SPLIT
Deletion of calcineurin from astrocytes reproduces proteome signature of Alzheimer’s disease and epilepsy and predisposes to seizures
Laura Tapella, Giulia Dematteis, Federico Alessandro Ruffinatti, Luisa Ponzoni, Fabio Fiordaliso, Alessandro Corbelli, Enrico Albanese, Beatrice Pistolato, Jessica Pagano, Elettra Barberis, Emilio Marengo, Claudia Balducci, Gianluigi Forloni, Chiara Verpelli, Carlo Sala, Carla Distasi, Mariaelvina Sala, Armando A Genazzani, Marcello Manfredf, Dmitry Lim
Calcineurin (CaN), acting downstream of intracellular calcium signals, orchestrates cellular remodelling in many cellular types. In astrocytes, principal homeostatic cells in the central nervous system (CNS), CaN is involved in neuroinflammation and gliosis, while its role in healthy CNS or in early neuro-pathogenesis is poorly understood. Here we report that in mice with conditional deletion of CaN from GFAP-expressing astrocytes (astroglial calcineurin KO, ACN-KO), at 1 month of age, transcription was not changed, while proteome was deranged in hippocampus and cerebellum. Gene ontology analysis revealed overrepresentation of annotations related to myelin sheath, mitochondria, ribosome and cytoskeleton. Overrepresented pathways were related to protein synthesis, oxidative phosphorylation, mTOR and neurological disorders, including Alzheimer’s disease (AD) and seizure disorder. Comparison with published proteomics datasets shows significant overlap with the proteome of a familial AD mouse model and of human subjects with drug-resistant epilepsy. Strikingly, beginning from about 5 months of age ACN-KO mice develop spontaneous tonic-clonic seizures with inflammatory signature of epileptic brain. Altogether, our data suggest that the deletion of astroglial CaN produces features of neurological disorders and predisposes mice to seizures. We suggest that calcineurin in astrocytes may serve as a novel Ca2+-sensitive switch which regulates protein expression and homeostasis in the central nervous system.
Features SPLIT RNA Extraction Kit and QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Loss of Tet2 affects proliferation and drug sensitivity through altered dynamics of cell-state transitions
Leanna Morinishi, Karl Kochanowski, Ross L. Levine, Lani F. Wu, Steven J. Altschuler
A persistent puzzle in cancer biology is how mutations, which neither alter canonical growth signaling pathways nor directly interfere with drug mechanism, can still recur and persist in tumors. One notable example is the loss-of-function mutation of the DNA demethylase Tet2 in acute myeloid leukemias (AMLs) that frequently persists from diagnosis through remission and relapse (Rothenberg-Thurley et al., 2018; Corces-Zimmerman et al., 2014; Nibourel et al., 2010), but whose fitness advantage in the setting of anti-leukemic chemotherapy is unclear. Here we use paired isogenic human AML cell lines to show that Tet2 loss-of-function alters the dynamics of transitions between differentiated and stem-like states. Mathematical modeling and experimental validation reveal that these altered cell-state dynamics can benefit the cell population by slowing population decay during drug treatment and lowering the number of survivor cells needed to re-establish the initial population. These studies shed light on the functional and phenotypic effects of a Tet2 loss-of-function in AML, illustrate how a single gene mutation can alter a cells’ phenotypic plasticity, and open up new avenues in the development of strategies to combat AML relapse.
Features SPLIT RNA Extraction Kit
LncRNA LUADT1 regulates miR‐34a/SIRT1 to participate in chondrocyte apoptosis
Su Ni, Chao Xu, Chao Zhuang, Gongyin Zhao, Chenkai Li, Yuji Wang, Xihu Qin
It is known that miR‐34a can promote the apoptosis of chondrocytes, which directly contribute to osteoarthritis (OA). Through bioinformatics analysis, we found that long noncoding RNA LUADT1 may interact with miR‐34a. We, therefore, further investigate the interactions between them in osteoarthritis. We found that LUADT1 was downregulated, while miR‐34a was upregulated in OA synovial fluid. Correlation analysis revealed no significant correlation between them. Overexpression experiment also revealed no significant effects of LUADT1 and miR‐34a on the expression of each other. However, the dual‐luciferase assay showed that LUADT1 and miR‐34a can directly interact with each other. Moreover, LUADT1 overexpression led to the upregulation of SIRT1, which is a downstream target of miR‐34a. Cell apoptosis showed that LUADT1 and SIRT1 overexpression led to decreased, while miR‐34a led to increased apoptotic rates of chondrocytes. Therefore, LUADT1 regulates miR‐34a/SIRT1 to participate in chondrocyte apoptosis.
Features SPLIT RNA Extraction Kit
Biphasic Response of Protein Kinase A to Cyclic Adenosine Monophosphate Triggers Distinct Epithelial Phenotypes
João Pedro Fonseca, Elham Aslankoohi, Hana El-Samad
Protein Kinase A (PKA) is an important cellular signaling hub whose activity has long been assumed to monotonically depend on the level of cyclic adenosine monophosphate (cAMP).
Using an optogenetic tool that can introduce precise amounts of cAMP in MDCKI cells, we demonstrate that PKA activity is instead characterized by a biphasic response, in which PKA activity increases and then decreases as a function of cAMP. We reveal that this behavior results from an elaborate integration by PKA of many cellular signals triggered by cAMP. In addition to the direct activation of PKA, cAMP also modulates the activity of p38 and ERK, which then converge on PKA to inhibit it. These interactions and their ensuing biphasic PKA profile have important physiological repercussions, triggering two distinct transcriptional programs elicited by low and high cAMP doses. These transcriptional responses in turn influence the ability of MDCKI cells to proliferate and form acini. Our data, supported by computational analyses, synthesize a set of network interconnections involving PKA and other important signaling pathways into a model that demonstrates how cells can capitalize on signal integration to create a diverse set of responses to cAMP concentration and produce complex input-output relationships.
Features SPLIT RNA Extraction Kit and QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina
Blockade of miR-140-3p prevents functional deterioration in afterload-enhanced engineered heart tissue
Tessa R. Werner, Ann-Cathrin Kunze, Justus Stenzig, Thomas Eschenhagen & Marc N. Hirt
Afterload enhancement (AE) of rat engineered heart tissue (EHT) in vitro leads to a multitude of changes that in vivo are referred to as pathological cardiac hypertrophy: e.g., cardiomyocyte hypertrophy, contractile dysfunction, reactivation of fetal genes and fibrotic changes. Moreover AE induced the upregulation of 22 abundantly expressed microRNAs. Here, we aimed at evaluating the functional effect of inhibiting 7 promising microRNAs (miR-21-5p, miR-146b-5p, miR-31a-5p, miR-322-5p, miR-450a-5p, miR-140-3p and miR-132-3p) in a small-range screen. Singular transfection of locked nucleic acid (LNA)-based anti-miRs at 100 nM (before the one week AE-procedure) led to a powerful reduction of the targeted microRNAs. Pretreatment with anti-miR-146b-5p, anti-miR-322-5p or anti-miR-450a-5p did not alter the AE-induced contractile decline, while anti-miR-31a-5p-pretreatment even worsened it. Anti-miR-21-5p and anti-miR-132-3p partially attenuated the AE-effect, confirming previous reports. LNA-anti-miR against miR-140-3p, a microRNA recently identified as a prognostic biomarker of cardiovascular disease, also attenuated the AE-effect. To simplify future in vitro experiments and to create an inhibitor for in vivo applications, we designed shorter miR-140-3p-inhibitors and encountered variable efficiency. Only the inhibitor that effectively repressed miR-140-3p was also protective against the AE-induced contractile decline. In summary, in a small-range functional screen, miR-140-3p evolved as a possible new target for the attenuation of afterload-induced pathological cardiac hypertrophy.
Features SPLIT RNA Extraction Kit
Magnetics-Based Approach for Fine-Tuning Afterload in Engineered Heart Tissues
Marita L. Rodriguez, Tessa R. Werner, Benjamin Becker, Thomas Eschenhagen, Marc N. Hirt
ACS Biomaterials Science & Engineering, doi:10.1021/acsbiomaterials.8b01568
Afterload plays important roles during heart development and disease progression; however, studying these effects in a laboratory setting is challenging. Current techniques lack the ability to precisely and reversibly alter afterload over time. Here, we describe a magnetics-based approach for achieving this control and present results from experiments in which this technique was employed to sequentially increase afterload applied to rat engineered heart tissues (rEHTs) over a 7-day period. Over the observation period, the contractile properties of rEHTs grown on control posts marginally increased. The average post deflection, fractional shortening, and twitch velocities measured for afterload-affected tissues initially followed this same trend but fell below control tissue values at high magnitudes of afterload. However, the average force, force production rate, and force relaxation rate for these rEHTs were consistently up to three-fold higher than for control tissues. Transcript levels of hypertrophic or fibrotic markers and cell size remained unaffected by afterload, suggesting that the increased force output was not accompanied by pathological remodeling. Accordingly, the increased force output was fully reversed to control levels during a stepwise decrease in afterload over 4 h. Afterload application did not affect systolic or diastolic tissue lengths, indicating that the afterload system was likely not a source of changes in preload strain. In summary, the afterload system developed herein is capable of fine-tuning EHT afterload while simultaneously allowing optical force measurements. Using this system, we found that small daily alterations in afterload can enhance the contractile properties of rEHTs, while larger increases can have temporarily undesirable effects. Overall, these findings demonstrate the significant role that afterload plays in cardiac force regulation. Future studies with this system may allow for novel insights into the mechanisms that underlie afterload-induced adaptations in cardiac force development.
Features SPLIT RNA Extraction Kit
Dido3-dependent SFPQ recruitment maintains efficiency in mammalian alternative splicing
Carmen Mora Gallardo, Ainhoa Sánchez de Diego, Julio Gutiérrez Hernández, Amaia Talavera-Gutiérrez, Thierry Fischer, Carlos Martínez-A, Karel H M van Wely
Features SPLIT RNA Extraction Kit
Hearing impairment due to Mir183/96/182 mutations suggests both loss and gain of function effects
Morag A. Lewis, Francesca Di Domenico, Neil J. Ingham, Haydn M. Prosser, Karen P. Steel
Features SPLIT RNA Extraction Kit
MATERIALS AND METHODS: Dual luciferase assay was performed to assess the targeted relationship between miR-142 and DJ-1. MiR-142, DJ-1, and PTEN expressions in SW1990 cells and drug-resistant SW1990/ADM cells were compared. SW1990/ADM cells were divided into five groups, including mimic-NC, miR-142 mimic, small interfere normal control (si-NC), si-DJ-1, and miR-142 mimic + si-DJ-1 groups. DJ-1, PTEN, phosphorylated-AKT (p-AKT), and Survivin expressions were tested. Cell apoptosis was determined by flow cytometry. Cell proliferation was evaluated by EdU staining.
RESULTS: MiR-142 targeted inhibited DJ-1 expression. MiR-142, PTEN, and cell apoptosis significantly down-regulated, while DJ-1, p-AKT, Survivin, and cell proliferation significantly elevated in SW1990/ADM cells compared with SW1990 cells. MiR-142 mimics and/or si-DJ-1 transfection markedly reduced DJ-1, p-AKT, and Survivin expressions enhanced PTEN level, attenuated cell proliferation, enhanced cell apoptosis, and weakened ADM resistance.
CONCLUSIONS: MiR-142 over-expression weakened ADM resistance in pancreatic cancer cells by targeting DJ-1 to enhance PTEN expression and attenuate PI3K/AKT signaling pathway activity.
Features SPLIT RNA Extraction Kit
Insights into the dynamics of memory, effector and apoptotic cytotoxic T lymphocytes in channel catfish, Ictalurus punctatus
David A. Spencera, Sylvie M.A. Quiniou, Jonathan Crider, Bryan Musungu, Eva Bengten, Melanie Wilson
Developmental & Comparative Immunology, doi:10.1016/j.dci.2018.11.001
Features SPLIT RNA Extraction Kit
Phytophthora infestans sporangia produced in culture and on tomato leaflet lesions show marked differences in indirect germination rates, aggressiveness, and global transcription profiles
William Fry, Sean P Patev, Kevin Meyers, Kan Bao, Zhangjun Fei
Molecular Plant-Microbe Interactions, doi: 10.1094/MPMI-09-18-0255-TA
Features SPLIT RNA Extraction Kit and SENSE mRNA-Seq Library Prep Kit
PATIENTS AND METHODS: Dual luciferase reporter gene assay was applied to confirm targeted regulation between miR-107 and mTOR. Tumor tissues were collected from glioma patients, in parallel with normal tissues after brain contusion surgery. Expressions of miR-107, mTOR and p-mTOR were compared. DDP-resistant cell line U251/DPP was generated. U251/DPP cells were further treated with miR-107 mimic or si-mTOR to examine the change of miR-107, mTOR, p-mTOR and survivin levels. Flow cytometry was used to quantify the effect of DDP treatment on cell proliferation or apoptosis.
RESULTS: Bioinformatics analysis revealed complementary binding sites between miR-107 and 3’-UTR of mTOR mRNA. Dual luciferase assay confirmed targeted regulation between miR-107 and mTOR. Compared to control group, in glioma tissues, mTOR and p-mTOR expressions were significantly elevated, while the level of miR-107 expression was markedly decreased. Of note, U251/DDP cells presented weakened apoptosis compared to U251 cells, with high levels of mTOR, p-mTOR and survivin and reduction of miR-107 expression. However, the transfection of miR-107 mimic and/or si-mTOR remarkably suppressed expressions of mTOR, p-mTOR and survivin in U251/DPP cells, weakened cell proliferation and enhanced apoptosis.
CONCLUSIONS: We demonstrated that the level of miR-107 was correlated with DDP resistance in glioma cells. Over-expression of miR-107 decreased DPP resistance of glioma cells via inhibition of mTOR, which provides academic basis for the future anti-glioma therapy.
Features SPLIT RNA Extraction Kit
MicroRNAs Establish Uniform Traits during the Architecture of Vertebrate Embryos
Dionna M. Kasper, Albertomaria Moro, Emma Ristori, Anand Narayanan, Guillermina Hill-Teran, Elizabeth Fleming, Miguel Moreno-Mateos, Charles E. Vejnar, Jing Zhang, Donghoon Lee, Mengting Gu, Mark Gerstein, Antonio Giraldez, Stefania Nicoli
Features SPLIT RNA Extraction Kit
GABAA receptor subunit deregulation in the hippocampus of human foetuses with Down syndrome
Ivan Milenkovic, Tamara Stojanovic, Eleonora Aronica, Livia Fülöp, Zsolt Bozsó, Zoltán Máté, Yuchio Yanagawa, Homa Adle-Biassette, Gert Lubec, Gábor Szabó, Tibor Harkany, Gábor G. Kovács, Erik Keimpema
Brain Structure and Function; doi: 10.1007/s00429-017-1563-3
Features SPLIT RNA Extraction Kit
MATERIALS AND METHODS: ASTN rat model was established in parallel with control group. Protein expressions of PTEN, p-AKT, PCNA, Cyclin D1 and Bcl-2 were quantified, along with glomerular mesangial cell (GMC) counting. Rat mesangial cell (RMC) was treated with 0 or 10 ng/mL IL-6, followed by flow cytometry analysis for apoptosis, cycle and PCNA expression. Expressions of PTEN, p-AKT, PCNA, Cyclin D1 and Bcl-2 were measured. RMC was treated with pSicoR-PTEN and/or LY294002, followed by the treatment of 10 ng/mL IL-6 for 48 h. Cell apoptosis, cycle, PCNA expression and protein expression were measured.
RESULTS: Lower PTEN expression was found in renal cortex of ATSN rats, along with increasing levels of p-AKT, PCNA, Cyclin D1, Bcl-2, and higher GMCs, compared to that in control rats. IL-6 treatment increased protein expression in RMC, facilitated cell proliferation and cycle progression and suppressed apoptosis. Over-expression of PTEN and/or LY294002 remarkably decreased protein expression in RMC, inhibited the effect of IL-6 on proliferation, and induced cell apoptosis and cycle arrest.
CONCLUSIONS: The down-regulation of PTEN played a role in enhancing PI3K/AKT pathway activity, facilitating GMC proliferation and MPGN pathogenesis.
Features SPLIT RNA Extraction Kit
Estrogen Regulates Bone Turnover by Targeting RANKL Expression in Bone Lining Cells
Carmen Streicher, Alexandra Heyny, Olena Andrukhova, Barbara Haigl, Svetlana Slavic, Christiane Schüler, Karoline Kollmann, Ingrid Kantner, Veronika Sexl, Miriam Kleiter, Lorenz C. Hofbauer, Paul J. Kostenuik & Reinhold G. Erben
Features SPLIT RNA Extraction Kit
Neuronal-expressed microRNA-targeted pseudogenes compete with coding genes in the human brain
S Barbash, A Simchovitz, A S Buchman, D A Bennett, S Shifman and H Soreq
Features QuantSeq 3’ mRNA-Seq Library Prep Kit for Illumina and SPLIT RNA Extraction Kit
Efficient and unbiased metagenomic recovery of RNA virus genomes from human plasma samples
Carmen F. Manso, David F. Bibby & Jean L. Mbisa
Features SPLIT RNA Extraction Kit
Alzheimer’s brains show inter-related changes in RNA and lipid metabolism
Shahar Barbash, Benjamin P. Garfinkel, Rotem Maoz, Alon Simchovitz, Bettina Nadorp, Alessandro Guffanti, Estelle R. Bennett, Courtney Nadeau, Andreas Türk, Lukas Paul, Torsten Reda, Yan Li, Aron S. Buchman, David S. Greenberg, Alexander Seitz, David A. Bennett, Patrick Giavalisco, Hermona Soreq
Features QuantSeq 3’ mRNA-Seq Library Prep Kit REV for Illumina and SPLIT RNA Extraction Kit
Features SENSE mRNA‐Seq Library Prep Kit
Features SPLIT RNA Extraction Kit
InFusion: Advancing Discovery of Fusion Genes and Chimeric Transcripts from Deep RNA-Sequencing Data
Konstantin Okonechnikov, Aki Imai-Matsushima, Lukas Paul, Alexander Seitz, Thomas F. Meyer, Fernando Garcia-Alcalde
Features SENSE mRNA‐Seq Library Prep Kit
Features SPLIT RNA Extraction Kit
Differences in DNA Repair Capacity, Cell Death and Transcriptional Response after Irradiation between a Radiosensitive and a Radioresistant Cell Line
Mireia Borràs-Fresneda, Joan-Francesc Barquinero, Maria Gomolka, Sabine Hornhardt, Ute Rössler, Gemma Armengol & Leonardo Barrios
Features QuantSeq 3′ mRNA-Seq Library Prep Kits
Features SPLIT RNA Extraction Kit
Alternative Splice Forms Influence Functions of Whirlin in Mechanosensory Hair Cell Stereocilia
Seham Ebrahim, Neil J. Ingham, Morag A. Lewis, Michael J.C. Rogers, Runjia Cui, Bechara Kachar, Johanna C. Pass, Karen P. Steel
Features SPLIT RNA Extraction Kit
Excessive Osteocytic Fgf23 Secretion Contributes to Pyrophosphate Accumulation and Mineralization Defect in Hyp Mice
Sathish K. Murali, Olena Andrukhova, Erica L. Clinkenbeard, Kenneth E. White, Reinhold G. Erben
PLoS Biol 14(4): e1002427. doi: 10.1371/journal.pbio.1002427
Features SPLIT RNA Extraction Kit
Amphiregulin lacks an essential role for the bone anabolic action of parathyroid hormone.
Freya F. Jay, Mithila Vaidya, Sabrina M. Porada, Olena Andrukhova, Marlon R. Schneider, Reinhold G. Erben
Mol Cell Endocrinol. 2015 Sep 28. pii: S0303-7207(15)30097-6. doi: 10.1016/j.mce.2015.09.031.
Features SPLIT RNA Extraction Kit
Nuclear accumulation of CDH1 mRNA in hepatocellular carcinoma cells.
Ghafoory S, Mehrabi A, Hafezi M, Cheng X, Breitkopf-Heinlein K, Hick M, Huichalaf M, Herbel V, Saffari A, Wölfl S.
Oncogenesis. 2015 Jun 1;4:e152. doi: 10.1038/oncsis.2015.11.
Features SPLIT RNA Extraction Kit