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Find video protocols related to scientific articles indexed in Pubmed.
The role of microRNAs in cellular senescence and age-related conditions of cartilage and bone.
Acta Orthop
PUBLISHED: 09-01-2014
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Background and purpose - We reviewed the current state of research on microRNAs in age-related diseases in cartilage and bone. Methods - PubMed searches were conducted using separate terms to retrieve articles on (1) the role of microRNAs on aging and tissue degeneration, (2) specific microRNAs that influence cellular and organism senescence, (3) microRNAs in age-related musculoskeletal conditions, and (4) the diagnostic and therapeutic potential of microRNAs in age-related musculoskeletal conditions. Results - An increasing number of studies have identified microRNAs associated with cellular aging and tissue degeneration. Specifically in regard to frailty, microRNAs have been found to influence the onset and course of age-related musculoskeletal conditions such as osteoporosis, osteoarthritis, and posttraumatic arthritis. Both intracellular and extracellular microRNAs may be suitable to function as diagnostic biomarkers. In particular Interpretation - The research data currently available suggest that microRNAs play an important role in orchestrating age-related processes and conditions of the musculoskeletal system. Further research may help to improve our understanding of the complexity of these processes at the cellular and extracellular level. The option to develop microRNA biomarkers and novel therapeutic agents for the degenerating diseases of bone and cartilage appears to be promising.
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MicroRNAs differentially present in the plasma of HIV elite controllers reduce HIV infection in vitro.
Sci Rep
PUBLISHED: 07-10-2014
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Elite controllers maintain HIV-1 viral loads below the limit of detection. The mechanisms responsible for this phenomenon are poorly understood. As microRNAs (miRNAs) are regulators of gene expression and some of them modulate HIV infection, we have studied the miRNA profile in plasma from HIV elite controllers and chronically infected individuals and compared against healthy donors. Several miRNAs correlate with CD4+ T cell count or with the known time of infection. No significant differences were observed between elite controllers and healthy donors; however, 16 miRNAs were different in the plasma of chronic infected versus healthy donors. In addition, levels of hsa-miR-29b-3p, hsa-miR-33a-5p and hsa-miR-146a-5p were higher in plasma from elite controllers than chronic infected and hsa-miR-29b-3p and hsa-miR-33a-5p overexpression significantly reduced the viral production in MT2 and primary T CD4+ cells. Therefore, levels of circulating miRNAs might be of diagnostic and/or prognostic value for HIV infection, and hsa-miR-29b-3p and miR-33a-5p may contribute to the design of new anti-HIV drugs.
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Identification of microRNAs specific for high producer CHO cell lines using steady-state cultivation.
Appl. Microbiol. Biotechnol.
PUBLISHED: 04-11-2014
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MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene expression. Hence, microRNAs are considered as potential targets for engineering of Chinese hamster ovary (CHO) cells to improve recombinant protein production. Here, we analyzed and compared the microRNA expression patterns of high, low, and non-producing recombinant CHO cell lines expressing two structurally different model proteins in order to identify microRNAs that are involved in heterologous protein synthesis and secretion and thus might be promising targets for cell engineering to increase productivity. To generate reproducible and comparable data, the cells were cultivated in a bioreactor under steady-state conditions. Global microRNA expression analysis showed that mature microRNAs were predominantly upregulated in the producing cell lines compared to the non-producer. Several microRNAs were significantly differentially expressed between high and low producers, but none of them commonly for both model proteins. The identification of target messenger RNAs (mRNAs) is essential to understand the biological function of microRNAs. Therefore, we negatively correlated microRNA and global mRNA expression data and combined them with computationally predicted and experimentally validated targets. However, statistical analysis of the identified microRNA-mRNA interactions indicated a considerable false positive rate. Our results and the comparison to published data suggest that the reaction of CHO cells to the heterologous protein expression is strongly product- and/or clone-specific. In addition, this study highlights the urgent need for reliable CHO-specific microRNA target prediction tools and experimentally validated target databases in order to facilitate functional analysis of high-throughput microRNA expression data in CHO cells.
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WNT Signaling Suppression in the Senescent Human Thymus.
J. Gerontol. A Biol. Sci. Med. Sci.
PUBLISHED: 03-22-2014
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Human thymus is completely developed in late fetal stages and its function peaks in newborns. After the first year of life, the thymus undergoes a progressive atrophy that dramatically decreases de novo T-lymphocyte maturation. Hormonal signaling and changes in the microRNA expression network are identified as underlying causes of human thymus involution. However, specific pathways involved in the age-related loss of thymic function remain unknown. In this study, we analyzed differential gene-expression profile and microRNA expression in elderly (70 years old) and young (less than 10 months old and 11 years old) human thymic samples. Our data have shown that WNT pathway deregulation through the overexpression of different inhibitors by the nonadipocytic component of the human thymus stimulates the age-related involution. These results are of particular interest because interference of WNT signaling has been demonstrated in both animal models and in vitro studies, with the three major hallmarks of thymic involution: (i) epithelial structure disruption, (ii) adipogenic process, and (iii) thymocyte development arrest. Thus, our results suggest that secreted inhibitors of the WNT pathway could be explored as a novel therapeutical target in the reversal of the age-related thymic involution.
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Controversial role of gamma-glutamyl transferase activity in cisplatin nephrotoxicity.
ALTEX
PUBLISHED: 03-14-2014
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Nephrotoxicity of chemotherapeutics is a major hindrance in the treatment of various tumors. Therefore, test systems that reflect mechanisms of human kidney toxicity are necessary, and to reduce animal testing cell culture based systems have to be developed. One cell type that is of specific interest in this regard are renal proximal tubular epithelial cells, as they reabsorb substances from human primary urine filtrates and thus are exposed to urinary excreted xenobiotics and are a major target of cisplatin toxicity. While animal studies using gamma glutamyl transferase (GGT) knock-out mice or GGT inhibitors show that GGT activity increases kidney toxicity of cisplatin, the use of various cell models gives contradictory results. We therefore used a cell panel of immortalized human renal proximal tubular epithelial (RPTECs) cell lines differing in GGT activity. Low GGT activity resulted in high cisplatin sensitivity, as observed in RPTEC-SV40 cells or after siRNA mediated knock-down of GGT in RPTEC/TERT1 cells that have high GGT activity. However, the addition of GGT did not rescue, but also increased cisplatin sensitivity and adding GGT inhibitor as well as substrate (glutathione) or product (cysteinyl-glycine) of GGT resulted in decreased sensitivity. While our data suggest that the use of cell panels are of value in toxicology and toxicogenomics, they also emphasize on the complex interplay of toxins with the intracellular and extracellular microenvironment. In addition, we hypothesize that especially epithelial barrier formation and polarity of RPTECs need to be considered in toxicity models to validly predict the in vivo situation.
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MicroRNA-663 induction upon oxidative stress in cultured human fibroblasts depends on the chronological age of the donor.
Biogerontology
PUBLISHED: 03-06-2014
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MicroRNAs, regulators of messenger RNA translation, have been observed to influence many physiological processes, amongst them the process of aging. Higher levels of microRNA-663 (miR-663) have previously been observed in human dermal fibroblasts subject to both replicative and stress-induced senescence compared to early passage cells. Also, higher levels of miR-663 have been found in memory T-cells and in human fibroblasts derived from older donors compared to younger donors. In previous studies we observed that dermal fibroblasts from donors of different chronological and biological age respond differentially to oxidative stress measured by markers of cellular senescence and apoptosis. In the present study we set out to study the association between miR-663 levels and chronological and biological age. Therefore we tested in a total of 92 human dermal fibroblast strains whether the levels of miR-663 in non-stressed and stressed conditions (fibroblasts were treated with 0.6 ?M rotenone in stressed conditions) were different in young, middle aged and old donors and whether they were different in middle aged donors dependent on their biological age, as indicated by the propensity for familial longevity. In non-stressed conditions the level of miR-663 did not differ between donors of different age categories and was not dependent on biological age. Levels of miR-663 did not differ dependent on biological age in stressed conditions either. However, for different age categories the level of miR-663 in stressed conditions did differ: the level of miR-663 was higher at higher age categories. Also, the ratio of miR-663 induction upon stress was significantly higher in donors from older age categories. In conclusion, we present evidence for an association of miR-663 upon stress and chronological age.
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Analysis of microRNA transcription and post-transcriptional processing by Dicer in the context of CHO cell proliferation.
J. Biotechnol.
PUBLISHED: 02-04-2014
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CHO cells are the mammalian cell line of choice for recombinant production of therapeutic proteins. However, their low rate of proliferation limits obtainable space-time yields due to inefficient biomass accumulation. We set out to correlate microRNA transcription to cell-specific growth-rate by microarray analysis of 5 CHO suspension cell lines with low to high specific growth rates. Global microRNA expression analysis and Pearson correlation studies showed that mature microRNA transcript levels are predominately up-regulated in a state of fast proliferation (46 positively correlated, 17 negatively correlated). To further validate this observation, the expression of three genes that are central to microRNA biogenesis (Dicer, Drosha and Dgcr8) was analyzed. The expression of Dicer, which mediates the final step in microRNA maturation, was found to be strongly correlated to growth rate. Accordingly, knockdown of Dicer impaired cell growth by reducing growth-correlating microRNA transcripts. Moderate ectopic overexpression of Dicer positively affected cell growth, while strong overexpression impaired growth, presumably due to the concomitant increase of microRNAs that inhibit cell growth. Our data therefore suggest that Dicer dependent microRNAs regulate CHO cell proliferation and that Dicer could serve as a potential surrogate marker for cellular proliferation.
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Novel human renal proximal tubular cell line for the production of complex proteins.
J. Biotechnol.
PUBLISHED: 01-31-2014
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Human host cell lines for the production of biopharmaceutical proteins are of interest due to differences in the glycosylation patterns of human and animal cell lines. Specifically, sialylation, which has a major impact on half-life and immunogenicity of recombinant biopharmaceuticals, differs markedly. Here, we established and characterized an immortalized well documented and serum-free host cell line, RS, from primary human renal proximal tubular epithelial cells (RPTEC). In order to test its capacity to produce complex glycosylated proteins, stable recombinant human erythropoietin (rhEpo) producing clones were generated. The clone with highest productivity, RS-1C9 was further characterized and showed stable productivity. Biological activity was observed in in vitro assays and 28% of rhEpo glyco-isoforms produced by RS-1C9 were in range and distribution of the biological reference standard (BRP) isoform, as compared to 11.5% of a CHO based rhEpo. Additionally, cellular ?-2,6 sialylation, Galactose-alpha-1,3-galactose (alpha-Gal) and N-glycolylneuraminic acid (NeuGc) patterns compare favourably to CHO cells. While productivity of RS still needs optimization, its amenability to upscaling in bioreactors, its production of glyco-isoforms that will increase yields after down-stream processing of about 2.5 fold, presence of sialylation and lack of Neu5Gc recommend RS as alternative human host cell line for production of biopharmaceuticals.
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Stable overexpression of miR-17 enhances recombinant protein production of CHO cells.
J. Biotechnol.
PUBLISHED: 01-29-2014
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miRNAs negatively regulate gene expression at post-transcriptional level, and consequently play an important role in the control of many cellular pathways. The use of miRNAs to engineer Chinese hamster ovary (CHO) cells is an emerging strategy to improve recombinant protein production. Here, we describe the effect of transient and stable miRNA overexpression on CHO cell phenotype. Using an established transient miRNA screening protocol, the effects of miR-17, miR-92a and cluster miR17-92a on CHO growth and protein productivity were studied and followed by analysis of cell pools with stable overexpression of these miRNAs. CHO cells stably engineered with miR-17 exhibited both enhanced growth performance and a 2-fold increase in specific productivity, which resulted in a 3-fold overall increase in EpoFc titer. While further studies of miRNA-mRNA interactions will be necessary to understand the molecular basis of this effect, these data provide valuable evidence for miR-17 as a cell engineering target to enhance CHO cell productivity.
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Characterization of a novel cell penetrating peptide derived from human Oct4.
Cell Regen (Lond)
PUBLISHED: 01-01-2014
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Oct4 is a transcription factor that plays a major role for the preservation of the pluripotent state in embryonic stem cells as well as for efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) or other progenitors. Protein-based reprogramming methods mainly rely on the addition of a fused cell penetrating peptide. This study describes that Oct4 inherently carries a protein transduction domain, which can translocate into human and mouse cells.
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Urine as a source of stem cells.
Adv. Biochem. Eng. Biotechnol.
PUBLISHED: 05-01-2013
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Traditionally, clinicians and researchers have relied on a skin biopsy or blood extraction as relatively accessible supplies for in vitro cell expansion and biological studies. Perhaps surprisingly, limited attention has been given to a totally noninvasive source, urine, which eliminates the discomfort associated with other procedures. This may arise from the perception that urine is merely a body waste. Yet, the analysis of urine is a longstanding fundamental test for diagnostic purposes and nowadays there is growing interest in using urine for detecting biomarkers. In addition, recent work including ours reinforces the idea that urine contains a variety of viable cell types with relevant applications. In this review, we describe those cell types and their potential uses.
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Delineation of the key aspects in the regulation of epithelial monolayer formation.
Mol. Cell. Biol.
PUBLISHED: 04-22-2013
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The formation, maintenance, and repair of epithelial barriers are of critical importance for whole-body homeostasis. However, the molecular events involved in epithelial tissue maturation are not fully established. To this end, we investigated the molecular processes involved in renal epithelial proximal-tubule monolayer maturation utilizing transcriptomic, metabolomic, and functional parameters. We uncovered profound dynamic alterations in transcriptional regulation, energy metabolism, and nutrient utilization over the maturation process. Proliferating cells exhibited high glycolytic rates and high transcript levels for fatty acid synthesis genes (FASN), whereas matured cells had low glycolytic rates, increased oxidative capacity, and preferentially expressed genes for beta oxidation. There were dynamic alterations in the expression and localization of several adherens (CDH1, -4, and -16) and tight junction (TJP3 and CLDN2 and -10) proteins. Genes involved in differentiated proximal-tubule function, cilium biogenesis (BBS1), and transport (ATP1A1 and ATP1B1) exhibited increased expression during epithelial maturation. Using TransAM transcription factor activity assays, we could demonstrate that p53 and FOXO1 were highly active in matured cells, whereas HIF1A and c-MYC were highly active in proliferating cells. The data presented here will be invaluable in the further delineation of the complex dynamic cellular processes involved in epithelial cell regulation.
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CHO microRNA engineering is growing up: Recent successes and future challenges.
Biotechnol. Adv.
PUBLISHED: 03-11-2013
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microRNAs with their ability to regulate complex pathways that control cellular behavior and phenotype have been proposed as potential targets for cell engineering in the context of optimization of biopharmaceutical production cell lines, specifically of Chinese Hamster Ovary cells. However, until recently, research was limited by a lack of genomic sequence information on this industrially important cell line. With the publication of the genomic sequence and other relevant data sets for CHO cells since 2011, the doors have been opened for an improved understanding of CHO cell physiology and for the development of the necessary tools for novel engineering strategies. In the present review we discuss both knowledge on the regulatory mechanisms of microRNAs obtained from other biological models and proof of concepts already performed on CHO cells, thus providing an outlook of potential applications of microRNA engineering in production cell lines.
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Identification of microRNA-mRNA functional interactions in UVB-induced senescence of human diploid fibroblasts.
BMC Genomics
PUBLISHED: 03-08-2013
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Cellular senescence can be induced by a variety of extrinsic stimuli, and sustained exposure to sunlight is a key factor in photoaging of the skin. Accordingly, irradiation of skin fibroblasts by UVB light triggers cellular senescence, which is thought to contribute to extrinsic skin aging, although molecular mechanisms are incompletely understood. Here, we addressed molecular mechanisms underlying UVB induced senescence of human diploid fibroblasts.
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Prediction of transcribed PIWI-interacting RNAs from CHO RNAseq data.
J. Biotechnol.
PUBLISHED: 02-27-2013
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Chinese hamster ovary (CHO) cells are currently the most important mammalian host for the manufacture of biopharmaceuticals. To enhance our understanding of cellular processes, pathways, and the genetic setup of CHO cell lines, we predicted PIWI interacting RNAs (piRNAs) from small RNA sequencing data. Although piRNAs are the least understood class of small non-coding RNAs that mediate RNA silencing, it is believed that they play a pivotal role in protecting genome integrity by repressing transposable elements. Since genomic integrity is the key to prolonged stability of recombinant CHO cell lines, we characterized piRNA sequences and expression in six CHO cell lines by computational analysis of an existing small RNA sequencing dataset using proTRAC and the published CHO genome as reference. Here we present the result of this analysis consisting of 25,626 piRNAs and 540 piRNA clusters. Moreover we provide first evidence for differential piRNA expression in adherent and suspension-adapted CHO-K1 and DUKXB11 host cell lines as well as their recombinant derivatives, indicating that piRNAs might be tools for cell line development and engineering.
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High levels of oncomiR-21 contribute to the senescence-induced growth arrest in normal human cells and its knock-down increases the replicative lifespan.
Aging Cell
PUBLISHED: 02-21-2013
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Cellular senescence of normal human cells has by now far exceeded its initial role as a model system for aging research. Many reports show the accumulation of senescent cells in vivo, their effect on their microenvironment and its double-edged role as tumour suppressor and promoter. Importantly, removal of senescent cells delays the onset of age-associated diseases in mouse model systems. To characterize the role of miRNAs in cellular senescence of endothelial cells, we performed miRNA arrays from HUVECs of five different donors. Twelve miRNAs, comprising hsa-miR-23a, hsa-miR-23b, hsa-miR-24, hsa-miR-27a, hsa-miR-29a, hsa-miR-31, hsa-miR-100, hsa-miR-193a, hsa-miR-221, hsa-miR-222 and hsa-let-7i are consistently up-regulated in replicatively senescent cells. Surprisingly, also miR-21 was found up-regulated by replicative and stress-induced senescence, despite being described as oncogenic. Transfection of early passage endothelial cells with miR-21 resulted in lower angiogenesis, and less cell proliferation mirrored by up-regulation of p21(CIP1) and down-regulation of CDK2. These two cell-cycle regulators are indirectly regulated by miR-21 via its validated direct targets NFIB (Nuclear factor 1 B-type), a transcriptional inhibitor of p21(CIP) (1) , and CDC25A, which regulates CDK2 activity by dephosphorylation. Knock-down of either NFIB or CDC25A shows a phenocopy of over-expressing miR-21 in regard to cell-cycle arrest. Finally, miR-21 over-epxression reduces the replicative lifespan, while stable knock-down by sponges extends the replicative lifespan of endothelial cells. Therefore, we propose that miR-21 is the first miRNA that upon its knock-down extends the replicative lifespan of normal human cells.
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Oncostatin M is a novel inhibitor of TGF-?1-induced matricellular protein expression.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 08-03-2011
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Matricellular proteins in the kidney have been associated with the development of tubulointerstitial fibrogenesis and the progression of renal disease. This study investigated potential antifibrotic effects of the cytokine oncostatin M (OSM) in human proximal tubule cells (PTC), particularly with regard to inhibition of profibrotic events initiated by TGF-?1. In human PTC, OSM diminished transforming growth factor (TGF)-?1-induced expression of the transcriptional epithelial-mesenchymal transition mediator FoxC2. Furthermore, exposure to OSM attenuated basal and TGF-?1-induced expression of the matricellular proteins SPARC, TSP-1, TNC, and CTGF regardless of the sequence of ligand administration. OSM was shown to result in rapid and sustained phosphorylation of both Stat1 and Stat3 and also in transient phosphorylation of Smad2/3 in contrast to TGF-?1, which demonstrated a gradually building phosphorylation of Smad2/3 and a brief phosphorylation of Smad1/5/8. Utilizing receptor-blocking molecules, we found the inhibitory effect of OSM on TGF-?1-induced CTGF mRNA expression occurs independently of Smad2/3 signaling and present evidence that this effect may be partially driven by OSM receptor-mediated Stat1 and/or Stat3 signaling pathways, thereby providing a mechanism whereby OSM can contribute to tubulointerstitial protection.
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Exo70, a subunit of the exocyst complex, interacts with SNEV(hPrp19/hPso4) and is involved in pre-mRNA splicing.
Biochem. J.
PUBLISHED: 06-07-2011
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The Cdc5L (cell division cycle 5-like) complex is a spliceosomal subcomplex that also plays a role in DNA repair. The complex contains the splicing factor hPrp19, also known as SNEV or hPso4, which is involved in cellular life-span regulation and proteasomal breakdown. In a recent large-scale proteomics analysis for proteins associated with this complex, proteins involved in transcription, cell-cycle regulation, DNA repair, the ubiquitin-proteasome system, chromatin remodelling, cellular aging, the cytoskeleton and trafficking, including four members of the exocyst complex, were identified. In the present paper we report that Exo70 interacts directly with SNEV(hPrp19/hPso4) and shuttles to the nucleus, where it associates with the spliceosome. We mapped the interaction site to the N-terminal 100 amino acids of Exo70, which interfere with pre-mRNA splicing in vitro. Furthermore, Exo70 influences the splicing of a model substrate as well as of its own pre-mRNA in vivo. In addition, we found that Exo70 is alternatively spliced in a cell-type- and cell-age- dependent way. These results suggest a novel and unexpected role of Exo70 in nuclear mRNA splicing, where it might signal membrane events to the splicing apparatus.
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Generation of induced pluripotent stem cells from urine.
J. Am. Soc. Nephrol.
PUBLISHED: 06-02-2011
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Forced expression of selected transcription factors can transform somatic cells into embryonic stem cell (ESC)-like cells, termed induced pluripotent stem cells (iPSCs). There is no consensus regarding the preferred tissue from which to harvest donor cells for reprogramming into iPSCs, and some donor cell types may be more prone than others to accumulation of epigenetic imprints and somatic cell mutations. Here, we present a simple, reproducible, noninvasive method for generating human iPSCs from renal tubular cells present in urine. This procedure eliminates many problems associated with other protocols, and the resulting iPSCs display an excellent ability to differentiate. These data suggest that urine may be a preferred source for generating iPSCs.
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Unraveling the Chinese hamster ovary cell line transcriptome by next-generation sequencing.
J. Biotechnol.
PUBLISHED: 05-09-2011
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The pyrosequencing technology from 454 Life Sciences and a novel assembly approach for cDNA sequences with the Newbler Assembler were used to achieve a major step forward to unravel the transcriptome of Chinese hamster ovary (CHO) cells. Normalized cDNA libraries originating from several cell lines and diverse culture conditions were sequenced and the resulting 1.84 million reads were assembled into 32,801 contiguous sequences, 29,184 isotigs, and 24,576 isogroups. A taxonomic classification of the isotigs showed that more than 70% of the assembled data is most similar to the transcriptome of Mus musculus, with most of the remaining isotigs being homologous to DNA sequences from Rattus norvegicus. Mapping of the CHO cell line contigs to the mouse transcriptome demonstrated that 9124 mouse transcripts, representing 6701 genes, are covered by more than 95% of their sequence length. Metabolic pathways of the central carbohydrate metabolism and biosynthesis routes of sugars used for protein N-glycosylation were reconstructed from the transcriptome data. All relevant genes representing major steps in the N-glycosylation pathway of CHO cells were detected. The present manuscript represents a data set of assembled and annotated genes for CHO cells that can now be used for a detailed analysis of the molecular functioning of CHO cell lines.
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Dynamic mRNA and miRNA profiling of CHO-K1 suspension cell cultures.
Biotechnol J
PUBLISHED: 03-01-2011
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In spite of the importance of Chinese hamster ovary (CHO) cells for recombinant protein production, very little is known about the molecular and gene regulatory mechanisms that control cellular phenotypes such as enhanced growth under serum-free conditions or high productivity. Most microarray analyses to this purpose are performed with samples taken during the exponential growth phase. However, the cellular transcriptome is dynamic, changing in response to external and internal stimuli and thus reflecting the current functional capacity of cells as well as their ability to adapt to a changing environment. Therefore, during batch or fed-batch cultivations it can be expected that the transcription pattern of genes will change and that such changes may give indications on the cellular state in terms of viability, growth, and productivity. In the current study we monitored the change in expression patterns of mRNAs and microRNAs (miRNA) during lag, exponential, and stationary phases in CHO-K1 suspension cell cultures. In total, over 1400 mRNAs and more than 100 miRNAs were differentially regulated (p<0.05) relative to the batch culture at the starting point. Functional clustering revealed groups of genes with similar expression patterns, which were subjected to functional pathway analysis. In addition, as miRNAs generally act as negative post-transcriptional regulators of mRNAs, we looked for changes in their expression that were inverse to those of their predicted target mRNAs.
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Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering.
J. Biotechnol.
PUBLISHED: 02-24-2011
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Chinese hamster ovary (CHO) cells are the predominant cell factory for the production of recombinant therapeutic proteins. Nevertheless, the lack in publicly available sequence information is severely limiting advances in CHO cell biology, including the exploration of microRNAs (miRNA) as tools for CHO cell characterization and engineering. In an effort to identify and annotate both conserved and novel CHO miRNAs in the absence of a Chinese hamster genome, we deep-sequenced small RNA fractions of 6 biotechnologically relevant cell lines and mapped the resulting reads to an artificial reference sequence consisting of all known miRNA hairpins. Read alignment patterns and read count ratios of 5 and 3 mature miRNAs were obtained and used for an independent classification into miR/miR* and 5p/3p miRNA pairs and discrimination of miRNAs from other non-coding RNAs, resulting in the annotation of 387 mature CHO miRNAs. The quantitative content of next-generation sequencing data was analyzed and confirmed using qPCR, to find that miRNAs are markers of cell status. Finally, cDNA sequencing of 26 validated targets of miR-17-92 suggests conserved functions for miRNAs in CHO cells, which together with the now publicly available sequence information sets the stage for developing novel RNAi tools for CHO cell engineering.
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GiSAO.db: a database for ageing research.
BMC Genomics
PUBLISHED: 02-16-2011
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Age-related gene expression patterns of Homo sapiens as well as of model organisms such as Mus musculus, Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster are a basis for understanding the genetic mechanisms of ageing. For an effective analysis and interpretation of expression profiles it is necessary to store and manage huge amounts of data in an organized way, so that these data can be accessed and processed easily.
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Identification of evolutionarily conserved genetic regulators of cellular aging.
Aging Cell
PUBLISHED: 10-28-2010
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To identify new genetic regulators of cellular aging and senescence, we performed genome-wide comparative RNA profiling with selected human cellular model systems, reflecting replicative senescence, stress-induced premature senescence, and distinct other forms of cellular aging. Gene expression profiles were measured, analyzed, and entered into a newly generated database referred to as the GiSAO database. Bioinformatic analysis revealed a set of new candidate genes, conserved across the majority of the cellular aging models, which were so far not associated with cellular aging, and highlighted several new pathways that potentially play a role in cellular aging. Several candidate genes obtained through this analysis have been confirmed by functional experiments, thereby validating the experimental approach. The effect of genetic deletion on chronological lifespan in yeast was assessed for 93 genes where (i) functional homologues were found in the yeast genome and (ii) the deletion strain was viable. We identified several genes whose deletion led to significant changes of chronological lifespan in yeast, featuring both lifespan shortening and lifespan extension. In conclusion, an unbiased screen across species uncovered several so far unrecognized molecular pathways for cellular aging that are conserved in evolution.
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Post-translational modification of cellular proteins by ubiquitin and ubiquitin-like molecules: role in cellular senescence and aging.
Adv. Exp. Med. Biol.
PUBLISHED: 10-05-2010
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Ubiquitination ofendogenous proteins is one of the key regulatory steps that guides protein degradation through regulation of proteasome activity. During the last years evidence has accumulated that proteasome activity is decreased during the aging process in various model systems and that these changes might be causally related to aging and age-associated diseases. Since in most instances ubiquitination is the primary event in target selection, the system ofubiquitination and deubiquitination might be of similar importance. Furthermore, ubiquitination and proteasomal degradation are not completely congruent, since ubiquitination confers also functions different from targeting proteins for degradation. Depending on mono- and polyubiquitination and on how ubiquitin chains are linked together, post-translational modifications of cellular proteins by covalent attachment of ubiquitin and ubiquitin-like proteins are involved in transcriptional regulation, receptor internalization, DNA repair, stabilization of protein complexes and autophagy. Here, we summarize the current knowledge regarding the ubiquitinome and the underlying ubiquitin ligases and deubiquitinating enzymes in replicative senescence, tissue aging as well as in segmental progeroid syndromes and discuss potential causes and consequences for aging.
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A new dominant peroxiredoxin allele identified by whole-genome re-sequencing of random mutagenized yeast causes oxidant-resistance and premature aging.
Aging (Albany NY)
PUBLISHED: 08-24-2010
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The combination of functional genomics with next generation sequencing facilitates new experimental strategies for addressing complex biological phenomena. Here, we report the identification of a gain-of-function allele of peroxiredoxin (thioredoxin peroxidase, Tsa1p) via whole-genome re-sequencing of a dominantSaccharomyces cerevisiae mutant obtained by chemical mutagenesis. Yeast strain K6001, a screening system for lifespan phenotypes, was treated with ethyl methanesulfonate (EMS). We isolated an oxidative stress-resistant mutant (B7) which transmitted this phenotype in a background-independent, monogenic and dominant way. By massive parallel pyrosequencing, we generated an 38.8 fold whole-genome coverage of the strains, which differed in 12,482 positions from the reference (S288c) genome. Via a subtraction strategy, we could narrow this number to 13 total and 4 missense nucleotide variations that were specific for the mutant. Via expression in wild type backgrounds, we show that one of these mutations, exchanging a residue in the peroxiredoxin Tsa1p, was responsible for the mutant phenotype causing background-independent dominant oxidative stress-resistance. These effects were not provoked by altered Tsa1p levels, nor could they be simulated by deletion, haploinsufficiency or over-expression of the wild-type allele. Furthermore, via both a mother-enrichment technique and a micromanipulation assay, we found a robust premature aging phenotype of this oxidant-resistant strain. Thus, TSA1-B7 encodes for a novel dominant form of peroxiredoxin, and establishes a new connection between oxidative stress and aging. In addition, this study shows that the re-sequencing of entire genomes is becoming a promising alternative for the identification of functional alleles in approaches of classic molecular genetics.
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miR-17-92 cluster: ups and downs in cancer and aging.
Biogerontology
PUBLISHED: 03-22-2010
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The miR-17-92 cluster encoding 6 single mature miRNAs was identified a couple of years ago to contain the first oncogenic miRNAs. Now, one of these 6 miRNAs, miR-19 has been identified as the key responsible for this oncogenic activity. This in turn reduces PTEN levels and in consequence activates the AKT/mTOR pathway that is also prominently involved in modulation of organismal life spans. In contrast, miR-19 and other members of the miR-17-92 cluster are found to be commonly downregulated in several human replicative and organismal aging models. Taken together, these findings suggest that miR-19 and the other members of the miR-17-92 cluster might be important regulators on the cross-roads between aging and cancer. Therefore, we here briefly summarize how this cluster is transcriptionally regulated, which target mRNAs have been confirmed so far and how this might be linked to modulation of organismal life-spans.
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miR-17, miR-19b, miR-20a, and miR-106a are down-regulated in human aging.
Aging Cell
PUBLISHED: 01-18-2010
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Aging is a multifactorial process where deterioration of body functions is driven by stochastic damage while counteracted by distinct genetically encoded repair systems. To better understand the genetic component of aging, many studies have addressed the gene and protein expression profiles of various aging model systems engaging different organisms from yeast to human. The recently identified small non-coding miRNAs are potent post-transcriptional regulators that can modify the expression of up to several hundred target genes per single miRNA, similar to transcription factors. Increasing evidence shows that miRNAs contribute to the regulation of most if not all important physiological processes, including aging. However, so far the contribution of miRNAs to age-related and senescence-related changes in gene expression remains elusive. To address this question, we have selected four replicative cell aging models including endothelial cells, replicated CD8(+) T cells, renal proximal tubular epithelial cells, and skin fibroblasts. Further included were three organismal aging models including foreskin, mesenchymal stem cells, and CD8(+) T cell populations from old and young donors. Using locked nucleic acid-based miRNA microarrays, we identified four commonly regulated miRNAs, miR-17 down-regulated in all seven; miR-19b and miR-20a, down-regulated in six models; and miR-106a down-regulated in five models. Decrease in these miRNAs correlated with increased transcript levels of some established target genes, especially the cdk inhibitor p21/CDKN1A. These results establish miRNAs as novel markers of cell aging in humans.
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Biogerontology in Austria.
Biogerontology
PUBLISHED: 01-08-2010
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In Austria significant progress in the field of biogerontology has been achieved in the past years. Biogerontological research is performed in academic and extramural institutions. The Institute for Biomedical Aging Research of the Austrian Academy of Science at Innsbruck is the largest institution dealing with biogerontology in Austria. Moreover, gerontologic research is performed at the Universities of Salzburg and Graz, the Medical Universities of Vienna, Innsbruck and Graz, the University of Veterinary Medicine (Vienna) and the University of Natural Resources and Applied Life Sciences (Vienna). This article describes the work of selected research groups involved in biogerontology in a geographic arrangement.
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Novel modulators of senescence, aging, and longevity: Small non-coding RNAs enter the stage.
Exp. Gerontol.
PUBLISHED: 01-08-2010
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During the last decade evidence has accumulated that the aging process is driven by limited allocation of energy to somatic maintenance resulting in accumulation of stochastic damage. This damage, affecting molecules, cells, and tissues, is counteracted by genetically programmed repair, the efficiency of which thus importantly determines the life and health span of organisms. Therefore, understanding the regulation of gene expression during cellular and organismal aging as well as upon exposure to various damaging events is important to understand the biology of aging and to positively influence the health span. The recent identification of small non-coding RNAs (ncRNAs), has added an additional layer of complexity to the regulation of gene expression with the classes of endogenous small inhibitory RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), QDE1-interacting RNAs (qiRNAs) and microRNAs (miRNAs). Some of these ncRNAs have not yet been identified in mammalian cells and are dependent on RNA-dependent RNA polymerases. The first mammalian enzyme with such activity has only now emerged and surprisingly consists of the catalytic subunit of telomerase (hTERT) together with RMPR, an alternative RNA component. The so far most studied small non-coding RNAs, miRNAs, however, are now increasingly found to operate in the complex network of cellular aging. Recent findings show that (i) miRNAs are regulated during cellular senescence in vitro, (ii) they contribute to tissue regeneration by regulation of stem cell function, and (iii) at least one miRNA modulates the life span of the model organism C. elegans. Additionally, (iv) they act as inhibitors of proteins mediating the insulin/IGF1 and target of rapamycin (TOR) signalling, both of which are conserved modulators of organism life span. Here we will give an overview on the current status of these topics. Since little is so far known on the functions of small ncRNAs in the context of aging and longevity, the entry of the RNA world into the field of biogerontology certainly holds additional surprises and promises. Even more so, as miRNAs are implicated in many age-associated pathologies, and as RNAi and miRNA based therapeutics are on their way to clinics.
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Blom7alpha is a novel heterogeneous nuclear ribonucleoprotein K homology domain protein involved in pre-mRNA splicing that interacts with SNEVPrp19-Pso4.
J. Biol. Chem.
PUBLISHED: 07-29-2009
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The removal of introns from pre-mRNA is performed by the spliceosome that stepwise assembles on the pre-mRNA before performing two catalytic steps. The spliceosome-associated CDC5L-SNEV(Prp19-Pso4) complex is implicated in activation of the second catalytic step of pre-mRNA splicing, and one of its members, SNEV(Prp19-Pso4), is also implicated in spliceosome assembly. To identify interaction partners of SNEVPrp19-Pso4, we have performed yeast two-hybrid screenings. Among the putative binding partners was a so far uncharacterized protein carrying two heterogeneous nuclear ribonucleoprotein K homology domains that we termed Blom7alpha. Blom7alpha is expressed in all tissues tested, and at least three splice variants exist. After confirming direct and physical interaction of SNEV and Blom7alpha, we investigated if it plays a functional role during pre-mRNA splicing. Indeed, Blom7alpha co-localizes and co-precipitates with splicing factors and pre-mRNA and is present in affinity-purified spliceosomes. More importantly, addition of Blom7alpha to HeLa nuclear extracts increased splicing activity in a dose-dependent manner. Furthermore, we tested if Blom7alpha influences splice site selection using two different minigene constructs. Indeed, both 5- as well as 3-site selection was altered upon Blom7alpha overexpression. Thus we suggest that Blom7alpha is a novel splicing factor of the K homology domain family that might be implicated in alternative splicing by helping to position the CDC5L-SNEV(Prp19-Pso4) complex at the splice sites.
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Acute adrenergic stress inhibits proliferation of murine hematopoietic progenitor cells via p38/MAPK signaling.
Stem Cells Dev.
PUBLISHED: 04-09-2009
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Acute adrenergic stress is a cause of hematopoietic failure that accompanies severe injury. Although the communication between neuronal and immune system is well documented and catecholamines are known as important regulators of homeostasis, the molecular mechanisms of hematopoietic failure are not well understood. To study the influence of adrenergic stress on hematopoietic progenitor cells (HPCs), which recently have been found to express adrenergic receptors, Lin(-),Sca(+), cells were isolated and treated with alpha- and beta-adrenergic agonists in vitro. Indeed, this stimulation resulted in significantly decreased colony formation capacity using granulocyte/macrophage colony-forming unit assays. This decline was dependent on the formation of reactive oxygen species (ROS) and activation of the p38/mitogen-activated protein kinase (MAPK) pathway, since the addition of antioxidants or a p38 inhibitor restored CFU formation. DNA damage by adrenergically induced ROS, however, does not seem to account for the reduction of colonies. Thus, catecholamine/p38/MAPK is identified as a key signal transduction pathway in HPCs besides those dependent on Wnt, Notch, and sonic hedgehog. Furthermore, a well-known target of p38 signaling, p16 is transcriptionally activated after adrenergic stimulation, suggesting that cell cycle arrest might importantly contribute to hematopoietic failure and immune dysfunctions after severe injury. Since increased levels of catecholamines are also observed in other conditions, such as during aging which is linked with decline of immune functions, adrenergic stress might as well contribute to the lowered immune defence in the elderly.
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Alterations in fatty acid utilization and an impaired antioxidant defense mechanism are early events in podocyte injury: a proteomic analysis.
Am. J. Pathol.
PUBLISHED: 03-05-2009
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Ultrastructural alterations of podocytes are closely associated with loss of glomerular filtration function. In the present study, we explored changes at the proteome level that paralleled the disturbances of podocyte architecture in the early stages of puromycin aminonucleoside (PA) nephrosis in vivo. Using two-dimensional fluorescence difference gel electrophoresis and vacuum matrix-assisted laser desorption/ionization mass spectrometry combined with postsource decay fragment ion analysis and high-energy collision-induced dissociation tandem mass spectrometry, 23 differentially expressed protein spots, corresponding to 16 glomerular proteins that are involved in various cellular functions, were unambiguously identified, and a subset was corroborated by Western blot analysis. The majority of these proteins were primarily related to fatty acid metabolism and redox regulation. Key enzymes of the mitochondrial beta-oxidation pathway and antioxidant enzymes were consistently down-regulated in PA nephrosis. These changes were paralleled by increased expression levels of CD36. PA treatment of murine podocytes in culture resembled these specific protein changes in vitro. In this cell system, the modulatory effects of albumin-bound fatty acids on the expression levels of Mn-superoxide dismutase in response to PA were demonstrated as well. Taken together, these results indicate that a disrupted fatty acid metabolism in concert with an impaired antioxidant defense mechanism in podocytes may play a role in the early stages of PA-induced lesions in podocytes.
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Large-scale production and characterization of novel CD4+ cytotoxic T cells with broad tumor specificity for immunotherapy.
Mol. Cancer Res.
PUBLISHED: 02-24-2009
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Immune-cell-based approaches using cytotoxic and dendritic cells are under constant scrutiny to design novel therapies for the treatment of tumors. These strategies are hampered by the lack of efficient and economical large-scale production methods for effector cells. Here we describe the propagation of large amounts of a unique population of CD4(+) cytotoxic T cells, which we termed tumor killer T cells (TKTC), because of their potent and broad antitumor cell activity. With this cultivation strategy, TKTCs from peripheral blood mononuclear cells are generated within a short period of time using a pulse with a stimulating cell line followed by continuous growth in serum-free medium supplemented with a mixture of interleukin-2 and cyclosporin A. Expression and functional profiling did not allow a classification of TKTCs to any thus far defined subtype of T cells. Cytotoxic assays showed that TKTCs kill a panel of tumor targets of diverse tissue origin while leaving normal cells unaffected. Blocking experiments revealed that TKTC killing was, to a significant extent, mediated by tumor necrosis factor-related apoptosis-inducing ligand and was independent of MHC restriction. These results suggest that TKTCs have a high potential as a novel tool in the adoptive immunotherapy of cancer.
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Telomerase immortalized human amnion- and adipose-derived mesenchymal stem cells: maintenance of differentiation and immunomodulatory characteristics.
Tissue Eng Part A
PUBLISHED: 01-08-2009
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Cell banking of mesenchymal stem cells (SCs) from various human tissues has significantly increased the feasibility of SC-based therapies. Sources such as adipose tissue and amnion offer outstanding possibilities for allogeneic transplantation due to their high differentiation potential and their ability to modulate immune reaction. Limitations, however, concern the reduced replicative potential as a result of progressive telomere erosion, which hampers scaleable production and long-term analysis of these cells. Here we report the establishment and characterization of two human amnion-derived and two human adipose-derived SC lines immortalized by ectopic expression of the catalytic subunit of human telomerase (hTERT). hTERT overexpression resulted in continuously growing SC lines that were largely unaltered concerning surface marker profile, morphology, karyotype, and immunosuppressive capacity with similar or enhanced differentiation potential for up to 87 population doublings. While all generated lines showed equal immunomodulation compared to the parental cells, one of the amnion-derived immortalized lines resulted in significantly increased immunogenicity. Although telomerase proves as important tool for immortalizing cells, our data emphasize the need for careful and standardized characterization of each individual cell population for cell banks.
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Secretion of microvesicular miRNAs in cellular and organismal aging.
Exp. Gerontol.
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Changes of factors circulating in the systemic environment during human aging have been investigated for a long time. Only recently however, miRNAs have been found to be secreted into the systemic and tissue environments where they are protected from RNAses by either carrier proteins or by being packaged into microvesicles. These miRNAs are then taken up by recipient cells, changing the cellular behavior by the classical miRNA induced silencing of target mRNAs. The origin of circulating miRNAs, however, is in most instances unclear, but senescent cells emerge as a possible source of such secreted miRNAs. Since differences in the circulating miRNAs have been found in a variety of age-associated diseases, and accumulation of senescent cells in the elderly emerges as a possible detrimental factor in aging, it is well conceivable that these miRNAs might contribute to the functional decline observed during aging of organisms. Therefore, we here give an overview on current knowledge on microvesicular secretion of miRNAs, changes of the systemic and tissue environments during aging of cells and organisms. Finally, we summarize current knowledge on miRNAs that are found to be specific for age-associated diseases.
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Inhibition of pre-mRNA splicing by a synthetic Blom7?-interacting small RNA.
PLoS ONE
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Originally the novel protein Blom7? was identified as novel pre-mRNA splicing factor that interacts with SNEV(Prp19/Pso4), an essential protein involved in extension of human endothelial cell life span, DNA damage repair, the ubiquitin-proteasome system, and pre-mRNA splicing. Blom7? belongs to the heteronuclear ribonucleoprotein K homology (KH) protein family, displaying 2 KH domains, a well conserved and widespread RNA-binding motif. In order to identify specific sequence binding motifs, we here used Systematic Evolution of Ligands by Exponential Enrichment (SELEX) with a synthetic RNA library. Besides sequence motifs like (U/A)(1-4) C(2-6) (U/A)(1-5), we identified an AC-rich RNA-aptamer that we termed AK48 (Aptamer KH-binding 48), binding to Blom7? with high affinity. Addition of AK48 to pre-mRNA splicing reactions in vitro inhibited the formation of mature spliced mRNA and led to a slight accumulation of the H complex of the spliceosome. These results suggest that the RNA binding activity of Blom7? might be required for pre-mRNA splicing catalysis. The inhibition of in-vitro splicing by the small RNA AK48 indicates the potential use of small RNA molecules in targeting the spliceosome complex as a novel target for drug development.
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Generation of human induced pluripotent stem cells from urine samples.
Nat Protoc
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Human induced pluripotent stem cells (iPSCs) have been generated with varied efficiencies from multiple tissues. Yet, acquiring donor cells is, in most instances, an invasive procedure that requires laborious isolation. Here we present a detailed protocol for generating human iPSCs from exfoliated renal epithelial cells present in urine. This method is advantageous in many circumstances, as the isolation of urinary cells is simple (30 ml of urine are sufficient), cost-effective and universal (can be applied to any age, gender and race). Moreover, the entire procedure is reasonably quick--around 2 weeks for the urinary cell culture and 3-4 weeks for the reprogramming--and the yield of iPSC colonies is generally high--up to 4% using retroviral delivery of exogenous factors. Urinary iPSCs (UiPSCs) also show excellent differentiation potential, and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases, including those affecting the kidney.
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Construction of a stability landscape of the CH3 domain of human IgG1 by combining directed evolution with high throughput sequencing.
J. Mol. Biol.
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One of the most important but still poorly understood issues in protein chemistry is the relationship between sequence and stability of proteins. Here, we present a method for analyzing the influence of each individual residue on the foldability and stability of an entire protein. A randomly mutated library of the crystallizable fragment of human immunoglobulin G class 1 (IgG1-Fc) was expressed on the surface of yeast, followed by heat incubation at 79°C and selection of stable variants that still bound to structurally specific ligands. High throughput sequencing allowed comparison of the mutation rate between the starting and selected library pools, enabling the generation of a stability landscape for the entire CH3 domain of human IgG1 at single residue resolution. Its quality was analyzed with respect to (i) the structure of IgG1-Fc, (ii) evolutionarily conserved positions and (iii) in silico calculations of the energy of unfolding of all variants in comparison with the wild-type protein. In addition, this new experimental approach allowed the assignment of functional epitopes of structurally specific ligands used for selection [Fc ?-receptor I (CD64) and anti-human CH2 domain antibody] to distinct binding regions in the CH2 domain.
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HIV-1 induces telomerase activity in monocyte-derived macrophages, possibly safeguarding one of its reservoirs.
J. Virol.
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Monocyte-derived macrophages (MDM) are widely distributed in all tissues and organs, including the central nervous system, where they represent the main part of HIV-infected cells. In contrast to activated CD4(+) T lymphocytes, MDM are resistant to cytopathic effects and survive HIV infection for a long period of time. The molecular mechanisms of how HIV is able to persist in macrophages are not fully elucidated yet. In this context, we have studied the effect of in vitro HIV-1 infection on telomerase activity (TA), telomere length, and DNA damage. Infection resulted in a significant induction of TA. This increase was directly proportional to the efficacy of HIV infection and was found in both nuclear and cytoplasmic extracts, while neither UV light-inactivated HIV nor exogenous addition of the viral protein Tat or gp120 affected TA. Furthermore, TA was not modified during monocyte-macrophage differentiation, MDM activation, or infection with vaccinia virus. HIV infection did not affect telomere length. However, HIV-infected MDM showed less DNA damage after oxidative stress than noninfected MDM, and this resistance was also increased by overexpressing telomerase alone. Taken together, our results suggest that HIV induces TA in MDM and that this induction might contribute to cellular protection against oxidative stress, which could be considered a viral strategy to make macrophages better suited as longer-lived, more resistant viral reservoirs. In the light of the clinical development of telomerase inhibitors as anticancer therapeutics, inhibition of TA in HIV-infected macrophages might also represent a novel therapeutic target against viral reservoirs.
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ATM-dependent phosphorylation of SNEVhPrp19/hPso4 is involved in extending cellular life span and suppression of apoptosis.
Aging (Albany NY)
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Defective DNA repair is widely acknowledged to negatively impact on healthy aging, since mutations in DNA repair factors lead to accelerated and premature aging. However, the opposite, namely if improved DNA repair will also increase the life or health span is less clear, and only few studies have tested if overexpression of DNA repair factors modulates life and health span in cells or organisms. Recently, we identified and characterized SNEVhPrp19/hPso4, a protein that plays a role in DNA repair and pre-mRNA splicing, and observed a doubling of the replicative life span upon ectopic overexpression, accompanied by lower basal DNA damage and apoptosis levels as well as an increased resistance to oxidative stress. Here we find that SNEVhPrp19/hPso4 is phosphorylated at S149 in an ataxia telangiectasia mutated protein (ATM)-dependent manner in response to oxidative stress and DNA double strand break inducing agents. By overexpressing wild-type SNEVhPrp19/hPso4 and a phosphorylation-deficient point-mutant, we found that S149 phosphorylation is necessary for mediating the resistance to apoptosis upon oxidative stress and is partially necessary for elongating the cellular life span. Therefore, ATM dependent phosphorylation of SNEVhPrp19/hPso4 upon DNA damage or oxidative stress might represent a novel axis capable of modulating cellular life span.
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Upregulation of miR-24 is associated with a decreased DNA damage response upon etoposide treatment in highly differentiated CD8(+) T cells sensitizing them to apoptotic cell death.
Aging Cell
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The life-long homeostasis of memory CD8(+) T cells as well as persistent viral infections have been shown to facilitate the accumulation of highly differentiated CD8(+) CD28(-) T cells, a phenomenon that has been associated with an impaired immune function in humans. However, the molecular mechanisms regulating homeostasis of CD8(+) CD28(-) T cells have not yet been elucidated. In this study, we demonstrate that the miR-23?24?27 cluster is up-regulated during post-thymic CD8(+) T-cell differentiation in humans. The increased expression of miR-24 in CD8(+) CD28(-) T cells is associated with decreased expression of the histone variant H2AX, a protein that plays a key role in the DNA damage response (DDR). Following treatment with the classic chemotherapeutic agent etoposide, a topoisomerase II inhibitor, apoptosis was increased in CD8(+) CD28(-) when compared to CD8(+) CD28(+) T cells and correlated with an impaired DDR in this cell type. The reduced capacity of CD8(+) CD28(-) T cell to repair DNA was characterized by the automated fluorimetric analysis of DNA unwinding (FADU) assay as well as by decreased phosphorylation of H2AX at Ser139, of ATM at Ser1981, and of p53 at Ser15. Interleukin (IL)-15 could prevent etoposide-mediated apoptosis of CD8(+) CD28(-) T cells, suggesting a role for IL-15 in the survival and the age-dependent accumulation of CD8(+) CD28(-) T cells in humans.
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A screening method to assess biological effects of microRNA overexpression in Chinese hamster ovary cells.
Biotechnol. Bioeng.
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MicroRNAs (miRNAs) are a novel class of short non-coding RNAs, which negatively regulate target gene expression at post-transcriptional level. They mediate an important layer of control in the global regulation of gene networks, controlling a broad range of physiological as well as patho-physiological pathways including development, cancer, metabolism, proliferation, and stress resistance. So far, more than 365 miRNA genes have been identified in CHO cells. The functional analysis of the physiological effect of such large numbers of miRNAs, however, requires an efficient functional screening method. In the current study, we therefore established and evaluated a protocol to perform miRNA overexpression and to screen their effect on bio-industrially relevant phenotypes, such as growth, viability and productivity, using a recombinant, Epo-Fc producing CHO cell line. For protocol optimization, four CHO miRNAs (cgr-miR-17, cgr-miR-221, cgr-miR-21, and cgr-miR-210) were cloned into small hairpin vectors including a GFP cassette and transfected. After transfection cells were analyzed for growth and productivity over a 4-day period. Even from this small set of four miRNAs, the overexpression of miR-17, one of the members of the oncogenic miR-17-92 cluster, gave proof of principle that this method enables the identification of miRNA engineering candidates as its overexpression increased the speed of cell proliferation without negatively impacting specific productivity. The here presented method is applicable for medium-throughput screening for microRNA, miR-sponge, siRNA, or mRNA overexpression along with detailed functional characterization using the same experimental set up. As the same procedure can be applied to different production cell lines, the protocol can also be used to test for individual, cell line specific responses to microRNAs. Thus our system represents a general platform to functionally screen candidates for rational cell factory design.
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Utilization and evaluation of CHO-specific sequence databases for mass spectrometry based proteomics.
Biotechnol. Bioeng.
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Recently released sequence information on Chinese hamster ovary (CHO) cells promises to not only facilitate our understanding of these industrially important cell factories through direct analysis of the sequence, but also to enhance existing methodologies and allow new tools to be developed. In this article we demonstrate the utilization of CHO specific sequence information to improve mass spectrometry (MS) based proteomic identification. The use of various CHO specific databases enabled the identification of 282 additional proteins, thus increasing the total number of identified proteins by 40-50%, depending on the sample source and methods used. In addition, a considerable portion of those proteins that were identified previously based on inter-species sequence homology were now identified by a larger number of peptides matched, thus increasing the confidence of identification. The new sequence information offers improved interpretation of proteomic analyses and will, in the years to come, prove vital to unraveling the CHO proteome.
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Computational identification of microRNA gene loci and precursor microRNA sequences in CHO cell lines.
J. Biotechnol.
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MicroRNAs (miRNAs) have recently entered Chinese hamster ovary (CHO) cell culture technology, due to their severe impact on the regulation of cellular phenotypes. Applications of miRNAs that are envisioned range from biomarkers of favorable phenotypes to cell engineering targets. These applications, however, require a profound knowledge of miRNA sequences and their genomic organization, which exceeds the currently available information of ~400 conserved mature CHO miRNA sequences. Based on these recently published sequences and two independent CHO-K1 genome assemblies, this publication describes the computational identification of CHO miRNA genomic loci. Using BLAST alignment, 415 previously reported CHO miRNAs were mapped to the reference genomes, and subsequently assigned to a distinct genomic miRNA locus. Sequences of the respective precursor-miRNAs were extracted from both reference genomes, folded in silico to verify correct structures and cross-compared. In the end, 212 genomic loci and pre-miRNA sequences representing 319 expressed mature miRNAs (approximately 50% of miRNAs represented matching pairs of 5 and 3 miRNAs) were submitted to the miRBase miRNA repository. As a proof-of-principle for the usability of the published genomic loci, four likely polycistronic miRNA cluster were chosen for PCR amplification using CHO-K1 and DHFR (-) genomic DNA. Overall, these data on the genomic context of miRNA expression in CHO will simplify the development of tools employing stable overexpression or deletion of miRNAs, allow the identification of miRNA promoters and improve detection methods such as microarrays.
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MicroRNA-26 family is required for human adipogenesis and drives characteristics of brown adipocytes.
Stem Cells
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Adipose tissue contains thermogenic adipocytes (i.e., brown and brite/beige) that oxidize nutrients at exceptionally high rates via non-shivering thermogenesis. Its recent discovery in adult humans has opened up new avenues to fight obesity and related disorders such as diabetes. Here we identified miR-26a and miR-26b as key regulators of human white and brite adipocyte differentiation. Both microRNAs are upregulated in early adipogenesis, and their inhibition prevented lipid accumulation while their overexpression accelerated it. Intriguingly, miR-26a significantly induced pathways related to energy dissipation, shifted mitochondrial morphology towards that seen in brown adipocytes, and promoted uncoupled respiration by markedly increasing the hallmark protein of brown fat, uncoupling protein 1 (UCP1). By combining in silico target prediction, transcriptomics, and an RNA interference screen, we identified the sheddase ADAM metallopeptidase domain 17 (ADAM17) as a direct target of miR-26 that mediated the observed effects on white and brite adipogenesis. These results point to a novel, critical role for the miR-26 family and its downstream effector ADAM17 in human adipocyte differentiation by promoting characteristics of energy-dissipating thermogenic adipocytes. Stem Cells 2013.
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Endogenous MicroRNA Clusters Outperform Chimeric Sequence Clusters in Chinese Hamster Ovary Cells.
Biotechnol J
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MicroRNAs (miRNA) are small non-coding RNAs (~22 nucleotides). They are transcribed as long primary transcripts, which are enzymatically processed in the nucleus (Drosha/Dgcr8) and cytoplasm (Dicer) into mature miRNAs, which regulate gene expression by silencing mRNA translation. The importance of miRNAs for coordinated gene expression is commonly accepted. Therefore, there is also a growing interest in the application of miRNAs to improve phenotypes of mammalian cell factories such as Chinese hamster ovary (CHO) cells. Few studies have reported the targeted over-expression of miRNAs in CHO cells using vector-based systems. These approaches were hampered by limited sequence availability, and required the design of "chimeric" miRNA genes, consisting of the mature CHO miRNA sequence encompassed by murine flanking and loop sequences. Here we show that the substitution of chimeric sequences with CHO-specific sequences for expression of miRNA clusters yields significantly higher expression levels of the mature miRNA in the case of miR-221/222 and miR-15b/16. Our data suggest that the Drosha/Dgcr8-mediated excision from primary transcripts is inhibited for chimeric miRNA sequences compared to the endogenous sequence. Overall, this study provides important guidelines for the targeted over-expression of clustered miRNAs in CHO cells.
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