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Find video protocols related to scientific articles indexed in Pubmed.
A quantitative look inside the body: minimally invasive infrared analysis in vivo.
Anal. Chem.
PUBLISHED: 10-22-2014
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Today's minimally invasive biosensors are often based on chemical reagents and suffer from, e.g., oxygen dependence, toxic reaction products, excess analyte consumption, and/or degradation of the reagents. Here, we show the first successful analyte quantification by means of a minimally invasive sensor in vivo, which does not use chemical reactions. The concentration of glucose is determined continuously in vivo using transcutaneous, fiber-based mid-infrared laser spectroscopy. When comparing the infrared data measured in vivo with the 127 reference readings of glucose obtained in vitro, an overall standard deviation of 17.5% and a median of the absolute values of the relative deviations of 11.0% are achieved. The encouraging results open up the path toward a reagent-free long-term implant for the continuous surveillance of metabolites. In addition, the high sampling rate facilitates important research in body metabolism as well as its application outside the field of medicine such as real-time analyte sensing during fermentation.
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CaM Kinase II mediates maladaptive post-infarct remodeling and pro-inflammatory chemoattractant signaling but not acute myocardial ischemia/reperfusion injury.
EMBO Mol Med
PUBLISHED: 09-05-2014
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CaMKII was suggested to mediate ischemic myocardial injury and adverse cardiac remodeling. Here, we investigated the roles of different CaMKII isoforms and splice variants in ischemia/reperfusion (I/R) injury by the use of new genetic CaMKII mouse models. Although CaMKII?C was upregulated 1 day after I/R injury, cardiac damage 1 day after I/R was neither affected in CaMKII?-deficient mice, CaMKII?-deficient mice in which the splice variants CaMKII?B and C were re-expressed, nor in cardiomyocyte-specific CaMKII?/? double knockout mice (DKO). In contrast, 5 weeks after I/R, DKO mice were protected against extensive scar formation and cardiac dysfunction, which was associated with reduced leukocyte infiltration and attenuated expression of members of the chemokine (C-C motif) ligand family, in particular CCL3 (macrophage inflammatory protein-1?, MIP-1?). Intriguingly, CaMKII was sufficient and required to induce CCL3 expression in isolated cardiomyocytes, indicating a cardiomyocyte autonomous effect. We propose that CaMKII-dependent chemoattractant signaling explains the effects on post-I/R remodeling. Taken together, we demonstrate that CaMKII is not critically involved in acute I/R-induced damage but in the process of post-infarct remodeling and inflammatory processes.
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A schematic workflow for collecting information about the interaction between copy number variants and microRNAs using existing resources.
Methods Mol. Biol.
PUBLISHED: 07-25-2014
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MicroRNAs (miRNAs) and copy number variations (CNVs) are two extensively studied genomic components in the field of modern biology-as they have been found to be associated with many disorders such as cancer, Alzheimer, pancreatitis, HIV susceptibility, beta-thalassemia, and glomerulonephritis. Several studies suggested that an alteration in CNV-miRNA interaction could result in some human diseases such as cancer. Therefore, the possible miRNA-binding site information within the CNV genes opens new avenues in understanding such disorders. In this chapter, we present a schematic approach for collecting the information on CNV-miRNA interactions using miRWalk and TargetScan databases.
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miRWalk database for miRNA-target interactions.
Methods Mol. Biol.
PUBLISHED: 07-25-2014
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miRWalk (http://mirwalk.uni-hd.de/) is a publicly available comprehensive resource, hosting the predicted as well as the experimentally validated microRNA (miRNA)-target interaction pairs. This database allows obtaining the possible miRNA-binding site predictions within the complete sequence of all known genes of three genomes (human, mouse, and rat). Moreover, it also integrates many novel features such as a comparative platform of miRNA-binding sites resulting from ten different prediction datasets, a holistic view of genetic networks of miRNA-gene pathway, and miRNA-gene-Online Mendelian Inheritance in Man disorder interactions, and unique experimentally validated information (e.g., cell lines, diseases, miRNA processing proteins). In this chapter, we describe a schematic workflow on how one can access the stored information from miRWalk and subsequently summarize its applications.
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Angiopoietin-1 is regulated by miR-204 and contributes to corneal neovascularization in KLEIP-deficient mice.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 06-12-2014
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Corneal neovascularization can cause loss of vision. The introduction of anti-VEGF therapy has been a major improvement in therapeutic options. Recently, we established Kelch-like Ect2-interacting protein (KLEIP/KLHL20) knockout mice as a model of spontaneous corneal neovascular dystrophy. The aim of the present study was to characterize corneal neovascularization in progressive corneal dystrophy in KLEIP(-/-) mice, to evaluate the efficacy of anti-VEGF therapy, and to identify novel molecular regulators in this experimental model.
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Transcutaneous assessment of glomerular filtration rate.
Stud Health Technol Inform
PUBLISHED: 05-24-2014
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Glomerular filtration rate (GFR) is considered the best parameter for the assessment of renal function, being usually determined on the basis of urine or plasma clearance of exogenous renal markers. The common methodology is invasive, time consuming and cumbersome, with multiple blood and/or urine sampling and following laboratory assays required. The method detailed here allows to transcutaneously determine the renal function in awake animals, in a non-invasive and efficient manner by using an electronic device which detects the fluorescence emitted through the skin from the renal marker FITC-Sinistrin. A crucial target has been to improve the fixation of the device, which is dependent on the skin structure. For validation, the technique has been compared with the classical clearance method, and its robustness has been demonstrated in healthy and diseased murine models. Moreover, the method allows sequential measurements in the same individual. Thus progression and recovery of renal failure can be followed. Therefore, its future application in humans would allow an accurate and appropriate prediction and monitoring of patients with established kidney disease over time. Furthermore, it will be possible to observe those patients under other pathological conditions with associated risk of developing renal problems.
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Prosurvival function of the cellular apoptosis susceptibility/importin-?1 transport cycle is repressed by p53 in liver cancer.
Hepatology
PUBLISHED: 05-02-2014
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Proteins of the karyopherin superfamily including importins and exportins represent an essential part of the nucleocytoplasmic transport machinery. However, the functional relevance and regulation of karyopherins in hepatocellular carcinoma (HCC) is poorly understood. Here we identified cellular apoptosis susceptibility (CAS, exportin-2) and its transport substrate importin-?1 (imp-?1) among significantly up-regulated transport factor genes in HCC. Disruption of the CAS/imp-?1 transport cycle by RNAi in HCC cell lines resulted in decreased tumor cell growth and increased apoptosis. The apoptotic phenotype upon CAS depletion could be recapitulated by direct knockdown of the X-linked inhibitor of apoptosis (XIAP) and partially reverted by XIAP overexpression. In addition, XIAP and CAS mRNA expression levels were correlated in HCC patient samples (r=0.463; P<0.01), supporting the in vivo relevance of our findings. Furthermore, quantitative mass spectrometry analyses of murine HCC samples (p53-/- versus p53+/+) indicated higher protein expression of CAS and imp-?1 in p53-/- tumors. Consistent with a role of p53 in regulating the CAS/imp-?1 transport cycle, we observed that both transport factors were repressed upon p53 induction in a p21-dependent manner.
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Overexpression of far upstream element (FUSE) binding protein (FBP)-interacting repressor (FIR) supports growth of hepatocellular carcinoma.
Hepatology
PUBLISHED: 03-17-2014
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The far upstream element binding protein (FBP) and the FBP-interacting repressor (FIR) represent molecular tools for transcriptional fine tuning of target genes. Strong overexpression of FBP in human hepatocellular carcinoma (HCC) supports tumor growth and correlates with poor patient prognosis. However, the role of the transcriptional repressor FIR in hepatocarcinogenesis remains poorly delineated. We show that overexpression of FIR correlates with tumor dedifferentiation and tumor cell proliferation in about 60% of primary HCCs. Elevated FIR levels are associated with genomic gains of the FIR gene locus at chromosome 8q24.3 in human HCC specimens. In vitro, nuclear enrichment of FIR supports HCC cell proliferation and migration. Expression profiling of HCC cells after small interfering RNA (siRNA)-mediated silencing of FIR identified the transcription factor DP-1 (TFDP1) as a transcriptional target of FIR. Surprisingly, FIR stimulates the expression of FBP in a TFDP1/E2F1-dependent manner. FIR splice variants lacking or containing exon 2 and/or exon 5 are expressed in the majority of HCCs but not in normal hepatocytes. Specific inhibition of FIR isoforms with and without exon 2 revealed that both groups of FIR splice variants facilitate tumor-supporting effects. This finding was confirmed in xenograft transplantation experiments with lentiviral-infected short hairpin RNA (shRNA) targeting all FIR variants as well as FIR with and without exon 2.
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MicroRNA profiling of rats with ochratoxin A nephrotoxicity.
BMC Genomics
PUBLISHED: 03-14-2014
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Nephrotoxicity is the most prominent one among the various toxicities of ochratoxin A (OTA). MicroRNAs (miRNAs) are small non-coding RNAs that have an impact on a wide range of biological processes by regulating gene expression at post-transcriptional level or protein systhesis level. The objective of this study is to analyze miRNA profiling in the kidneys of rats gavaged with OTA.
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Resistance of ferroportin to hepcidin binding causes exocrine pancreatic failure and fatal iron overload.
Cell Metab.
PUBLISHED: 02-28-2014
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The regulatory axis between the iron hormone hepcidin and its receptor, the iron exporter ferroportin (FPN), is central to iron homeostasis. Mutations preventing hepcidin-mediated degradation of FPN cause systemic iron overload. We have introduced a point mutation (C326S) into the murine Fpn locus, resembling human hereditary hemochromatosis type 4, including elevated plasma iron and ferritin levels, high transferrin saturation, hepatic iron overload, and iron depletion of duodenal enterocytes and reticuloendothelial macrophages. Unlike other mouse models of iron overload, homozygous C326S mice die between 7 and 14 months of age. Pancreatic acinar cells display marked iron accumulation, oxidative damage and degeneration, associated with failure of the exocrine pancreas and severe body weight loss. Rescue experiments reveal iron overload and exocrine pancreatic failure as leading causes of death. This work uncovers the critical importance of the hepcidin-ferroportin regulatory axis for life and unveils the sensitivity of the exocrine pancreas to iron overload.
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Neisseria meningitidis elicits a pro-inflammatory response involving I?B? in a human blood-cerebrospinal fluid barrier model.
J Neuroinflammation
PUBLISHED: 02-25-2014
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The human-specific, Gram-negative bacterium Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis worldwide. The blood-cerebrospinal fluid barrier (BCSFB), which is constituted by the epithelial cells of the choroid plexus (CP), has been suggested as one of the potential entry sites of Nm into the CSF and can contribute to the inflammatory response during infectious diseases of the brain. Toll-like receptors (TLRs) are involved in mediating signal transduction caused by the pathogens.
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Quantum cascade laser-based hyperspectral imaging of biological tissue.
J Biomed Opt
PUBLISHED: 02-20-2014
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The spectroscopy of analyte-specific molecular vibrations in tissue thin sections has opened up a path toward histopathology without the need for tissue staining. However, biomedical vibrational imaging has not yet advanced from academic research to routine histopathology due to long acquisition times for the microscopic hyperspectral images and/or cost and availability of the necessary equipment. Here we show that the combination of a fast-tuning quantum cascade laser with a microbolometer array detector allows for a rapid image acquisition and bares the potential for substantial cost reduction. A 3.1 x 2.8 mm2 unstained thin section of mouse jejunum has been imaged in the 9.2 to 9.7 ?m wavelength range (spectral resolution ~1 cm(-1)) within 5 min with diffraction limited spatial resolution. The comparison of this hyperspectral imaging approach with standard Fourier transform infrared imaging or mapping of the identical sample shows a reduction in acquisition time per wavenumber interval and image area by more than one or three orders of magnitude, respectively.
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Direct acute tubular damage contributes to Shigatoxin-mediated kidney failure.
J. Pathol.
PUBLISHED: 01-07-2014
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The pathogenesis and therapy of Shigatoxin 2 (Stx2)-mediated kidney failure remain controversial. Our aim was to test whether, during an infection with Stx2-producing E. coli (STEC), Stx2 exerts direct effects on renal tubular epithelium and thereby possibly contributes to acute renal failure. Mice represent a suitable model because they, like humans, express the Stx2-receptor Gb3 in the tubular epithelium but, in contrast to humans, not in glomerular endothelia, and are thus free of glomerular thrombotic microangiopathy (TMA). In wild-type mice, Stx2 caused acute tubular dysfunction with consequent electrolyte disturbance, which was most likely the cause of death. Tubule-specific depletion of Gb3 protected the mice from acute renal failure. In vitro, Stx2 induced secretion of proinflammatory cytokines and apoptosis in human tubular epithelial cells, thus implicating a direct effect of Stx2 on the tubular epithelium. To correlate these results to human disease, kidney biopsies and outcome were analysed in patients with Stx2-associated kidney failure (n = 11, aged 22-44 years). The majority of kidney biopsies showed different stages of an ongoing TMA; however, no glomerular complement activation could be demonstrated. All biopsies, including those without TMA, showed severe acute tubular damage. Due to these findings, patients were treated with supportive therapy without complement-inhibiting antibodies (eculizumab) or immunoadsorption. Despite the severity of the initial disease [creatinine 6.34 (1.31-17.60) mg/dl, lactate dehydrogenase 1944 (753-2792) U/l, platelets 33 (19-124)/nl and haemoglobin 6.2 (5.2-7.8) g/dl; median (range)], all patients were discharged after 33 (range 19-43) days with no neurological symptoms and no dialysis requirement [creatinine 1.39 (range 0.84-2.86) mg/dl]. The creatinine decreased further to 0.90 (range 0.66-1.27) mg/dl after 24 months. Based on these data, one may surmise that acute tubular damage represents a separate pathophysiological mechanism, importantly contributing to Stx2-mediated acute kidney failure. Specifically in young adults, an excellent outcome can be achieved by supportive therapy only.
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Forced arm use is superior to voluntary training for motor recovery and brain plasticity after cortical ischemia in rats.
Exp Transl Stroke Med
PUBLISHED: 01-07-2014
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Both the immobilization of the unaffected arm combined with physical therapy (forced arm use, FAU) and voluntary exercise (VE) as model for enriched environment are promising approaches to enhance recovery after stroke. The genomic mechanisms involved in long-term plasticity changes after different means of rehabilitative training post-stroke are largely unexplored. The present investigation explored the effects of these physical therapies on behavioral recovery and molecular markers of regeneration after experimental ischemia.
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Systemic treatment with erythropoietin protects the neurovascular unit in a rat model of retinal neurodegeneration.
PLoS ONE
PUBLISHED: 01-01-2014
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Rats expressing a transgenic polycystic kidney disease (PKD) gene develop photoreceptor degeneration and subsequent vasoregression, as well as activation of retinal microglia and macroglia. To target the whole neuroglialvascular unit, neuro- and vasoprotective Erythropoietin (EPO) was intraperitoneally injected into four-week old male heterozygous PKD rats three times a week at a dose of 256 IU/kg body weight. For comparison EPO-like peptide, lacking unwanted side effects of EPO treatment, was given five times a week at a dose of 10 µg/kg body weight. Matched EPO treated Sprague Dawley and water-injected PKD rats were held as controls. After four weeks of treatment the animals were sacrificed and analysis of the neurovascular morphology, glial cell activity and pAkt localization was performed. The number of endothelial cells and pericytes did not change after treatment with EPO or EPO-like peptide. There was a nonsignificant reduction of migrating pericytes by 23% and 49%, respectively. Formation of acellular capillaries was significantly reduced by 49% (p<0.001) or 40% (p<0.05). EPO-treatment protected against thinning of the central retina by 10% (p<0.05), a composite of an increase of the outer nuclear layer by 12% (p<0.01) and in the outer segments of photoreceptors by 26% (p<0.001). Quantification of cell nuclei revealed no difference. Microglial activity, shown by gene expression of CD74, decreased by 67% (p<0.01) after EPO and 36% (n.s.) after EPO-like peptide treatment. In conclusion, EPO safeguards the neuroglialvascular unit in a model of retinal neurodegeneration and secondary vasoregression. This finding strengthens EPO in its protective capability for the whole neuroglialvascular unit.
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Identification of novel SHOX target genes in the developing limb using a transgenic mouse model.
PLoS ONE
PUBLISHED: 01-01-2014
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Deficiency of the human short stature homeobox-containing gene (SHOX) has been identified in several disorders characterized by reduced height and skeletal anomalies such as Turner syndrome, Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia as well as isolated short stature. SHOX acts as a transcription factor during limb development and is expressed in chondrocytes of the growth plates. Although highly conserved in vertebrates, rodents lack a SHOX orthologue. This offers the unique opportunity to analyze the effects of human SHOX expression in transgenic mice. We have generated a mouse expressing the human SHOXa cDNA under the control of a murine Col2a1 promoter and enhancer (Tg(Col2a1-SHOX)). SHOX and marker gene expression as well as skeletal phenotypes were characterized in two transgenic lines. No significant skeletal anomalies were found in transgenic compared to wildtype mice. Quantitative and in situ hybridization analyses revealed that Tg(Col2a1-SHOX), however, affected extracellular matrix gene expression during early limb development, suggesting a role for SHOX in growth plate assembly and extracellular matrix composition during long bone development. For instance, we could show that the connective tissue growth factor gene Ctgf, a gene involved in chondrogenic and angiogenic differentiation, is transcriptionally regulated by SHOX in transgenic mice. This finding was confirmed in human NHDF and U2OS cells and chicken micromass culture, demonstrating the value of the SHOX-transgenic mouse for the characterization of SHOX-dependent genes and pathways in early limb development.
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Evidence for Activation of the Unfolded Protein Response in Collagen IV Nephropathies.
J. Am. Soc. Nephrol.
PUBLISHED: 11-21-2013
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Thin-basement-membrane nephropathy (TBMN) and Alport syndrome (AS) are progressive collagen IV nephropathies caused by mutations in COL4A3/A4/A5 genes. These nephropathies invariably present with microscopic hematuria and frequently progress to proteinuria and CKD or ESRD during long-term follow-up. Nonetheless, the exact molecular mechanisms by which these mutations exert their deleterious effects on the glomerulus remain elusive. We hypothesized that defective trafficking of the COL4A3 chain causes a strong intracellular effect on the cell responsible for COL4A3 expression, the podocyte. To this end, we overexpressed normal and mutant COL4A3 chains (G1334E mutation) in human undifferentiated podocytes and tested their effects in various intracellular pathways using a microarray approach. COL4A3 overexpression in the podocyte caused chain retention in the endoplasmic reticulum (ER) that was associated with activation of unfolded protein response (UPR)-related markers of ER stress. Notably, the overexpression of normal or mutant COL4A3 chains differentially activated the UPR pathway. Similar results were observed in a novel knockin mouse carrying the Col4a3-G1332E mutation, which produced a phenotype consistent with AS, and in biopsy specimens from patients with TBMN carrying a heterozygous COL4A3-G1334E mutation. These results suggest that ER stress arising from defective localization of collagen IV chains in human podocytes contributes to the pathogenesis of TBMN and AS through activation of the UPR, a finding that may pave the way for novel therapeutic interventions for a variety of collagenopathies.
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MALDI imaging MS reveals candidate lipid markers of polycystic kidney disease.
J. Lipid Res.
PUBLISHED: 07-12-2013
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Autosomal recessive polycystic kidney disease (ARPKD) is a severe, monogenetically inherited kidney and liver disease. PCK rats carrying the orthologous mutant gene serve as a model of human disease, and alterations in lipid profiles in PCK rats suggest that defined subsets of lipids may be useful as molecular disease markers. Whereas MALDI protein imaging mass spectrometry (IMS) has become a promising tool for disease classification, widely applicable workflows that link MALDI lipid imaging and identification as well as structural characterization of candidate disease-classifying marker lipids are lacking. Here, we combine selective MALDI imaging of sulfated kidney lipids and Fisher discriminant analysis (FDA) of imaging data sets for identification of candidate markers of progressive disease in PCK rats. Our study highlights strong increases in lower mass lipids as main classifiers of cystic disease. Structure determination by high-resolution mass spectrometry identifies these altered lipids as taurine-conjugated bile acids. These sulfated lipids are selectively elevated in the PCK rat model but not in models of related hepatorenal fibrocystic diseases, suggesting that they be molecular markers of the disease and that a combination of MALDI imaging with high-resolution MS methods and Fisher discriminant data analysis may be applicable for lipid marker discovery.
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Quantitative characterization of tissue globotetraosylceramides in a rat model of polycystic kidney disease by PrimaDrop sample preparation and indirect high-performance thin layer chromatography-matrix-assisted laser desorption/ionization-time-of-flight-
Anal. Chem.
PUBLISHED: 06-11-2013
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Glycosphingolipids (GSL) have been associated with a variety of diseases, including cancer and autosomal dominant polycystic kidney disease (ADPKD). In contrast to glucosylceramide and gangliosides, alterations in complex neutral GSLs such as globotetraosylceramide (Gb4Cer) have not been investigated in ADPKD yet, and mass spectrometry analysis of Gb4Cer from tissue extracts remains challenging. To this end, we introduce PrimaDrop as an improved and widely applicable sample preparation method for automated matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) analysis of lipid extracts, which promotes homogeneous cocrystallization and enables relative quantification by indirect thin layer chromatography (TLC)-MALDI-time-of-flight (TOF)-MS against an internal bradykinin standard. Application of the method for detailed investigation of Gb4Cer isoforms in kidneys of an ADPKD rat model revealed increased levels of sphingoid base-containing isoforms in cystic kidneys, whereas changes were subtle for Gb4Cer-containing phytosphingoid bases. We furthermore established an absolute LC-ESI-MS/MS quantification method and demonstrate that absolute quantities of Gb4Cer correlate well with relative quantities obtained by indirect TLC-MALDI-TOF-MS. Taken together, our study proposes an effective sample preparation method for automated analysis of lipid extracts and TLC eluates and suggests that indirect high-performace (HP)TLC-MALDI-TOF-MS with automated data acquisition is a viable option for analysis of neutral glycosphingolipids and that Gb4Cer may play a role in the pathogenesis of ADPKD.
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Differentiation of epidermal keratinocytes is dependent on glucosylceramide:ceramide processing.
Hum. Mol. Genet.
PUBLISHED: 06-06-2013
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Skin barrier function is primarily assigned to the outer epidermal layer, the stratum corneum (SC), mainly composed of corneocytes and lipid-enriched extracellular matrix. Epidermal ceramides (Cers) are essential barrier lipids, containing ultra-long-chain (ULC) fatty acids (FAs) with a unique ?-hydroxy group, which is necessary for binding to corneocyte proteins. In the SC, Cers are believed to derive from glucosylated intermediates, namely glucosylceramides (GlcCers), as surmised from human Gauchers disease and related mouse models. Tamoxifen (TAM)-induced deletion of the endogenous GlcCer-synthesizing enzyme UDP-glucose:ceramide glucosyltransferase (UGCG) in keratin K14-positive cells resulted in epidermal GlcCer depletion. Although free extractable Cers were elevated in total epidermis and as well in SC, protein-bound Cers decreased significantly in Ugcg(f/fK14CreERT2) mice, indicating glucosylation to be required for regular Cer processing as well as arrangement and extrusion of lipid lamellae. The almost complete loss of protein-bound Cers led to a disruption of the water permeability barrier (WPB). UGCG-deficient mice developed an ichthyosis-like skin phenotype marked by impaired keratinocyte differentiation associated with delayed wound healing. Gene expression profiling of Ugcg-mutant skin revealed a subset of differentially expressed genes involved in lipid signaling and epidermal differentiation/proliferation, correlating to human skin diseases such as psoriasis and atopic dermatitis. Peroxisome proliferator-activated receptor beta/delta (PPAR?/?), a Cer-sensitive transcription factor was identified as potential mediator of the altered gene sets.
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Progression of glomerular filtration rate reduction determined in conscious Dahl salt-sensitive hypertensive rats.
Hypertension
PUBLISHED: 04-29-2013
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Sequential changes in glomerular filtration rate during development of hypertension in the conscious Dahl salt-sensitive rats were determined using a new method for measurement. Using a miniaturized device, disappearance curves of fluorescein isothiocyanate-sinistrin were measured by transcutaneous excitation and real-time detection of the emitted light through the skin. Rats with implanted femoral venous catheters (dye injection and sampling) and carotid catheters (mean arterial pressure by telemetry) were studied, while maintained on a 0.4% NaCl diet and on days 2, 5, 7, 14, and 21 after switching to 4.0% (high-salt [HS]) diet. A separate group of rats were maintained on 0.4% for 21 days as a time control. Mean arterial pressure rose progressively from the last day of 0.4% (130±2 mm Hg) reaching significance by day 5 of HS and averaged 162±7 mm Hg by day 21. Urine albumin excretion was significantly elevated (×3) by day 7 of HS in Dahl salt-sensitive rats. Glomerular filtration rate reduced on day 14 of HS falling from 1.53±0.06 mL/min per 100 g body weight to 1.27±0.04. By day 21, glomerular filtration rate had fallen 28% to 1.1±0.04 mL/min per 100 g (t(1/2) 28.4±1.1 minute.) No significant reductions of creatinine clearance were observed throughout the study in response to HS demonstrating the insensitivity of creatinine clearance measurements even with creatinine measured using mass spectrometry. We conclude that the observed reduction of glomerular filtration rate was a consequence and not a cause of the hypertension and that this noninvasive approach could be used in these conscious Dahl salt-sensitive rats for a longitudinal assessment of renal function.
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Inhibition of Comt with tolcapone slows progression of polycystic kidney disease in the more severely affected PKD/Mhm (cy/+) substrain of the Hannover Sprague-Dawley rat.
Nephrol. Dial. Transplant.
PUBLISHED: 03-29-2013
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Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human inherited diseases. Modifier genes seem to modulate the disease progression and might therefore be promising drug targets. Although a number of modifier loci have been already identified, no modifier gene has been proven to be a real modifier yet.
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The emerging role of MRI in quantitative renal glomerular morphology.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 03-20-2013
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Techniques to measure morphological parameters, such as glomerular (and thereby nephron) number, glomerular size, and kidney volume, have been vital to understanding factors contributing to chronic kidney disease (CKD). These techniques have also been important to understanding the associations between CKD and other systemic and cardiovascular diseases and have led to the identification of developmental risk factors for these pathologies. However, existing techniques in quantitative kidney morphology are resource- and time-consuming and are destructive to the organ. This review discusses the emerging generation of techniques to study kidney morphology quantitatively using magnetic resonance imaging (MRI) using the intravenous injection of the superparamagnetic nanoparticle cationic ferritin, which binds to the glomerular basement membrane. A primary advantage of MRI over previously established techniques is the ability to quantify morphology in the intact organ with minimal sample preparation. We highlight areas of research where MRI-based morphological measurements will be helpful in animal models and possibly diagnostic clinical nephrology, discuss technical challenges in light of the progress in MRI techniques to date, and identify novel measurements that may be possible using MRI, both ex vivo and in vivo.
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Bone morphogenetic protein-9 induces epithelial to mesenchymal transition in hepatocellular carcinoma cells.
Cancer Sci.
PUBLISHED: 02-13-2013
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Epithelial-mesenchymal transition (EMT) is an important mechanism to initiate cancer invasion and metastasis. Bone morphogenetic protein (BMP)-9 is a member of the transforming growth factor (TGF)-? superfamily. It has been suggested to play a role in cancer development in some non-hepatic tumors. In the present study, two hepatocellular carcinoma (HCC) lines, HLE and HepG2, were treated with BMP-9 in vitro, and phenotypic changes and cell motility were analyzed. In situ hybridization (ISH) and immunohistochemical analyses were performed with human HCC tissue samples in order to assess expression levels of BMP-9. In vivo, BMP-9 protein and mRNA were expressed in all the tested patients to diverse degrees. At the protein level, mildly positive (1 + ) BMP-9 staining could be observed in 25/41 (61%), and moderately to strongly positive (2 + ) in 16/41 (39%) of the patients. In 27/41 (65%) patients, the BMP-9 protein expression level was consistent with the mRNA expression level as measured by ISH. In those patients with 2 + protein level, nuclear pSmad1 expression in cancer cells was also significantly increased. Expression of BMP-9 was positively related to nuclear Snail expression and reversely correlated to cell surface E-cadherin expression, although this did not reach statistical significance. Expression levels of BMP-9 were significantly associated with the T stages of the investigated tumors and high levels of BMP-9 were detected by immunofluorescence especially at the tumor borders in samples from an HCC mouse model. In vitro, BMP-9 treatment caused a reduction of E-cadherin and ZO-1 and an induction of Vimentin and Snail expression. Furthermore, cell migration was enhanced by BMP-9 in both HCC cell lines. These results imply that EMT induced by BMP-9 is related to invasiveness of HCC.
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Islet1 is a direct transcriptional target of the homeodomain transcription factor Shox2 and rescues the Shox2-mediated bradycardia.
Basic Res. Cardiol.
PUBLISHED: 02-08-2013
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The hearts rhythm is initiated and regulated by a group of specialized cells in the sinoatrial node (SAN), the primary pacemaker of the heart. Abnormalities in the development of the SAN can result in irregular heart rates (arrhythmias). Although several of the critical genes important for SAN formation have been identified, our understanding of the transcriptional network controlling SAN development remains at a relatively early stage. The homeodomain transcription factor Shox2 is involved in the specification and patterning of the SAN. While the Shox2 knockout in mice results in embryonic lethality due to severe cardiac defects including improper SAN development, Shox2 knockdown in zebrafish causes a reduced heart rate (bradycardia). In order to gain deeper insight into molecular pathways involving Shox2, we compared gene expression levels in right atria of wildtype and Shox2 (-/-) hearts using microarray experiments and identified the LIM homeodomain transcription factor Islet1 (Isl1) as one of its putative target genes. The downregulation of Isl1 expression in Shox2 (-/-) hearts was confirmed and the affected region narrowed down to the SAN by whole-mount in situ hybridization. Using luciferase reporter assays and EMSA studies, we identified two specific SHOX2 binding sites within intron 2 of the ISL1 locus. We also provide functional evidence for Isl1 as a transcriptional target of Shox2 by rescuing the Shox2-mediated bradycardia phenotype with Isl1 using zebrafish as a model system. Our findings demonstrate a novel epistatic relationship between Shox2 and Isl1 in the heart with important developmental consequences for SAN formation and heart beat.
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Bilateral kidney sodium-MRI: Enabling accurate quantification of renal sodium concentration through a two-element phased array system.
J Magn Reson Imaging
PUBLISHED: 02-06-2013
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To develop a sodium-MRI ((23) Na-MRI) method for bilateral renal sodium concentration (RSC) measurements in rat kidneys at 9.4 Tesla (T).
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Transcriptional cofactor TBLR1 controls lipid mobilization in white adipose tissue.
Cell Metab.
PUBLISHED: 02-05-2013
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Lipid mobilization (lipolysis) in white adipose tissue (WAT) critically controls lipid turnover and adiposity in humans. While the acute regulation of lipolysis has been studied in detail, the transcriptional determinants of WAT lipolytic activity remain still largely unexplored. Here we show that the genetic inactivation of transcriptional cofactor transducin beta-like-related 1(TBLR1) blunts the lipolytic response of white adipocytes through the impairment of cAMP-dependent signal transduction. Indeed, mice lacking TBLR1 in adipocytes are defective in fasting-induced lipid mobilization and, when placed on a high-fat-diet, show aggravated adiposity, glucose intolerance, and insulin resistance. TBLR1 levels are found to increase under lipolytic conditions in WAT of both human patients and mice, correlating with serum free fatty acids (FFAs). As a critical regulator of WAT cAMP signaling and lipid mobilization, proper activity of TBLR1 in adipocytes might thus represent a critical molecular checkpoint for the prevention of metabolic dysfunction in subjects with obesity-related disorders.
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Yes-associated protein up-regulates Jagged-1 and activates the Notch pathway in human hepatocellular carcinoma.
Gastroenterology
PUBLISHED: 02-04-2013
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Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP activity are unclear.
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Neuronal expression of glucosylceramide synthase in central nervous system regulates body weight and energy homeostasis.
PLoS Biol.
PUBLISHED: 01-31-2013
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Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.
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In-Silico Algorithms for the Screening of Possible microRNA Binding Sites and Their Interactions.
Curr. Genomics
PUBLISHED: 01-25-2013
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MicroRNAs (miRNAs) comprise a recently discovered class of small, non-coding RNA molecules of 21-25 nucleotides in length that regulate the gene expression by base-pairing with the transcripts of their targets i.e. protein-coding genes, leading to down-regulation or repression of the target genes. However, target gene activation has also been described. miRNAs are involved in diverse regulatory pathways, including control of developmental timing, apoptosis, cell proliferation, cell differentiation, modulation of immune response to macrophages, and organ development and are associated with many diseases, such as cancer. Computational prediction of miRNA targets is much more challenging in animals than in plants, because animal miRNAs often perform imperfect base-pairing with their target sites, unlike plant miRNAs which almost always bind their targets with near perfect complementarity. In the past years, a large number of target prediction programs and databases on experimentally validated information have been developed for animal miRNAs to fulfil the need of experimental scientists conducting miRNA research. In this review we first succinctly describe the prediction criteria (rules or principles) adapted by prediction algorithms to generate possible miRNA binding site interactions and introduce most relevant algorithms, and databases. We then summarize their applications with the help of some previously published studies. We further provide experimentally validated functional binding sites outside 3-UTR region of target mRNAs and the resources which offer such predictions. Finally, the issue of experimental validation of miRNA binding sites will be briefly discussed.
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Lipid droplet accumulation is associated with an increase in hyperglycemia-induced renal damage: prevention by liver X receptors.
Am. J. Pathol.
PUBLISHED: 01-12-2013
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Dyslipidemia is a frequent component of the metabolic disorder of diabetic patients contributing to organ damage. Herein, in low-density lipoprotein receptor-deficient hyperlipidemic and streptozotozin-induced diabetic mice, hyperglycemia and hyperlipidemia acted reciprocally, accentuating renal injury and altering renal function. In hyperglycemic-hyperlipidemic kidneys, the accumulation of Tip47-positive lipid droplets in glomeruli, tubular epithelia, and macrophages was accompanied by the concomitant presence of the oxidative stress markers xanthine oxidoreductase and nitrotyrosine, findings that could also be evidenced in renal biopsy samples of diabetic patients. As liver X receptors (LXR?,?) regulate genes linked to lipid and carbohydrate homeostasis and inhibit inflammatory gene expression in macrophages, the effects of systemic and macrophage-specific LXR activation were analyzed on renal damage in hyperlipidemic-hyperglycemic mice. LXR stimulation by GW3965 up-regulated genes involved in cholesterol efflux and down-regulated proinflammatory/profibrotic cytokines, inhibiting the pathomorphology of diabetic nephropathy, renal lipid accumulation, and improving renal function. Xanthine oxidoreductase and nitrotyrosine levels were reduced. In macrophages, GW3965 or LXR? overexpression significantly suppressed glycated or acetylated low-density lipoprotein-induced cytokines and reactive oxygen species. Specifically, in mice, transgenic expression of LXR? in macrophages significantly ameliorated hyperlipidemic-hyperglycemic nephropathy. The results demonstrate the presence of lipid droplet-induced oxidative mechanisms and the pathophysiologic role of macrophages in diabetic kidneys and indicate the potent regulatory role of LXRs in preventing renal damage in diabetes.
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TSC22D4 is a molecular output of hepatic wasting metabolism.
EMBO Mol Med
PUBLISHED: 01-11-2013
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In mammals, proper storage and distribution of lipids in and between tissues is essential for the maintenance of energy homeostasis. Here, we show that tumour growth triggers hepatic metabolic dysfunction as part of the cancer cachectic phenotype, particularly by reduced hepatic very-low-density-lipoprotein (VLDL) secretion and hypobetalipoproteinemia. As a molecular cachexia output pathway, hepatic levels of the transcription factor transforming growth factor beta 1-stimulated clone (TSC) 22 D4 were increased in cancer cachexia. Mimicking high cachectic levels of TSC22D4 in healthy livers led to the inhibition of hepatic VLDL release and lipogenic genes, and diminished systemic VLDL levels under both normal and high fat dietary conditions. Liver-specific ablation of TSC22D4 triggered hypertriglyceridemia through the induction of hepatic VLDL secretion. Furthermore, hepatic TSC22D4 expression levels were correlated with the degree of body weight loss and VLDL hypo-secretion in cancer cachexia, and TSC22D4 deficiency rescued tumour cell-induced metabolic dysfunction in hepatocytes. Therefore, hepatic TSC22D4 activity may represent a molecular rationale for peripheral energy deprivation in subjects with metabolic wasting diseases, including cancer cachexia.
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Parallel analysis of mRNA and microRNA microarray profiles to explore functional regulatory patterns in polycystic kidney disease: using PKD/Mhm rat model.
PLoS ONE
PUBLISHED: 01-10-2013
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Autosomal polycystic kidney disease (ADPKD) is a frequent monogenic renal disease, characterised by fluid-filled cysts that are thought to result from multiple deregulated pathways such as cell proliferation and apoptosis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of many genes associated with such biological processes and human pathologies. To explore the possible regulatory role of miRNAs in PKD, the PKD/Mhm (cy/+) rat, served as a model to study human ADPKD. A parallel microarray-based approach was conducted to profile the expression changes of mRNAs and miRNAs in PKD/Mhm rats. 1,573 up- and 1,760 down-regulated genes were differentially expressed in PKD/Mhm. These genes are associated with 17 pathways (such as focal adhesion, cell cycle, ECM-receptor interaction, DNA replication and metabolic pathways) and 47 (e.g., cell proliferation, Wnt and Tgf? signaling) Gene Ontologies. Furthermore, we found the similar expression patterns of deregulated genes between PKD/Mhm (cy/+) rat and human ADPKD, PKD1(L3/L3), PKD1(-/-), Hnf1?-deficient, and Glis2(lacZ/lacZ) models. Additionally, several differentially regulated genes were noted to be target hubs for miRNAs. We also obtained 8 significantly up-regulated miRNAs (rno-miR-199a-5p, -214, -146b, -21, -34a, -132, -31 and -503) in diseased kidneys of PKD/Mhm rats. Additionally, the binding site overrepresentation and pathway enrichment analyses were accomplished on the putative targets of these 8 miRNAs. 7 out of these 8 miRNAs and their possible interactions have not been previously described in ADPKD. We have shown a strong overlap of functional patterns (pathways) between deregulated miRNAs and mRNAs in the PKD/Mhm (cy/+) rat model. Our findings suggest that several miRNAs may be associated in regulating pathways in ADPKD. We further describe novel miRNAs and their possible targets in ADPKD, which will open new avenues to understand the pathogenesis of human ADPKD. Furthermore they could serve as a useful resource for anti-fibrotic therapeutics.
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Iron regulatory proteins control a mucosal block to intestinal iron absorption.
Cell Rep
PUBLISHED: 01-07-2013
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Mammalian iron metabolism is regulated systemically by the hormone hepcidin and cellularly by iron regulatory proteins (IRPs) that orchestrate a posttranscriptional regulatory network. Through ligand-inducible genetic ablation of both IRPs in the gut epithelium of adult mice, we demonstrate that IRP deficiency impairs iron absorption and promotes mucosal iron retention via a ferritin-mediated "mucosal block." We show that IRP deficiency does not interfere with intestinal sensing of body iron loading and erythropoietic iron need, but rather alters the basal expression of the iron-absorption machinery. IRPs thus secure sufficient iron transport across absorptive enterocytes by restricting the ferritin "mucosal block" and define a basal set point for iron absorption upon which IRP-independent systemic regulatory inputs are overlaid.
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CNVs-microRNAs Interactions Demonstrate Unique Characteristics in the Human Genome. An Interspecies in silico Analysis.
PLoS ONE
PUBLISHED: 01-01-2013
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MicroRNAs (miRNAs) and copy number variations (CNVs) represent two classes of newly discovered genomic elements that were shown to contribute to genome plasticity and evolution. Recent studies demonstrated that miRNAs and CNVs must have co-evolved and interacted in an attempt to maintain the balance of the dosage sensitive genes and at the same time increase the diversity of dosage non-sensitive genes, contributing to species evolution. It has been previously demonstrated that both the number of miRNAs that target genes found in CNV regions as well as the number of miRNA binding sites are significantly higher than those of genes found in non-CNV regions. These findings raise the possibility that miRNAs may have been created under evolutionary pressure, as a mechanism for increasing the tolerance to genome plasticity. In the current study, we aimed in exploring the differences of miRNAs-CNV functional interactions between human and seven others species. By performing in silico whole genome analysis in eight different species (human, chimpanzee, macaque, mouse, rat, chicken, dog and cow), we demonstrate that miRNAs targeting genes located within CNV regions in humans have special functional characteristics that provide an insight into the differences between humans and other species.
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Simultaneous Measurement of Kidney Function by Dynamic Contrast Enhanced MRI and FITC-Sinistrin Clearance in Rats at 3 Tesla: Initial Results.
PLoS ONE
PUBLISHED: 01-01-2013
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Glomerular filtration rate (GFR) is an essential parameter of kidney function which can be measured by dynamic contrast enhanced magnetic resonance imaging (MRI-GFR) and transcutaneous approaches based on fluorescent tracer molecules (optical-GFR). In an initial study comparing both techniques in separate measurements on the same animal, the correlation of the obtained GFR was poor. The goal of this study was to investigate if a simultaneous measurement was feasible and if thereby, the discrepancies in MRI-GFR and optical-GFR could be reduced. For the experiments healthy and unilateral nephrectomised (UNX) Sprague Dawley (SD) rats were used. The miniaturized fluorescent sensor was fixed on the depilated back of an anesthetized rat. A bolus of 5 mg/100 g b.w. of FITC-sinistrin was intravenously injected. For dynamic contrast enhanced perfusion imaging (DCE-MRI) a 3D time-resolved angiography with stochastic trajectories (TWIST) sequence was used. By means of a one compartment model the excretion half-life (t1/2) of FITC-sinistrin was calculated and converted into GFR. GFR from DCE-MRI was calculated by fitting pixel-wise a two compartment renal filtration model. Mean cortical GFR and GFR by FITC-sinistrin were compared by Bland-Altman plots and pair-wise t-test. Results show that a simultaneous GFR measurement using both techniques is feasible. Mean optical-GFR was 4.34±2.22 ml/min (healthy SD rats) and 2.34±0.90 ml/min (UNX rats) whereas MRI-GFR was 2.10±0.64 ml/min (SD rats) and 1.17±0.38 ml/min (UNX rats). Differences between healthy and UNX rats were significant (p<0.05) and almost equal percentage difference (46.1% and 44.3%) in mean GFR were assessed with both techniques. Overall mean optical-GFR values were approximately twice as high compared to MRI-GFR values. However, compared to a previous study, our results showed a higher agreement. In conclusion, the possibility to use the transcutaneous method in MRI may have a huge impact in improving and validating MRI methods for GFR assessment in animal models.
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The Raf kinase inhibitor PLX5568 slows cyst proliferation in rat polycystic kidney disease but promotes renal and hepatic fibrosis.
Nephrol. Dial. Transplant.
PUBLISHED: 07-29-2011
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Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of renal failure. Aberrant epithelial cell proliferation is a major cause of progressive cyst enlargement in ADPKD. Since activation of the Ras/Raf signaling system has been detected in cyst-lining epithelia, inhibition of Raf kinase has been proposed as an approach to retard the progression of ADPKD. Methods and results. PLX5568, a novel selective small molecule inhibitor of Raf kinases, attenuated proliferation of human ADPKD cyst epithelial cells. It reduced in vitro cyst growth of Madin-Darby Canine Kidney cells and of human ADPKD cells within a collagen gel. In male cy/+ rats with polycystic kidneys, PLX5568 inhibited renal cyst growth along with a significant reduction in the number of proliferating cell nuclear antigen- and phosphorylated extracellular signal-regulated kinase-positive cyst-lining epithelial cells. Furthermore, treated animals showed increased capacity to concentrate urine. However, PLX5568 did not lead to a consistent improvement of renal function. Moreover, although relative cyst volume was decreased, total kidney-to-body weight ratio was not significantly reduced by PLX5568. Further analyses revealed a 2-fold increase of renal and hepatic fibrosis in animals treated with PLX5568.
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Validation of a new non-invasive blood pressure measurement method on mice via pulse wave propagation time measurement on a cuff.
Biomed Tech (Berl)
PUBLISHED: 06-11-2011
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In the present article, we describe the validation of a new non-invasive method for measuring blood pressure (BP) which also enables to determine the three BP values: systolic, diastolic and mean value. Our method is based on the pulse transit time (PTT) measurement along an artery directly at the BP cuff. The accuracy of this method was evaluated by comparison with the direct simultaneous measurement of blood pressure from 40 anesthetized female mice. Close correlation between the gained data from these two methods was observed.
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Division of labor by dual feedback regulators controls JAK2/STAT5 signaling over broad ligand range.
Mol. Syst. Biol.
PUBLISHED: 06-10-2011
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Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear. Based on extensive time-resolved data sets in primary erythroid progenitor cells, we established a dynamic pathway model to dissect the roles of the two transcriptional negative feedback regulators of the suppressor of cytokine signaling (SOCS) family, CIS and SOCS3, in JAK2/STAT5 signaling. Facilitated by the model, we calculated the STAT5 response for experimentally unobservable Epo concentrations and provide a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. Model predictions show that the two feedbacks CIS and SOCS3 are most effective at different ligand concentration ranges due to their distinct inhibitory mechanisms. This divided function of dual feedback regulation enables control of STAT5 responses for Epo concentrations that can vary 1000-fold in vivo. Our modeling approach reveals dose-dependent feedback control as key property to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations.
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Quantification of glomerular number and size distribution in normal rat kidneys using magnetic resonance imaging.
Nephrol. Dial. Transplant.
PUBLISHED: 06-03-2011
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Glomerular number and size are important risk factors for chronic kidney disease (CKD) and cardiovascular disease and have traditionally been estimated using invasive techniques. Here, we report a novel technique to count and size every glomerulus in the rat kidney using magnetic resonance imaging (MRI).
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miRWalk--database: prediction of possible miRNA binding sites by "walking" the genes of three genomes.
J Biomed Inform
PUBLISHED: 04-08-2011
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MicroRNAs are small, non-coding RNA molecules that can complementarily bind to the mRNA 3-UTR region to regulate the gene expression by transcriptional repression or induction of mRNA degradation. Increasing evidence suggests a new mechanism by which miRNAs may regulate target gene expression by binding in promoter and amino acid coding regions. Most of the existing databases on miRNAs are restricted to mRNA 3-UTR region. To address this issue, we present miRWalk, a comprehensive database on miRNAs, which hosts predicted as well as validated miRNA binding sites, information on all known genes of human, mouse and rat. All mRNAs, mitochondrial genes and 10 kb upstream flanking regions of all known genes of human, mouse and rat were analyzed by using a newly developed algorithm named miRWalk as well as with eight already established programs for putative miRNA binding sites. An automated and extensive text-mining search was performed on PubMed database to extract validated information on miRNAs. Combined information was put into a MySQL database. miRWalk presents predicted and validated information on miRNA-target interaction. Such a resource enables researchers to validate new targets of miRNA not only on 3-UTR, but also on the other regions of all known genes. The Validated Target module is updated every month and the Predicted Target module is updated every 6 months. miRWalk is freely available at http://mirwalk.uni-hd.de/.
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Two non-invasive GFR-estimation methods in rat models of polycystic kidney disease: 3.0 Tesla dynamic contrast-enhanced MRI and optical imaging.
Nephrol. Dial. Transplant.
PUBLISHED: 03-28-2011
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The aim of this study was the assessment of kidney morphology and glomerular filtration rate (GFR) in rat models of polycystic kidney disease and a healthy control group of Sprague-Dawley rats (SD rats). The performance of two non-invasive GFR estimation methods-3.0 Tesla magnetic resonance imaging (MRI) and optical imaging were investigated. Data of GFR assessment was compared to surrogate markers of kidney function and renal histology.
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Increased number of microRNA target sites in genes encoded in CNV regions. Evidence for an evolutionary genomic interaction.
Mol. Biol. Evol.
PUBLISHED: 03-25-2011
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MicroRNAs (miRNAs) and copy number variations (CNVs) are two newly discovered genetic elements that have revolutionized the field of molecular biology and genetics. By performing in silico whole genome analysis, we demonstrate that both the number of miRNAs that target genes found in CNV regions as well as the number of miRNA-binding sites are significantly higher than those of genes found in non-CNV regions. This suggests that miRNAs may have acted as equilibrators of gene expression during evolution in an attempt to regulate aberrant gene expression and to increase the tolerance to genome plasticity.
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Comparative transcriptome profiling of amyloid precursor protein family members in the adult cortex.
BMC Genomics
PUBLISHED: 03-24-2011
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The ?-amyloid precursor protein (APP) and the related ?-amyloid precursor-like proteins (APLPs) undergo complex proteolytic processing giving rise to several fragments. Whereas it is well established that A? accumulation is a central trigger for Alzheimers disease, the physiological role of APP family members and their diverse proteolytic products is still largely unknown. The secreted APPs? ectodomain has been shown to be involved in neuroprotection and synaptic plasticity. The ?-secretase-generated APP intracellular domain (AICD) functions as a transcriptional regulator in heterologous reporter assays although its role for endogenous gene regulation has remained controversial.
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Inducible renal principal cell-specific mineralocorticoid receptor gene inactivation in mice.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 03-09-2011
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To investigate the role of the mineralocorticoid receptor (MR) in renal ENaC-mediated sodium reabsorption, we have previously used the Cre-loxP system to generate mice with principal-cell specific MR ablation (MR(AQP2Cre) mice). To restrict Cre expression to principal cells, we have used the regulatory elements of the mouse aquaporin-2 (AQP2) gene to drive Cre expression. Since AQP2 is already expressed during renal development, MR ablation took place long before the analysis performed at the adult stage. To investigate whether the early onset of MR ablation affected the adult renal sodium handling, we developed a transgene expressing the CreER(T2) fusion protein under control of the regulatory elements of the AQP2 gene (AQP2CreER(T2)). Immunofluorescence revealed MR loss in the collecting duct (CD) and late connecting tubule after induction of MR ablation by tamoxifen in MR(AQP2CreERT2) mice that equals the MR loss in MR(AQP2Cre) mice. Surprisingly, tamoxifen-independent MR loss is observed in CDs of noninduced mutants without affecting circulating aldosterone levels. Under a low-salt diet, the induced ablation of MR at the adult stage recapitulates the renal sodium wasting observed in mice with constitutive early-onset MR ablation. The AQP2CreER(T2) transgene is a new tool for investigating in vivo the function of genes downstream of MR in renal ENaC-mediated sodium reabsorption by inducible somatic gene inactivation.
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Transcutaneous assessment of renal function in conscious rats with a device for measuring FITC-sinistrin disappearance curves.
Kidney Int.
PUBLISHED: 03-02-2011
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Determination of the urinary or plasma clearance of exogenous renal markers, such as inulin or iohexol, is considered to be the gold standard for glomerular filtration rate (GFR) measurement. Here, we describe a technique allowing determination of renal function based on transcutaneously measured elimination kinetics of fluorescein isothiocyanate (FITC)-sinistrin, the FITC-labeled active pharmaceutical ingredient of a commercially available marker of GFR. A low cost device transcutaneously excites FITC-sinistrin at 480? nm and detects the emitted light through the skin at 520 ?nm. A radio-frequency transmission allows remote monitoring and real-time analysis of FITC-sinistrin excretion as a marker of renal function. Due to miniaturization, the whole device fits on the back of freely moving rats, and requires neither blood sampling nor laboratory assays. As proof of principle, comparative measurements of transcutaneous and plasma elimination kinetics of FITC-sinistrin were compared in freely moving healthy rats, rats showing reduced kidney function due to unilateral nephrectomy and PKD/Mhm rats with cystic kidney disease. Results show highly comparable elimination half-lives and GFR values in all animal groups. Bland-Altman analysis of enzymatically compared with transcutaneously measured GFR found a mean difference (bias) of 0.01 and a -0.30 to 0.33?ml/min per 100?g body weight with 95% limit of agreement. Thus, with this device, renal function can be reliably measured in freely moving rats eliminating the need for and influence of anesthesia on renal function.
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Suppression of chronic damage in renal allografts by Liver X receptor (LXR) activation relevant contribution of macrophage LXR?.
Am. J. Pathol.
PUBLISHED: 02-22-2011
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Liver X receptors (LXR)-?,? regulate intracellular cholesterol homeostasis and inhibit inflammatory gene expression. We studied the effects of the LXR?,?-agonist GW3965 on acute and chronic organ damage in the F344-LEW rat kidney transplantation model. In addition, to gain LXR isoform and cell-specific insights BALB/c kidneys were transplanted into mice with macrophage overexpression of LXR? (mLXR?-tg) and evaluated 7 and 42 days after transplantation. After 56 days GW3965 improved significantly function and morphology of rat kidney allografts by substantial reduction of mononuclear cell infiltrate and fibrosis; in vitro GW3965 reduced inflammatory activity of bone marrow-derived macrophages (BMDMs) and alloreactivity of T cells. Kidneys transplanted into mLXR?-tg mice were also protected from development of chronic allograft dysfunction. Similarly to GW3965-activated BMDMs, mLXR?-tg macrophages secreted significantly less monocyte chemoattractant protein 1 and macrophage inflammatory protein 1?. Interestingly, 7 days after transplantation, when the total number of intragraft macrophages did not differ, evidently more arginase 1- and mannose receptor C type 1-positive cells were found in LXR rat and mice kidney allografts; in vitro both LXR activation by GW3965 and mLXR? overexpression accentuated the induction of alternative activation of BMDMs by IL-4/IL-13, suggesting an additional mechanism by LXRs to prevent graft damage. The results highlight the relevance of macrophage LXR? in allograft rejection and prevention of fibrosis.
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Genome-wide screening in human growth plates during puberty in one patient suggests a role for RUNX2 in epiphyseal maturation.
J. Endocrinol.
PUBLISHED: 02-09-2011
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In late puberty, estrogen decelerates bone growth by stimulating growth plate maturation. In this study, we analyzed the mechanism of estrogen action using two pubertal growth plate specimens of one girl at Tanner stage B2 and Tanner stage B3. Histological analysis showed that progression of puberty coincided with characteristic morphological changes: a decrease in total growth plate height (P=0.002), height of the individual zones (P<0.001), and an increase in intercolumnar space (P<0.001). Microarray analysis of the specimens identified 394 genes (72% upregulated and 28% downregulated) that changed with the progression of puberty. Overall changes in gene expression were small (average 1.38-fold upregulated and 1.36-fold downregulated genes). The 394 genes mapped to 13 significantly changing pathways (P<0.05) associated with growth plate maturation (e.g. extracellular matrix, cell cycle, and cell death). We next scanned the upstream promoter regions of the 394 genes for the presence of evolutionarily conserved binding sites for transcription factors implicated in growth plate maturation such as estrogen receptor (ER), androgen receptor, ELK1, STAT5B, cyclic AMP response element (CREB), and RUNX2. High-quality motif sites for RUNX2 (87 genes), ELK1 (43 genes), and STAT5B (31 genes), but not ER, were evolutionarily conserved, indicating their functional relevance across primates. Moreover, we show that some of these sites are direct target genes of these transcription factors as shown by ChIP assays.
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Continuous glucose monitoring by means of mid-infrared transmission laser spectroscopy in vitro.
Analyst
PUBLISHED: 01-27-2011
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The continuous surveillance of glucose concentration reduces short-term risks and long-term complications for people with diabetes mellitus, a disorder of glucose metabolism. As a first step towards the continuous monitoring of glucose, reagent-free transmission spectroscopy in the mid-infrared region has been carried out in vitro using a quantum cascade laser and an optical silver halide fiber. A 30 ?m gap in the fiber allowed for transmission spectroscopy of aqueous glucose solutions at a wavelength of 9.69 ?m, which is specific to a molecular vibration of glucose. A noise-equivalent concentration as low as 4 mg/dL was achieved at an average power of 1.8 mW and an integration time of 50 s. This is among the most precise of glucose measurements using mid-infrared spectroscopy. Even with the very low average laser power of 0.07 mW the sensitivity of previous results (using a fiber optical evanescent field analysis) has been improved upon by almost one order of magnitude. Finally, the impact of potentially interfering substances such as other carbohydrates was analyzed.
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First multimodal embolization particles visible on x-ray/computed tomography and magnetic resonance imaging.
Invest Radiol
PUBLISHED: 01-26-2011
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Embolization therapy is gaining importance in the treatment of malignant lesions, and even more in benign lesions. Current embolization materials are not visible in imaging modalities. However, it is assumed that directly visible embolization material may provide several advantages over current embolization agents, ranging from particle shunt and reflux prevention to improved therapy control and follow-up assessment. X-ray- as well as magnetic resonance imaging (MRI)-visible embolization materials have been demonstrated in experiments. In this study, we present an embolization material with the property of being visible in more than one imaging modality, namely MRI and x-ray/computed tomography (CT). Characterization and testing of the substance in animal models was performed.
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Hepatic deficiency in transcriptional cofactor TBL1 promotes liver steatosis and hypertriglyceridemia.
Cell Metab.
PUBLISHED: 01-20-2011
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The aberrant accumulation of lipids in the liver ("fatty liver") is tightly associated with several components of the metabolic syndrome, including type 2 diabetes, coronary heart disease, and atherosclerosis. Here we show that the impaired hepatic expression of transcriptional cofactor transducin beta-like (TBL) 1 represents a common feature of mono- and multigenic fatty liver mouse models. Indeed, the liver-specific ablation of TBL1 gene expression in healthy mice promoted hypertriglyceridemia and hepatic steatosis under both normal and high-fat dietary conditions. TBL1 deficiency resulted in inhibition of fatty acid oxidation due to impaired functional cooperation with its heterodimerization partner TBL-related (TBLR) 1 and the nuclear receptor peroxisome proliferator-activated receptor (PPAR) ?. As TBL1 expression levels were found to also inversely correlate with liver fat content in human patients, the lack of hepatic TBL1/TBLR1 cofactor activity may represent a molecular rationale for hepatic steatosis in subjects with obesity and the metabolic syndrome.
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Gene expression profiling of vasoregression in the retina--involvement of microglial cells.
PLoS ONE
PUBLISHED: 01-17-2011
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Vasoregression is a hallmark of vascular eye diseases but the mechanisms involved are still largely unknown. We have recently characterized a rat ciliopathy model which develops primary photoreceptor degeneration and secondary vasoregression. To improve the understanding of secondary vasoregression in retinal neurodegeneration, we used microarray techniques to compare gene expression profiles in this new model before and after retinal vasoregression. Differential gene expression was validated by quantitative RT-PCR, Western blot and immunofluorescence. Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression. Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression. Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.
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New insights into the influence of cigarette smoking on urothelial carcinogenesis: smoking-induced gene expression in tumor-free urothelium might discriminate muscle-invasive from nonmuscle-invasive urothelial bladder cancer.
Mol. Carcinog.
PUBLISHED: 01-07-2011
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Smoking is the main risk factor for urothelial bladder cancer. In former smokers the risk decreases but does not reach the low level of never smokers. This indicates reversible and permanent smoking-derived genetic alterations. Transcriptional changes may point to mechanisms, how smoking promotes urothelial bladder cancer. To identify smoking-derived transcriptional changes we performed gene expression profiling in current, former, and never smokers, using tumor and tumor-free urothelium from patients with nonmuscle-invasive urothelial bladder cancer (NMIBC) or muscle-invasive urothelial bladder cancer (MIBC). Smoking turned out to influence gene expression much less than tumor stage (NMIBC or MIBC) and tumor transformation (tumor-free or tumor). Smoking seemed to influence gene expression in patients with MIBC more strongly compared to those with NMIBC. The least irreversible changes after smoking cessation were proposed in tumor-free urothelium from patients with NMIBC. Growth factors and oncogenes were up-regulated in tumor-free urothelium from smokers with MIBC but not from smokers with NMIBC. A panel of genes up-regulated in smokers have potential for early detection and distinction of MIBC from NMIBC using tumor-free tissue.
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Selective downregulation of retinoic acid-inducible gene I within the intestinal epithelial compartment in Crohns disease.
Inflamm. Bowel Dis.
PUBLISHED: 01-06-2011
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A defective innate immune response may contribute to the pathogenesis of Crohns disease (CD) and ulcerative colitis (UC). Employing a global gene expression analysis, this study was aimed at identifying specifically regulated genes within the epithelial compartment in inflammatory bowel disease (IBD).
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Dynamic mathematical modeling of IL13-induced signaling in Hodgkin and primary mediastinal B-cell lymphoma allows prediction of therapeutic targets.
Cancer Res.
PUBLISHED: 12-02-2010
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Primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) share a frequent constitutive activation of JAK (Janus kinase)/STAT signaling pathway. Because of complex, nonlinear relations within the pathway, key dynamic properties remained to be identified to predict possible strategies for intervention. We report the development of dynamic pathway models based on quantitative data collected on signaling components of JAK/STAT pathway in two lymphoma-derived cell lines, MedB-1 and L1236, representative of PMBL and cHL, respectively. We show that the amounts of STAT5 and STAT6 are higher whereas those of SHP1 are lower in the two lymphoma cell lines than in normal B cells. Distinctively, L1236 cells harbor more JAK2 and less SHP1 molecules per cell than MedB-1 or control cells. In both lymphoma cell lines, we observe interleukin-13 (IL13)-induced activation of IL4 receptor ?, JAK2, and STAT5, but not of STAT6. Genome-wide, 11 early and 16 sustained genes are upregulated by IL13 in both lymphoma cell lines. Specifically, the known STAT-inducible negative regulators CISH and SOCS3 are upregulated within 2 hours in MedB-1 but not in L1236 cells. On the basis of this detailed quantitative information, we established two mathematical models, MedB-1 and L1236 model, able to describe the respective experimental data. Most of the model parameters are identifiable and therefore the models are predictive. Sensitivity analysis of the model identifies six possible therapeutic targets able to reduce gene expression levels in L1236 cells and three in MedB-1. We experimentally confirm reduction in target gene expression in response to inhibition of STAT5 phosphorylation, thereby validating one of the predicted targets.
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Transgenic overexpression of Anks6(p.R823W) causes polycystic kidney disease in rats.
Am. J. Pathol.
PUBLISHED: 12-02-2010
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The PKD/Mhm(cy/+) rat is a widely used animal model for the study of human autosomal dominant polycystic kidney disease, one of the most common genetic disorders, affecting one in 1000 individuals. We identified a new gene, Anks6, which is mutated (Anks6((p.R823W))) in PKD/Mhm(cy/+) rats. The evidence for a causal link between Anks6((p.R823W)) and cystogenesis is still lacking, and the function of Anks6 is presently unknown. This study presents a novel transgenic rat model that overexpresses the mutated 2.8-kb Anks6((p.R823W)) cDNA in the renal tubular epithelium. The transgenic Anks6((p.R823W)) acts in a dominant-negative fashion and causes a predictable polycystic phenotype that largely mimics the general characteristics of the PKD/Mhm(cy/+) rats. Cyst development is accompanied by enhanced c-myc expression and continuous proliferation, apoptosis, and de-differentiation of the renal tubular epithelium as well as by a lack of translational up-regulation of p21 during aging. Using Northern blot analysis and in situ hybridization studies, we identified the first 10 days of age as the period during which transgene expression precedes and initiates cystic growth. Thus, we not only provide the first in vivo evidence for a causal link between the novel Anks6((p.R823W)) gene mutation and polycystic kidney disease, but we also developed a new transgenic rat model that will serve as an important resource for further exploration of the still unknown function of Anks6.
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Combining theoretical analysis and experimental data generation reveals IRF9 as a crucial factor for accelerating interferon ?-induced early antiviral signalling.
FEBS J.
PUBLISHED: 10-21-2010
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Type I interferons (IFN) are important components of the innate antiviral response. A key signalling pathway activated by IFN? is the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Major components of the pathway have been identified. However, critical kinetic properties that facilitate accelerated initiation of intracellular antiviral signalling and thereby promote virus elimination remain to be determined. By combining mathematical modelling with experimental analysis, we show that control of dynamic behaviour is not distributed among several pathway components but can be primarily attributed to interferon regulatory factor 9 (IRF9), constituting a positive feedback loop. Model simulations revealed that increasing the initial IRF9 concentration reduced the time to peak, increased the amplitude and enhanced termination of pathway activation. These model predictions were experimentally verified by IRF9 over-expression studies. Furthermore, acceleration of signal processing was linked to more rapid and enhanced expression of IFN? target genes. Thus, the amount of cellular IRF9 is a crucial determinant for amplification of early dynamics of IFN?-mediated signal transduction.
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Knowledge-based matrix factorization temporally resolves the cellular responses to IL-6 stimulation.
BMC Bioinformatics
PUBLISHED: 08-13-2010
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External stimulations of cells by hormones, cytokines or growth factors activate signal transduction pathways that subsequently induce a re-arrangement of cellular gene expression. The analysis of such changes is complicated, as they consist of multi-layered temporal responses. While classical analyses based on clustering or gene set enrichment only partly reveal this information, matrix factorization techniques are well suited for a detailed temporal analysis. In signal processing, factorization techniques incorporating data properties like spatial and temporal correlation structure have shown to be robust and computationally efficient. However, such correlation-based methods have so far not be applied in bioinformatics, because large scale biological data rarely imply a natural order that allows the definition of a delayed correlation function.
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Zyxin mediation of stretch-induced gene expression in human endothelial cells.
Circ. Res.
PUBLISHED: 08-12-2010
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Prolonged exposure to enhanced stretch, such as in hypertension, triggers endothelial dysfunction, a hallmark of pathological vascular remodeling processes. Despite its clinical relevance, little is known about stretch-induced gene expression in endothelial cells.
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Inhibition of mTOR with sirolimus does not attenuate progression of liver and kidney disease in PCK rats.
Nephrol. Dial. Transplant.
PUBLISHED: 07-07-2010
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Activation of the mTOR pathway has been implicated in the mediation of the progression of polycystic kidney disease (PKD). Whereas targeted inhibition of mTOR has been proven to be effective in various animal models of autosomal dominant PKD, its efficacy in autosomal recessive PKD (ARPKD) remains to be elucidated. We examined the effects of sirolimus in PCK rats, an orthologous animal model of human ARPKD.
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Cyst formation in the PKD2 (1-703) transgenic rat precedes deregulation of proliferation-related pathways.
BMC Nephrol
PUBLISHED: 05-14-2010
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Polycystic Kidney Disease is characterized by the formation of large fluid-filled cysts that eventually destroy the renal parenchyma leading to end-stage renal failure. Although remarkable progress has been made in understanding the pathologic mechanism of the disease, the precise orchestration of the early events leading to cyst formation is still unclear. Abnormal cellular proliferation was traditionally considered to be one of the primary irregularities leading to cyst initiation and growth. Consequently, many therapeutic interventions have focused on targeting this abnormal proliferation, and some have even progressed to clinical trials. However, the role of proliferation in cyst development was primarily examined at stages where cysts are already visible in the kidneys and therefore at later stages of disease development.
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Genome-wide comparison between IL-17 and combined TNF-alpha/IL-17 induced genes in primary murine hepatocytes.
BMC Genomics
PUBLISHED: 04-07-2010
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Cytokines such as TNF-alpha and IL-1 beta are known for their contribution to inflammatory processes in liver. In contrast, the cytokine IL-17 has not yet been assigned a role in liver diseases. IL-17 can cooperate with TNF-alpha to induce a synergistic response on several target genes in different cell lines, but no data exist for primary hepatocytes. To enhance our knowledge on the impact of IL-17 alone and combined with TNF-alpha in primary murine hepatocytes a comprehensive microarray study was designed. IL-1 beta was included as this cytokine is suggested to act in a similar manner as the combination of TNF-alpha and IL-17, especially with respect to its role in mRNA stabilization.
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Peroxisome proliferator-activated receptor (PPAR)gamma can inhibit chronic renal allograft damage.
Am. J. Pathol.
PUBLISHED: 04-02-2010
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Chronic inflammation and fibrosis are the leading causes of chronic allograft failure. The nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma is a transcription factor known to have antidiabetogenic and immune effects, and PPARgamma forms obligate heterodimers with the retinoid X receptor (RXR). We have reported that a retinoic acid (RAR)/RXR-agonist can potently influence the course of renal chronic allograft dysfunction. In this study, in a Fischer to Lewis rat renal transplantation model, administration of the PPARgamma-agonist, rosiglitazone, independent of dose (3 or 30 mg/kgBW/day), lowered serum creatinine, albuminuria, and chronic allograft damage with a chronic vascular damage score as follows: 35.0 +/- 5.8 (controls) vs. 8.1 +/- 2.4 (low dose-Rosi; P < 0.05); chronic tubulointerstitial damage score: 13.6 +/- 1.8 (controls) vs. 2.6 +/- 0.4 (low dose-Rosi; P < 0.01). The deposition of extracellular matrix proteins (collagen, fibronectin, decorin) was strikingly lower. The expression of transforming growth factor-beta1 was inhibited, whereas that of bone morphogenic protein-7 (BMP-7) was increased. Intragraft mononuclear cells and activated fibroblast numbers were reduced by 50%. In addition, the migratory and proliferative activity of these cells was significantly inhibited in vitro. PPARgamma activation diminished the number of cells expressing the proinflammatory and fibrogenic proteoglycan biglycan. In macrophages its secretion was blocked by rosiglitazone in a predominantly PPARgamma-dependent manner. The combination of PPARgamma- and RAR/RXR-agonists resulted in additive effects in the inhibition of fibrosis. In summary, PPARgamma activation was potently immunosuppressive and antifibrotic in kidney allografts, and these effects were enhanced by a RAR/RXR-agonist.
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CD95-ligand on peripheral myeloid cells activates Syk kinase to trigger their recruitment to the inflammatory site.
Immunity
PUBLISHED: 02-11-2010
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Injury to the central nervous system initiates an uncontrolled inflammatory response that results in both tissue repair and destruction. Here, we showed that, in rodents and humans, injury to the spinal cord triggered surface expression of CD95 ligand (CD95L, FasL) on peripheral blood myeloid cells. CD95L stimulation of CD95 on these cells activated phosphoinositide 3-kinase (PI3K) and metalloproteinase-9 (MMP-9) via recruitment and activation of Syk kinase, ultimately leading to increased migration. Exclusive CD95L deletion in myeloid cells greatly decreased the number of neutrophils and macrophages infiltrating the injured spinal cord or the inflamed peritoneum after thioglycollate injection. Importantly, deletion of myeloid CD95L, but not of CD95 on neural cells, led to functional recovery of spinal injured animals. Our results indicate that CD95L acts on peripheral myeloid cells to induce tissue damage. Thus, neutralization of CD95L should be considered as a means to create a controlled beneficial inflammatory response.
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The proteoglycan biglycan regulates expression of the B cell chemoattractant CXCL13 and aggravates murine lupus nephritis.
J. Clin. Invest.
PUBLISHED: 01-04-2010
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CXCL13 is a key B cell chemoattractant and marker of disease activity in patients with SLE; however, the mechanism of its induction has not been identified yet. Here, we have shown that the proteoglycan biglycan triggers CXCL13 expression via TLR2/4 in macrophages and dendritic cells. In vivo, levels of biglycan were markedly elevated in the plasma and kidneys of human SLE patients and lupus-prone (MRL/lpr) mice. Overexpression of soluble biglycan in MRL/lpr mice raised plasma and renal levels of CXCL13 and caused accumulation of B cells with an enhanced B1/B cell ratio in the kidney, worsening of organ damage, and albuminuria. Importantly, biglycan also triggered CXCL13 expression and B cell infiltration in the healthy kidney. Conversely, biglycan deficiency improved systemic and renal outcome in lupus-prone mice, with lower levels of autoantibodies, less enlargement of the spleen and lymph nodes, and reduction in renal damage and albuminuria. This correlated with a marked decline in circulating and renal CXCL13 and a reduction in the number of B cells in the kidney. Collectively, our results describe what we believe to be a novel mechanism for the regulation of CXCL13 by biglycan, a host-derived ligand for TLR2/4. Blocking biglycan-TLR2/4 interactions might be a promising strategy for the management of SLE and other B cell-mediated inflammatory disease entities.
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Recipient Toll-like receptors contribute to chronic graft dysfunction by both MyD88- and TRIF-dependent signaling.
Dis Model Mech
PUBLISHED: 12-28-2009
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Toll-like receptors (TLRs) recognize specific molecular patterns derived from microbial components (exogenous ligands) or stressed cells (endogenous ligands). Stimulation of these receptors leads to a pronounced inflammatory response in a variety of acute animal models. Chronic allograft dysfunction (CAD) was regarded as a candidate disease to test whether TLRs influence chronic fibrosing inflammation. Potential endogenous renal TLR ligands, specifically for TLR2 and TLR4, have now been detected by a significant upregulation of glucose regulated protein (GRP)-94, fibrinogen, heat shock protein (HSP)-60, HSP-70, biglycan (Bgn) and high-mobility group box chromosomal protein 1 (HMGB1) in the acute and chronic transplant setting. In a genetic approach to define the contribution of TLR2 and TLR4, and their adaptor proteins MyD88 and TRIF [Toll/interleukin (IL)-1 receptor domain-containing adaptor-protein inducing interferon beta], to CAD, kidney transplantation of TLR wild-type grafts to recipients who were deficient in TLR2, TLR4, TLR2/4, MyD88 and TRIF was performed. TLR and adaptor protein deficiencies significantly improved the excretory function of chronic kidney grafts by between 65% and 290%, and histopathologic signs of chronic allograft damage were significantly ameliorated. T cells, dendritic cells (DCs) and foremost macrophages were reduced in grafts by up to 4.5-fold. The intragraft concentrations of IL-6, IL-10, monocyte chemotactic protein-1 (MCP-1) and IL-12p70 were significantly lower. TLR-, MyD88- and TRIF-deficient recipients showed a significant reduction in fibrosis. alpha-smooth muscle actin (alpha-SMA)-positive cells were decreased by up to ninefold, and collagen I and III were reduced by up to twofold. These findings highlight the functional relevance of TLRs and their two major signaling pathways in graft-infiltrating mononuclear cells in the pathophysiology of CAD. A TLR signaling blockade may be a therapeutic option for the prevention of CAD.
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Telomere shortening reduces regenerative capacity after acute kidney injury.
J. Am. Soc. Nephrol.
PUBLISHED: 12-03-2009
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Telomeres of most somatic cells progressively shorten, compromising the regenerative capacity of human tissues during aging and chronic diseases and after acute injury. Whether telomere shortening reduces renal regeneration after acute injury is unknown. Here, renal ischemia-reperfusion injury led to greater impairment of renal function and increased acute and chronic histopathologic damage in fourth-generation telomerase-deficient mice compared with both wild-type and first-generation telomerase-deficient mice. Critically short telomeres, increased expression of the cell-cycle inhibitor p21, and more apoptotic renal cells accompanied the pronounced damage in fourth-generation telomerase-deficient mice. These mice also demonstrated significantly reduced proliferative capacity in tubular, glomerular, and interstitial cells. These data suggest that critical telomere shortening in the kidney leads to increased senescence and apoptosis, thereby limiting regenerative capacity in response to injury.
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A novel CXCL8 protein-based antagonist in acute experimental renal allograft damage.
Mol. Immunol.
PUBLISHED: 07-15-2009
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Acute renal allograft damage is caused by early leukocyte infiltration which is mediated in part by chemokines presented by glycosaminoglycan (GAG) structures on endothelial surfaces. CXCL8 can recruit neutrophils and induce the firm arrest of monocytes on activated endothelial cells. A human CXCL8-based antagonist (dnCXCL8) designed to generate a dominant-negative mutant protein with enhanced binding to GAG structures and reduced CXCR1/2 receptor binding ability was tested in models of early allograft injury. The agent displayed enhanced binding to GAG structures in vitro and could antagonize CXCL8-induced firm adhesion of monocytes as well as neutrophils to activated microvascular endothelium in physiologic flow assays. In a rat model of acute renal damage, dnCXCL8 treatment limited proximal tubular damage and reduced granulocyte infiltration. In a Fischer 344 (RT1(lvl)) to Lewis (RT1(l)) rat acute renal allograft model, dnCXCL8 was found to reduce monocyte and CD8+ T-cell infiltration into glomeruli and to limit tubular interstitial inflammation and tubulitis in vivo. Early treatment of allografts with agents like dnCXCL8 may help reduce acute allograft damage and preserve renal morphology and thereby help limit chronic dysfunction.
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Vasoregression linked to neuronal damage in the rat with defect of polycystin-2.
PLoS ONE
PUBLISHED: 06-05-2009
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Neuronal damage is correlated with vascular dysfunction in the diseased retina, but the underlying mechanisms remain controversial because of the lack of suitable models in which vasoregression related to neuronal damage initiates in the mature retinal vasculature. The aim of this study was to assess the temporal link between neuronal damage and vascular patency in a transgenic rat (TGR) with overexpression of a mutant cilia gene polycystin-2.
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