bluuebunny/crossref_metadata_2025_split huggingface dataset:
Muscle‐derived exosome/miRNA‐26a attenuates skeletal muscle wasting and cardiomyopathy in chronic kidney disease (CKD) mice
Authors: Wang Bin , Liu Bi cheng 
Published: June 2021

IntroductionUremic cardiomyopathy and muscle atrophy contribute to CKD‐induced morbidity and mortality. Exosomes, natural carriers of many signal molecules including microRNA (miR), mediate organ to organ communication. We hypothesized that miR‐26 would benefit both CKD‐induced muscle wasting and cardiomyopathy through exosome‐mediated muscle‐heart crosstalk.MethodsWe used an engineered exosome vector which contains an exosomal membrane protein gene Lamp2b fused with muscle specific surface peptide for targeting delivery. Exosome encapsulated miR‐26a precursor RNA (Exo/miR26) were generated in muscle satellite cells and injected into the tibialis anterior (TA) muscle of CKD mice (5/6 subtotal nephrectomy) for 12 weeks. NanoSight instrument was used to quantify exosomes. A miR deep sequencing assay and qPCR were used to identify microRNA expression. Cardiac ultrasound was used to detect heart size and function. In‐Vivo Xtreme camera system was used to detect exosome in vivo.ResultsmiR‐26a was decreased in skeletal muscle and heart of CKD mice. Uremic serum enhanced secretion of miR‐26a exosomes in cultured C2C12 skeletal and H9C2 cardiac muscle cells. The intervention of Exo/miR26a increased the expression of miR‐26a in skeletal muscle and heart, as well as increased muscle cross‐section area and decreased CKD‐induced upregulation of atrogin‐1 and MuRF1. Curiously, cardiac fibrosis lesion was partially depressed, and FoxO1, α‐SMA, connect tissue growth factor (CTGF), fibronectin and collagen1α were decreased in CKD mice with intramuscular injection of Exo/miR‐26a. Echocardiography showed that the percentage of ejection fraction was increased in CKD mice treated with Exo/miR26a. Using fluorescence dye labeled Exo/miR26a, we found that the fluorescence intensity in heart was correlated with skeletal muscle, examined by linear regression. We found that miR‐26a directly inhibits FoxO1 and CTGF, which provided mechanism for inhibition of muscle atrophy and cardiac fibrosis by Exo/miR26a.ConclusionsOverexpression of miR‐26a in muscle prevents CKD‐induced muscle loss and attenuates cardiac fibrosis via exosome‐mediated muscle‐heart crosstalk.Support or Funding InformationNational Natural Science Foundation of China (81700618), National Key Research Program (2018YFC130046, 2018YFC1314000).
imageThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Postganglionic Parasympathetic but Not Postganglionic Sympathetic Neuron Derived Exosomes Inhibit Hyperglycemia Induced Apoptosis in H9c2 Cells
Authors: Singla Reetish Kumar , Garner Kaley , Singla Dinender K. , Cheng Zixi (Jack) 
Published: June 2021

Diabetic cardiomyopathy involves the two main types of cell death: apoptosis and necrosis. It is unclear, however, on whether apoptosis induced by hyperglycemia in a cell culture model can be inhibited by postganglionic parasympathetic neuron derived exosomes (PPN‐Exos). Postganglionic parasympathetic and postganglionic sympathetic neurons were isolated, cell cultured, and exosomes were isolated. H9c2 cells were utilized to induce apoptosis via hyperglycemia, which were then divided into 4 groups: 1. Control, 2. H9c2 + Glucose, 3. H9c2 + Glucose + PPN‐Exos, 4. H9c2 + Glucose + postganglionic sympathetic neuron‐derived exosomes (PSN‐Exos). An MTT assay kit was utilized to determine cell proliferation and viability, while apoptosis was determined using TUNEL staining and cell death detection (CDD) ELISA kit. The H9c2 cells that were exposed to glucose displayed significantly (p<0.05) reduced cell viability, and a significant (p<0.05) increase in TUNEL staining and CDD ELISA was present in hyperglycemic H9c2 cells compared to controls and this reduced cell viability and increased apoptosis was reversed with PPN‐Exos but not with PSN‐Exos, suggesting a cell protecting components are present in these PPN‐Exos. Our data on pro‐apoptotic gene expression of caspase‐3 and BAX was significantly (p<0.05) increased in glucose treated H9c2 cells compared with control. These pro‐apoptotic genes were attenuated with PPN‐Exos treatment. Anti‐apoptotic protein Bcl‐2 levels were significantly (p<0.05) reduced in hyperglycemia group and this decrease in Bcl‐2 was improved following PPN‐Exos treatment. In conclusion, our data demonstrate that apoptosis is present in hyperglycemic H9c2 cells mediated through caspase‐3 and BAX pathway is inhibited with PPN‐Exos but not with PSN‐Exos. Our data also suggest that PPN‐Exos must be containing a unique set of protective factors that needs further investigation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Histones Activate Toll‐Like Receptor 9 to Trigger Calcium Oscillations in Rat Pancreatic Acinar Cells
Authors: Cui Zong Jie , Guo Hai Yan 
Published: June 2021

In acute pancreatitis histones are released by infiltrating neutrophils, but how histones modulate pancreatic acinar cells has not been investigated. We have examined histone modulation of rat pancreatic acini and pancreatic acinar tumor cell AR4‐2J by calcium imaging. Histones were found to have no effect on calcium in pancreatic acini but blocked calcium oscillations induced by CCK or ACh. Both mixed (Hx) and individual (H1, H2A, H2B, H3, H4) histones induced calcium oscillations in AR4‐2J. RT‐PCR and Western blot verified expression of histone‐targeted TLR2, 4, 9. Immunocytochemistry identified TLR2/TLR4 on apical plasma membrane and TLR9 in zymogen granule regions in pancreatic acini. TLR2 was found on neighboring and TLR9 on peripheral plasma membranes but TLR4 was in nucleus in AR4‐2J clusters. Neither TLR2 agonist zymosan‐A nor TLR4 agonist lipopolysaccharide had any effect on calcium, but TLR9 agonist ODN1826 induced calcium oscillations; TLR9 antagonist ODN2088 blocked H4‐induced calcium oscillations in AR4‐2J, which also disappeared after treatment of AR4‐2J with glucocorticoid dexamethasone (Dex), with concurrent TLR9 migration from plasma membrane to cell interiors. TLR9 down regulation with siRNA completely suppressed H4‐induced calcium oscillations. These data together suggest that extracellular histones activate plasma membrane TLR9 to trigger calcium oscillations in AR4‐2J cells. TLR9 protein shuttling between plasma and endosomal membranes determines whether calcium signaling is triggered. Ongoing work will outline the signaling pathway from plasma membrane TLR9 activation to cytosolic calcium oscillations and its correlation with the canonical MyD88‐dependent pathway of TLR9 activation.Support or Funding InformationThis work was supported by The Natural Science Foundation of China (31670856).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Matrix Fibrous Proteins and Cells in Central Vein Fibrosis of Cadaveric Livers
Authors: Mak Ki M 
Published: June 2021

The central vein (CV) is defined as a hepatic vein at the center of the classic liver lobule. It has a cross‐sectional diameter not exceeding 125 μm. The endothelium lining of the vein is thin and has openings that drain every sinusoid of a liver lobule. The rim or wall of the CV is less than 2 μm in thickness with sparse matrix fibrous proteins and cells. Fibrosis of the CV (CVF), also is called perivenular fibrosis, is marked by thickening of the rim of the vein. The fibrotic tissue deposited in the CV may act like a “pervenular fibrous dam” that obliterates the sinusoidal openings into the vein, resulting in sinusoidal portal hypertension. CVF occurs early in hepatic fibrogenesis of rats induced by CCl4 or porcine serum administration. CVF often accompanies perisinusoidal fibrosis of the centrilobular area in alcoholic liver disease and nonalcoholic fatty liver disease. In the liver of elderly cadavers (mean age, 82.1±10.4), we have observed a 49.2 % incidence (31/63 livers) of CVF, but the matrix components of the fibrotic veins are not fully characterized. This study assessed fibrous proteins and cells in CVF of cadaveric livers. A total of 72 fibrotic veins out of 315 veins from 63 livers were studied. The diameters of the veins ranged from 25.4 to 123.7 μm (mean, 75.5 ± 18.3 μm). CVF was considered present when the rim thickness exceeded 9 μm (determined on Sirius red stained sections) coincident with fibrous tissue extension into the perivenous parenchyma. Immunoperoxidase staining revealed that the CV endothelium was positive for factor VIII‐related antigen, a vascular endothelial phenotypic marker. It also stained for CD31 and D34. Staining of basement membrane collagen IV was continuous around the CV endothelium, while laminin, another principal basement membrane protein, was not detected in the CV matrix. Thus, the CV appeared to lack a continuous endothelial basement membrane and a diffusion barrier between the CV circulation and perivenous parenchyma. Fibrillar collagens I, III and V and filamentous collagen VI were robust in the rim of the fibrotic vein. The codistribution of these collagens rendered thickening of the CV as well as provided strength to the CV matrix scaffold. Elastic fibers were noted occasionally among collagen bundles. Myofibroblasts, identified by a‐smooth muscle actin, were the dominant fibrogenic cells producing the fibrous matrix. Few mononuclear cells were also present. The deposition of collagenous fibrous tissue in the rim could occlude the CV endothelial openings, reducing the efficiency and increasing the resistance of sinusoidal blood flow into the CV. This may cause sinusoidal hypertension, impair hepatocyte functions and enhance disease susceptibility of the aged people.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CYP2C9 and CYP2C19: Massively Parallel Characterization of the Function of Genomic Missense Variants
Authors: Zhang Lingxin , Sarangi Vivekananda , Moon Irene , Devarajan Sandhya , Reid Joel M. , Wang Liewei , Weinshilboum Richard 
Published: June 2021

Single nucleotide variants in the open reading frames (ORFs) of pharmacogenes are important causes of interindividual variability in drug response. Because of the very large number of missense variants in ORFs, it is difficult to link the genotypes of these variants to their phenotypes using “one at a time” expression systems. Therefore, the functional characterization of variants of unknown significance in clinically important pharmacogenes remains a major challenge in pharmacogenomics. Deep mutational scanning (DMS) is a high‐throughput technique that make it possible to analyze hundreds of different missense variants in a parallel and scalable fashion. Engineered “landing pad” HEK 293T cells were used as a platform to integrate pooled variant libraries, resulting in one variant per cell for further assessment. We adapted this DMS system to analyze the function of missense variants in the ORFs of CYP2C9 and CYP2C19. The ORFs of CYP2C9 and CYP2C19 were fused to green fluorescent protein, and individual variants with different fluorescence intensities were used to indicate different levels of protein expression. Pooled variant libraries were generated by nicking mutagenesis, and we then used recombinase to integrate the libraries into landing pad cells, one per cell. Multiplexed functional selection performed with Fluorescence Activated Cell Sorting separated cells into different bins by fluorescence readout at the single–cell level. Amplicon sequencing of DNA collected in each bin, followed by computational analysis of the frequency of variants appearing in each bin, was used to determine levels of protein expression. To identify potentially severely damaging variants for these two important pharmacogenes, we studied 243 missense variants in the CYP2C9 and CYP2C19 ORFs that had been identified by the Exome Aggregation Consortium Project and by the Mayo Clinic RIGHT 1K Study. We found that 13 of 115 CYP2C9 and 25 of 128 CYP2C19 missense variants displayed less than approximately 25% of the wildtype protein expression, a level that may have clinical relevance for drug metabolism. Variant calling by the DMS method for the severely damaging variants was compared with the results of individual variant assay by western blot and with the predictions of computational algorithms such as Polyphen and SIFT. Our results suggest that DMS is an efficient method for the high‐throughput identification of damaging ORF variants that might have potential clinical application in pharmacogenomics.Support or Funding InformationFunded by NIH grant U19 GM61388 (The Pharmacogenomics Research Network), R01 GM28157, R01 GM125633, T32 GM08685 and by the Mayo Clinic Center for Individualized MedicineThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization is Essential to Increase Transfection Efficiency and Cell Viability in Primary and Difficult‐to‐Transfect cell lines
Authors: Jordan Elizabeth T. , Collins Michelle , Terefe Joseph , Pineda Maxinne , Ugozzoli Luis , Rubio Teresa 
Published: June 2021

Electroporation is a valuable tool for nucleic acid delivery because it can be used for a wide variety of cell types. Many scientists are shifting towards the use of cell types of increasing scientific interest, including lineage‐specific cell types, bone marrow cells and stem cells, but these cells can be difficult to transfect. The ability to transfect these cell types with nucleic acid molecules of interest at a relatively high efficiency while maintaining cell viability would offer a new tool towards the elucidation of the pathway(s) in which a gene product is involved.In this poster, we present data demonstrating that optimization of electroporation parameters is essential to deliver molecules in a highly efficient manner. We display results of transfection of several difficult to transfect cell types, including Human Primary Fibroblasts, HUVEC, Jurkat and (mouse) Neuro‐2A neural cells with plasmid DNAs and siRNAs. We used multiple detection methods to determine transfection efficiency, including flow cytometry, real‐time PCR and the luciferase assay. Gene Pulser electroporation buffer and square or exponential wave pulses were used to convey molecules of interest into the cells. Optimization of electroporation parameters was carried out using the high throughput Gene Pulser MXcell electroporation system. Just 4 hours post electroporation with a GAPDH siRNA, Jurkat cells expressed 88% less GAPDH mRNA than controls transfected with scramble siRNA. The same degree of silencing persisted at 24 and 48 hours post electroporation. Transfection of cells with fluorescently labeled siRNA resulted in 75% transfection efficiency for Neuro‐2A, 93% for HPF and 98% for HUVEC cells, as analyzed by flow cytometry. We provide optimized electroporation conditions for these and other difficult to transfect cells.
Differential placental gene expression in preeclampsia
Authors: Enquobahrie Daniel A , Meller Margaret , Rice Kenneth , Psaty Bruce M , Siscovick David S , Williams Michelle A 
Published: June 2021

Objective: Candidate genes associated with preeclampsia have not been fully described. We conducted a microarray study to investigate global placental gene expression in preeclampsia.Study design: Cases (n=18) and controls (n=18) were selected among patients receiving care at Swedish Medical Center, Seattle, WA. Oligonucleotide probes representing 22,000 genes were used to measure gene expression. Differentially expressed genes were selected using criteria based on Students T‐test, fold change and Significance Analysis of Microarrays (SAM) analysis. Functions and functional relationships of differentially expressed genes were evaluated using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Ingenuity Pathway Analysis (IPA) tools.Results: Genes (n=58) participating in immune system, inflammation, oxidative stress, signaling, growth and development pathways were differentially expressed in preeclampsia. These genes include previously described candidate genes (such as leptin, LEP), potential candidate genes with related functions (such as cytochrome P450 family 11 subfamily A, CYP11A) and novel genes (such as cyclin‐dependent kinase inhibitor 1C, CDKN1C).Conclusion: Expression of genes (both candidate and novel) with diverse functions is associated with preeclampsia risk, reflecting the complex pathogenesis.
Characterization of the metabolic remodeling in liver by genetic mutation of ALDH2 in muscles
Authors: Hishiki Takako , Adachi Takeshi , Sano Motoaki , Katayama Takaharu , Endo Jin , Fukuda Keiichi , Suematsu Makoto 
Published: June 2021

Objective: Aldehyde dehydrogenases 2 (ALDH2) is the detox enzyme to catalyze aldehydes‐carboxylation, which modulates reactive oxygen species in mitochondria. We created transgenic (TG) mice expressing mutant ALDH2 in muscles, which exhibited the phenocopy of senescence. To test the mechanism of detoxification against the systemic aldehydes toxicity, we characterized the compensational metabolic remodeling in liver from TG mice.Methods: ALDH2 expression and phosphorylation of acetyl‐CoA calboxylase (P‐ACC) were assessed with immuno‐blotting. Gene chip analysis and metabolome analysis with capillary electrophoresis mass spectrometry were performed.Results: In TG mice, the mutant protein was highly expressed in heart and skeletal muscles, but not in liver. The upregulation of enzymes for fatty acid oxidation and an increase in the P‐ACC caused the elevation of citrate levels. The inhibition of phosphofructokinase by citrate caused the elevation of glucose 1‐phosphate (G1P) levels and the reduction of metabolites in glycolysis below fructose 1,6‐bisphosphate. The elevated G1P levels could increase the utilization for glucuronide conjugation to detoxify aldehydes.Conclusion: We concluded that the metabolic remodeling of fatty acid oxidation and glycolysis to augment the flux for glucuronide conjugation contributed to the detoxification in liver against the systemic aldehyde toxicity.
Inhibition of fatty acid synthase activates the unfolded protein response in tumor cells
Authors: Kridel Steven J. 
Published: June 2021

Fatty acid synthase (FAS), the enzyme that catalyzes the de novo synthesis of palmitate, is highly expressed in most tumors, with little or no expression in the corresponding normal tissue. Considering the strong link between FAS expression, tumor progression and disease recurrence, it is critical to develop FAS inhibitors and understand their anti‐tumor mechanisms. Because of previous links between lipid homeostasis and endoplasmic reticulum (ER) function in multiple models systems, we hypothesized that the unfolded protein response (UPR) plays a role in the anti‐tumor mechanism of FAS inhibitors. Indeed, FAS inhibitors induce 1) a robust PERK‐dependent phosphorylation of the translation initiation factor eIF2α and 2) the IRE1‐mediated processing of XBP1 specifically in tumor cells, consistent with UPR signal transduction. Induction of the UPR precedes activation of the canonical markers of apoptosis. Inhibition of FAS also antagonizes turnover of ubiquitin‐modified proteins. Interestingly, the proteasome inhibitor Velcade, an FDA‐approved drug that also induces the UPR, induces fatty acid synthesis by up‐regulation of acetyl‐CoA carboxylase (ACC) expression and synergizes with FAS inhibitors to increase UPR signaling. These data provide a link between the fatty acid synthesis pathway and ER homeostasis in tumor cells and demonstrates crosstalk between the fatty acid synthesis and proteasome pathways. These data also suggest that combining FAS inhibitors with other drugs that affect ER homeostasis may translate into more effective tumor therapies.Research supported by NIH CA114104 and the Department of Defense Prostate Cancer Research Program W81XWH‐07‐1‐0024.
Signaling from β1‐ and β2‐adrenergic receptors is defined by differential interactions with PDE4
Authors: Richter Wito , Day Peter , Agrawal Rani , Bruss Matthew D. , Granier Sébastien , Wang Yvonne L. , Rasmussen Søren G.F. , Horner Kathleen , Wang Ping , Lei Tao , Patterson Andrew J. , Kobilka Brian , Conti Marco 
Published: June 2021

β1‐ and β2‐adrenergic receptors (βARs) are highly homologous, yet they play clearly distinct roles in cardiac physiology and pathology. Myocyte contraction, for instance, is readily stimulated by β1AR but not β2AR signaling and chronic stimulation of the two receptors has opposing effects on myocyte apoptosis and cell survival. Differences in the assembly of macromolecular signaling complexes may explain the distinct biological outcomes. Here we demonstrate that β1AR forms a signaling complex with a cAMP‐specific phosphodiesterase (PDE) in a manner inherently different from a β2AR/β‐arrestin/PDE complex reported previously. The β1AR binds a PDE variant, PDE4D8, in a direct manner and occupancy of the receptor by an agonist causes dissociation of this complex. Conversely, agonist binding to the β2AR is a prerequisite for the recruitment of a complex consisting of β‐arrestin and the PDE4D variant, PDE4D5, to the receptor. We propose that the distinct modes of interaction with PDEs result in divergent cAMP signals in the vicinity of the two receptors, thus, providing an additional layer of complexity to enforce the specificity of β1‐ and β2‐adrenoceptor signaling.This research was supported by Fondation Leducq and NIH grants HD20788 to M.C. and HL71078‐01 to B.K.
Localized PKD activity at PDZ domain‐containing scaffolds
Authors: Kunkel Maya T. , Garcia Erin L. , Hall Randy A. , Newton Alexandra C. 
Published: June 2021

The protein kinase D (PKD) family plays a role in many cellular functions including the regulation of Golgi sorting, apoptosis, cell growth and cell survival. The mammalian PKD family consists of three isoforms, PKD1, PKD2 and PKD3, and of these, PKD1 and PKD2 contain PDZ‐binding motifs at their C‐terminus. Using a proteomic array containing 96 purified PDZ domains, we have identified domains that interact with the 25 C‐terminal residues of PKD1 or PKD2. We show that PKD activity localized at the PDZ domain scaffold is significantly greater than bulk cytosolic activity. Specifically, a FRET‐based kinase activity reporter designed to report PKD signaling, DKAR (D Kinase Activity Reporter), when targeted to the scaffold protein, reveals enhanced amplitude and duration of signaling compared to cytosol. Thus, this study unveils novel protein scaffolds that localize PKD activity.This work was supported by NIH P01 DK54441