A

Abstract Purpose of Review Serum amyloid A (SAA) is a highly sensitive acute phase reactant that has been linked to a number of chronic inflammatory diseases. During a systemic inflammatory response, liver-derived SAA is primarily found on high-density lipoprotein (HDL). The purpose of this review is to discuss recent literature addressing the pathophysiological functions of SAA and the significance of its association with HDL. Recent Findings Studies in gene-targeted mice establish that SAA contributes to atherosclerosis and some metastatic cancers. Accumulating evidence indicates that the lipidation state of SAA profoundly affects its bioactivities, with lipid-poor, but not HDL-associated, SAA capable of inducing inflammatory responses in vitro and in vivo. Factors that modulate the equilibrium between lipid-free and HDL-associated SAA have been identified. Summary HDL may serve to limit SAA’s bioactivities in vivo. Understanding the factors leading to the release of systemic SAA from HDL may provide insights into chronic disease mechanisms.

Sign in / Sign up

Export Citation Format

Share Document