Efficient Generation of iPS Cells from Skeletal Muscle Stem Cells

PubMed Central

Tan, Kah Yong; Eminli, Sarah; Hettmer, Simone; Hochedlinger, Konrad; Wagers, Amy J.

2011-01-01

Reprogramming of somatic cells into inducible pluripotent stem cells generally occurs at low efficiency, although what limits reprogramming of particular cell types is poorly understood. Recent data suggest that the differentiation status of the cell targeted for reprogramming may influence its susceptibility to reprogramming as well as the differentiation potential of the induced pluripotent stem (iPS) cells that are derived from it. To assess directly the influence of lineage commitment on iPS cell derivation and differentiation, we evaluated reprogramming in adult stem cell and mature cell populations residing in skeletal muscle. Our data using clonal assays and a second-generation inducible reprogramming system indicate that stem cells found in mouse muscle, including resident satellite cells and mesenchymal progenitors, reprogram with significantly greater efficiency than their more differentiated daughters (myoblasts and fibroblasts). However, in contrast to previous reports, we find no evidence of biased differentiation potential among iPS cells derived from myogenically committed cells. These data support the notion that adult stem cells reprogram more efficiently than terminally differentiated cells, and argue against the suggestion that “epigenetic memory” significantly influences the differentiation potential of iPS cells derived from distinct somatic cell lineages in skeletal muscle. PMID:22028872

Alkaline phosphatase and OCT-3/4 as useful markers for predicting susceptibility of human deciduous teeth-derived dental pulp cells to reprogramming factor-induced iPS cells.

PubMed

Inada, Emi; Saitoh, Issei; Kubota, Naoko; Soda, Miki; Matsueda, Kazunari; Murakami, Tomoya; Sawami, Tadashi; Kagoshima, Akiko; Yamasaki, Youichi; Sato, Masahiro

2017-11-01

The aim of the present study was to prove that primary cells enriched with stem cells are more easily reprogrammed to generate induced pluripotent stem (iPS) cells than those with scarce numbers of stem cells. We surveyed the alkaline phosphatase (ALP) activity in five primarily-isolated human deciduous teeth-derived dental pulp cells (HDDPC) with cytochemical staining to examine the possible presence of stem cells. Next, the expression of stemness-specific factors, such as OCT(Octumer-binding transcription factor)3/4, NANOG, SOX2(SRY (sex determining region Y)-box 2), CD90, muscle segment homeodomain homeobox (MSX) 1, and MSX2, was assessed with a reverse transcription polymerase chain reaction method. Finally, these isolated HDDPC were transfected with plasmids carrying genes coding Yamanaka factors to determine whether these cells could be reprogrammed to generate iPS cells. Of the five primarily-isolated HDDPC, two (HDDPC-1 and -5) exhibited higher degrees of ALP activity. OCT-3/4 expression was also prominent in those two lines. Furthermore, these two lines proliferated faster than the other three lines. The transfection of HDDPC with Yamanaka factors resulted in the generation of iPS cells from HDDPC-1 and -5. The number of cells with the stemness property of HDDPC differs among individuals, which suggests that HDDPC showing an increased expression of both ALP and OCT-3/4 can be more easily reprogrammed to generate iPS cells after the forced expression of reprogramming factors. © 2016 John Wiley & Sons Australia, Ltd.

Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts

PubMed Central

Wu, Yuehong; Li, Ouyang; He, Chengwen; Li, Yong; Li, Min; Liu, Xiaoming; Wang, Yujiong; He, Yulong

2017-01-01

Induced pluripotent stem cells (iPS) represent an important tool to develop disease-modeling assays, drug testing assays and cell-based replacement therapies. The application of iPS in these fields requires the development of suitable animal models. Of the suitable species, guinea pigs are particularly important and offer significant advantages. Successful iPS generation has been accomplished in a number of species; however, it has not been reported in the guinea pig. The present study successfully generated iPS from guinea pigs (giPS) using single polycistronic virus transduction with mouse octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), Kruppel-like factor 4 and c-Myc. The giPS cell lines were cultured in media containing leukemia inhibitory factor and guinea pig fibroblast cells were used as feeder cells. These cultures were expanded under feeder-free culture conditions using ESGRO Complete Plus Clonal Grade medium containing 15% fetal bovine serum on gelatin-coated dishes. The resultant cells had a normal karyotype, exhibited alkaline phosphatase activity and expressed the pluripotency markers Oct4, Sox2 and Nanog. The cells differentiated in vivo to form teratomas that contained all three germ layers of the tissue cells. The generation of giPS may facilitate future studies investigating the mechanisms underlying innate immunity, particularly for tuberculosis. These experiments provide proof of principle that iPS technology may be adapted to use the guinea pig as a model of human diseases. PMID:28393187

Inositol pyrophosphates promote tumor growth and metastasis by antagonizing liver kinase B1

PubMed Central

Rao, Feng; Xu, Jing; Fu, Chenglai; Cha, Jiyoung Y.; Gadalla, Moataz M.; Xu, Risheng; Barrow, James C.; Snyder, Solomon H.

2015-01-01

The inositol pyrophosphates, molecular messengers containing an energetic pyrophosphate bond, impact a wide range of biologic processes. They are generated primarily by a family of three inositol hexakisphosphate kinases (IP6Ks), the principal product of which is diphosphoinositol pentakisphosphate (IP7). We report that IP6K2, via IP7 synthesis, is a major mediator of cancer cell migration and tumor metastasis in cell culture and in intact mice. IP6K2 acts by enhancing cell-matrix adhesion and decreasing cell–cell adhesion. This action is mediated by IP7-elicited nuclear sequestration and inactivation of the tumor suppressor liver kinase B1 (LKB1). Accordingly, inhibitors of IP6K2 offer promise in cancer therapy. PMID:25617365

Induced pluripotent stem (iPS) cells: a new source for cell-based therapeutics?

PubMed

de Lázaro, Irene; Yilmazer, Açelya; Kostarelos, Kostas

2014-07-10

The generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of defined transcription factors has provided the regenerative medicine field with a new tool for cell replacement strategies. The advantages that these pluripotent cells can offer in comparison to other sources of stem cells include the generation of patient-derived cells and the lack of embryonic tissue while maintaining a versatile differentiation potential. The promise of iPS cell derivatives for therapeutic applications is encouraging albeit very early in development, with the first clinical study currently ongoing in Japan. Many challenges are yet to be circumvented before this technology can be clinically translated widely though. The delivery and expression of the reprogramming factors, the genomic instability, epigenetic memory and impact of cell propagation in culture are only some of the concerns. This article aims to critically discuss the potential of iPS cells as a new source of cell therapeutics. Copyright © 2014 Elsevier B.V. All rights reserved.

Generation and characterization of induced pluripotent stem cells from guinea pig fetal fibroblasts.

PubMed

Wu, Yuehong; Li, Ouyang; He, Chengwen; Li, Yong; Li, Min; Liu, Xiaoming Liu; Wang, Yujiong; He, Yulong

2017-06-01

Induced pluripotent stem cells (iPS) represent an important tool to develop disease‑modeling assays, drug testing assays and cell‑based replacement therapies. The application of iPS in these fields requires the development of suitable animal models. Of the suitable species, guinea pigs are particularly important and offer significant advantages. Successful iPS generation has been accomplished in a number of species; however, it has not been reported in the guinea pig. The present study successfully generated iPS from guinea pigs (giPS) using single polycistronic virus transduction with mouse octamer‑binding transcription factor 4 (Oct4), sex determining region Y‑box 2 (Sox2), Kruppel‑like factor 4 and c‑Myc. The giPS cell lines were cultured in media containing leukemia inhibitory factor and guinea pig fibroblast cells were used as feeder cells. These cultures were expanded under feeder‑free culture conditions using ESGRO Complete Plus Clonal Grade medium containing 15% fetal bovine serum on gelatin‑coated dishes. The resultant cells had a normal karyotype, exhibited alkaline phosphatase activity and expressed the pluripotency markers Oct4, Sox2 and Nanog. The cells differentiated in vivo to form teratomas that contained all three germ layers of the tissue cells. The generation of giPS may facilitate future studies investigating the mechanisms underlying innate immunity, particularly for tuberculosis. These experiments provide proof of principle that iPS technology may be adapted to use the guinea pig as a model of human diseases.

Generation of iPS-derived model cells for analyses of hair shaft differentiation.

PubMed

Kido, Takumi; Horigome, Tomoatsu; Uda, Minori; Adachi, Naoki; Hirai, Yohei

2017-09-01

Biological evaluation of hair growth/differentiation activity in vitro has been a formidable challenge, primarily due to the lack of relevant model cell systems. To solve this problem, we generated a stable model cell line in which successive differentiation via epidermal progenitors to hair components is easily inducible and traceable. Mouse induced pluripotent stem (iPS) cell-derived cells were selected to stably express a tetracycline (Tet)-inducible bone morphogenic protein-4 (BMP4) expression cassette and a luciferase reporter driven by a hair-specific keratin 31 gene (krt31) promoter (Tet-BMP4-KRT31-Luc iPS). While Tet- BMP4-KRT31-Luc iPS cells could be maintained as stable iPS cells, the cells differentiated to produce luciferase luminescence in the presence of all-trans retinoic acid (RA) and doxycycline (Dox), and addition of a hair differentiation factor significantly increased luciferase fluorescence. Thus, this cell line may provide a reliable cell-based screening system to evaluate drug candidates for hair differentiation activity.

Generation of an ICF syndrome model by efficient genome editing of human induced pluripotent stem cells using the CRISPR system.

PubMed

Horii, Takuro; Tamura, Daiki; Morita, Sumiyo; Kimura, Mika; Hatada, Izuho

2013-09-30

Genome manipulation of human induced pluripotent stem (iPS) cells is essential to achieve their full potential as tools for regenerative medicine. To date, however, gene targeting in human pluripotent stem cells (hPSCs) has proven to be extremely difficult. Recently, an efficient genome manipulation technology using the RNA-guided DNase Cas9, the clustered regularly interspaced short palindromic repeats (CRISPR) system, has been developed. Here we report the efficient generation of an iPS cell model for immunodeficiency, centromeric region instability, facial anomalies syndrome (ICF) syndrome using the CRISPR system. We obtained iPS cells with mutations in both alleles of DNA methyltransferase 3B (DNMT3B) in 63% of transfected clones. Our data suggest that the CRISPR system is highly efficient and useful for genome engineering of human iPS cells.

Generation of Healthy Mice from Gene-Corrected Disease-Specific Induced Pluripotent Stem Cells

PubMed Central

Rittelmeyer, Ina; Sharma, Amar Deep; Sgodda, Malte; Zaehres, Holm; Bleidißel, Martina; Greber, Boris; Gentile, Luca; Han, Dong Wook; Rudolph, Cornelia; Steinemann, Doris; Schambach, Axel; Ott, Michael; Schöler, Hans R.; Cantz, Tobias

2011-01-01

Using the murine model of tyrosinemia type 1 (fumarylacetoacetate hydrolase [FAH] deficiency; FAH −/− mice) as a paradigm for orphan disorders, such as hereditary metabolic liver diseases, we evaluated fibroblast-derived FAH −/−-induced pluripotent stem cells (iPS cells) as targets for gene correction in combination with the tetraploid embryo complementation method. First, after characterizing the FAH −/− iPS cell lines, we aggregated FAH −/−-iPS cells with tetraploid embryos and obtained entirely FAH −/−-iPS cell–derived mice that were viable and exhibited the phenotype of the founding FAH −/− mice. Then, we transduced FAH cDNA into the FAH −/−-iPS cells using a third-generation lentiviral vector to generate gene-corrected iPS cells. We could not detect any chromosomal alterations in these cells by high-resolution array CGH analysis, and after their aggregation with tetraploid embryos, we obtained fully iPS cell–derived healthy mice with an astonishing high efficiency for full-term development of up to 63.3%. The gene correction was validated functionally by the long-term survival and expansion of FAH-positive cells of these mice after withdrawal of the rescuing drug NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione). Furthermore, our results demonstrate that both a liver-specific promoter (transthyretin, TTR)-driven FAH transgene and a strong viral promoter (from spleen focus-forming virus, SFFV)-driven FAH transgene rescued the FAH-deficiency phenotypes in the mice derived from the respective gene-corrected iPS cells. In conclusion, our data demonstrate that a lentiviral gene repair strategy does not abrogate the full pluripotent potential of fibroblast-derived iPS cells, and genetic manipulation of iPS cells in combination with tetraploid embryo aggregation provides a practical and rapid approach to evaluate the efficacy of gene correction of human diseases in mouse models. PMID:21765802

HSP90 regulates cell survival via inositol hexakisphosphate kinase-2

PubMed Central

Chakraborty, Anutosh; Koldobskiy, Michael A.; Sixt, Katherine M.; Juluri, Krishna R.; Mustafa, Asif K.; Snowman, Adele M.; van Rossum, Damian B.; Patterson, Randen L.; Snyder, Solomon H.

2008-01-01

Heat-shock proteins (HSPs) are abundant, inducible proteins best known for their ability to maintain the conformation of proteins and to refold damaged proteins. Some HSPs, especially HSP90, can be antiapoptotic and the targets of anticancer drugs. Inositol hexakisphosphate kinase-2 (IP6K2), one of a family of enzymes generating the inositol pyrophosphate IP7 [diphosphoinositol pentakisphosphate (5-PP-IP5)], mediates apoptosis. Increased IP6K2 activity sensitizes cancer cells to stressors, whereas its depletion blocks cell death. We now show that HSP90 physiologically binds IP6K2 and inhibits its catalytic activity. Drugs and selective mutations that abolish HSP90–IP6K2 binding elicit activation of IP6K2, leading to cell death. Thus, the prosurvival actions of HSP90 reflect the inhibition of IP6K2, suggesting that selectively blocking this interaction could provide effective and safer modes of chemotherapy. PMID:18195352

Ataxia-telangiectasia mutated (ATM) deficiency decreases reprogramming efficiency and leads to genomic instability in iPS cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kinoshita, Taisuke; Nagamatsu, Go, E-mail: gonag@sc.itc.keio.ac.jp; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012

2011-04-08

Highlights: {yields} iPS cells were induced with a fluorescence monitoring system. {yields} ATM-deficient tail-tip fibroblasts exhibited quite a low reprogramming efficiency. {yields} iPS cells obtained from ATM-deficient cells had pluripotent cell characteristics. {yields} ATM-deficient iPS cells had abnormal chromosomes, which were accumulated in culture. -- Abstract: During cell division, one of the major features of somatic cell reprogramming by defined factors, cells are potentially exposed to DNA damage. Inactivation of the tumor suppressor gene p53 raised reprogramming efficiency but resulted in an increased number of abnormal chromosomes in established iPS cells. Ataxia-telangiectasia mutated (ATM), which is critical in the cellularmore » response to DNA double-strand breaks, may also play an important role during reprogramming. To clarify the function of ATM in somatic cell reprogramming, we investigated reprogramming in ATM-deficient (ATM-KO) tail-tip fibroblasts (TTFs). Although reprogramming efficiency was greatly reduced in ATM-KO TTFs, ATM-KO iPS cells were successfully generated and showed the same proliferation activity as WT iPS cells. ATM-KO iPS cells had a gene expression profile similar to ES cells and WT iPS cells, and had the capacity to differentiate into all three germ layers. On the other hand, ATM-KO iPS cells accumulated abnormal genome structures upon continuous passages. Even with the abnormal karyotype, ATM-KO iPS cells retained pluripotent cell characteristics for at least 20 passages. These data indicate that ATM does participate in the reprogramming process, although its role is not essential.« less

Generation of Isogenic Human iPS Cell Line Precisely Corrected by Genome Editing Using the CRISPR/Cas9 System.

PubMed

Grobarczyk, Benjamin; Franco, Bénédicte; Hanon, Kevin; Malgrange, Brigitte

2015-10-01

Genome engineering and human iPS cells are two powerful technologies, which can be combined to highlight phenotypic differences and identify pathological mechanisms of complex diseases by providing isogenic cellular material. However, very few data are available regarding precise gene correction in human iPS cells. Here, we describe an optimized stepwise protocol to deliver CRISPR/Cas9 plasmids in human iPS cells. We highlight technical issues especially those associated to human stem cell culture and to the correction of a point mutation to obtain isogenic iPS cell line, without inserting any resistance cassette. Based on a two-steps clonal isolation protocol (mechanical picking followed by enzymatic dissociation), we succeed to select and expand corrected human iPS cell line with a great efficiency (more than 2% of the sequenced colonies). This protocol can also be used to obtain knock-out cell line from healthy iPS cell line by the NHEJ pathway (with about 15% efficiency) and reproduce disease phenotype. In addition, we also provide protocols for functional validation tests after every critical step.

Common genetic variation drives molecular heterogeneity in human iPSCs.

PubMed

Kilpinen, Helena; Goncalves, Angela; Leha, Andreas; Afzal, Vackar; Alasoo, Kaur; Ashford, Sofie; Bala, Sendu; Bensaddek, Dalila; Casale, Francesco Paolo; Culley, Oliver J; Danecek, Petr; Faulconbridge, Adam; Harrison, Peter W; Kathuria, Annie; McCarthy, Davis; McCarthy, Shane A; Meleckyte, Ruta; Memari, Yasin; Moens, Nathalie; Soares, Filipa; Mann, Alice; Streeter, Ian; Agu, Chukwuma A; Alderton, Alex; Nelson, Rachel; Harper, Sarah; Patel, Minal; White, Alistair; Patel, Sharad R; Clarke, Laura; Halai, Reena; Kirton, Christopher M; Kolb-Kokocinski, Anja; Beales, Philip; Birney, Ewan; Danovi, Davide; Lamond, Angus I; Ouwehand, Willem H; Vallier, Ludovic; Watt, Fiona M; Durbin, Richard; Stegle, Oliver; Gaffney, Daniel J

2017-06-15

Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.

Excision of a viral reprogramming cassette by delivery of synthetic Cre mRNA

PubMed Central

Loh, Yuin-Han; Yang, Jimmy Chen; De Los Angeles, Alejandro; Guo, Chunguang; Cherry, Anne; Rossi, Derrick J.; Park, In-Hyun; Daley, George Q.

2012-01-01

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy, in vitro modeling of human disease, and drug screening. To date, most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively, transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This report describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector, which includes the coding sequences of human OCT4, SOX2, KLF4, and cMYC linked with picornaviral 2A plasmids. Moreover, after reprogramming has been achieved, this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells. PMID:22605648

Practical Integration-Free Episomal Methods for Generating Human Induced Pluripotent Stem Cells.

PubMed

Kime, Cody; Rand, Tim A; Ivey, Kathryn N; Srivastava, Deepak; Yamanaka, Shinya; Tomoda, Kiichiro

2015-10-06

The advent of induced pluripotent stem (iPS) cell technology has revolutionized biomedicine and basic research by yielding cells with embryonic stem (ES) cell-like properties. The use of iPS-derived cells for cell-based therapies and modeling of human disease holds great potential. While the initial description of iPS cells involved overexpression of four transcription factors via viral vectors that integrated within genomic DNA, advances in recent years by our group and others have led to safer and higher quality iPS cells with greater efficiency. Here, we describe commonly practiced methods for non-integrating induced pluripotent stem cell generation using nucleofection of episomal reprogramming plasmids. These methods are adapted from recent studies that demonstrate increased hiPS cell reprogramming efficacy with the application of three powerful episomal hiPS cell reprogramming factor vectors and the inclusion of an accessory vector expressing EBNA1. Copyright © 2015 John Wiley & Sons, Inc.

Induced pluripotent stem cells: Mechanisms, achievements and perspectives in farm animals

PubMed Central

Kumar, Dharmendra; Talluri, Thirumala R; Anand, Taruna; Kues, Wilfried A

2015-01-01

Pluripotent stem cells are unspecialized cells with unlimited self-renewal, and they can be triggered to differentiate into desired specialized cell types. These features provide the basis for an unlimited cell source for innovative cell therapies. Pluripotent cells also allow to study developmental pathways, and to employ them or their differentiated cell derivatives in pharmaceutical testing and biotechnological applications. Via blastocyst complementation, pluripotent cells are a favoured tool for the generation of genetically modified mice. The recently established technology to generate an induced pluripotency status by ectopic co-expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc allows to extending these applications to farm animal species, for which the derivation of genuine embryonic stem cells was not successful so far. Most induced pluripotent stem (iPS) cells are generated by retroviral or lentiviral transduction of reprogramming factors. Multiple viral integrations into the genome may cause insertional mutagenesis and may increase the risk of tumour formation. Non-integration methods have been reported to overcome the safety concerns associated with retro and lentiviral-derived iPS cells, such as transient expression of the reprogramming factors using episomal plasmids, and direct delivery of reprogramming mRNAs or proteins. In this review, we focus on the mechanisms of cellular reprogramming and current methods used to induce pluripotency. We also highlight problems associated with the generation of iPS cells. An increased understanding of the fundamental mechanisms underlying pluripotency and refining the methodology of iPS cell generation will have a profound impact on future development and application in regenerative medicine and reproductive biotechnology of farm animals. PMID:25815117

Macrophages induce differentiation of plasma cells through CXCL10/IP-10

PubMed Central

Joo, HyeMee; Clayton, Sandra; Dullaers, Melissa; Herve, Marie-Cecile; Blankenship, Derek; De La Morena, Maria Teresa; Balderas, Robert; Picard, Capucine; Casanova, Jean-Laurent; Pascual, Virginia; Oh, SangKon; Banchereau, Jacques

2012-01-01

In tonsils, CD138+ plasma cells (PCs) are surrounded by CD163+ resident macrophages (Mϕs). We show here that human Mϕs (isolated from tonsils or generated from monocytes in vitro) drive activated B cells to differentiate into CD138+CD38++ PCs through secreted CXCL10/IP-10 and VCAM-1 contact. IP-10 production by Mϕs is induced by B cell–derived IL-6 and depends on STAT3 phosphorylation. Furthermore, IP-10 amplifies the production of IL-6 by B cells, which sustains the STAT3 signals that lead to PC differentiation. IP-10–deficient mice challenged with NP-Ficoll show a decreased frequency of NP-specific PCs and lower titers of antibodies. Thus, our results reveal a novel dialog between Mϕs and B cells, in which IP-10 acts as a PC differentiation factor. PMID:22987802

Ethanol potentiates the genotoxicity of the food-derived mammary carcinogen PhIP in human estrogen receptor-positive mammary cells: mechanistic support for lifestyle factors (cooked red meat and ethanol) associated with mammary cancer.

PubMed

Malik, Durr-E-Shahwar; David, Rhiannon M; Gooderham, Nigel J

2018-04-01

Consumption of cooked/processed meat and ethanol are lifestyle risk factors in the aetiology of breast cancer. Cooking meat generates heterocyclic amines such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Epidemiology, mechanistic and animal studies indicate that PhIP is a mammary carcinogen that could be causally linked to breast cancer incidence; PhIP is DNA damaging, mutagenic and oestrogenic. PhIP toxicity involves cytochrome P450 (CYP1 family)-mediated metabolic activation to DNA-damaging species, and transcriptional responses through Aryl hydrocarbon receptor (AhR) and estrogen-receptor-α (ER-α). Ethanol consumption is a modifiable lifestyle factor strongly associated with breast cancer risk. Ethanol toxicity involves alcohol dehydrogenase metabolism to reactive acetaldehyde, and is also a substrate for CYP2E1, which when uncoupled generates reactive oxygen species (ROS) and DNA damage. Here, using human mammary cells that differ in estrogen-receptor status, we explore genotoxicity of PhIP and ethanol and mechanisms behind this toxicity. Treatment with PhIP (10 -7 -10 -4 M) significantly induced genotoxicity (micronuclei formation) preferentially in ER-α positive human mammary cell lines (MCF-7, ER-α+) compared to MDA-MB-231 (ER-α-) cells. PhIP-induced CYP1A2 in both cell lines but CYP1B1 was selectively induced in ER-α(+) cells. ER-α inhibition in MCF-7 cells attenuated PhIP-mediated micronuclei formation and CYP1B1 induction. PhIP-induced CYP2E1 and ROS via ER-α-STAT-3 pathway, but only in ER-α (+) MCF-7 cells. Importantly, simultaneous treatments of physiological concentrations ethanol (10 -3 -10 -1 M) with PhIP (10 -7 -10 -4 M) increased oxidative stress and genotoxicity in MCF-7 cells, compared to the individual chemicals. Collectively, these data offer a mechanistic basis for the increased risk of breast cancer associated with dietary cooked meat and ethanol lifestyle choices.

Future perspective of induced pluripotent stem cells for diagnosis, drug screening and treatment of human diseases.

PubMed

Lian, Qizhou; Chow, Yenyen; Esteban, Miguel Angel; Pei, Duanqing; Tse, Hung-Fat

2010-07-01

Recent advances in stem cell biology have transformed the understanding of cell physiology and developmental biology such that it can now play a more prominent role in the clinical application of stem cell and regenerative medicine. Success in the generation of human induced pluripotent stem cells (iPS) as well as related emerging technology on the iPS platform provide great promise in the development of regenerative medicine. Human iPS cells show almost identical properties to human embryonic stem cells (ESC) in pluripotency, but avoid many of their limitations of use. In addition, investigations into reprogramming of somatic cells to pluripotent stem cells facilitate a deeper understanding of human stem cell biology. The iPS cell technology has offered a unique platform for studying the pathogenesis of human disease, pharmacological and toxicological testing, and cell-based therapy. Nevertheless, significant challenges remain to be overcome before the promise of human iPS cell technology can be realised.

Stably Fluorescent Cell Line of Human Ovarian Epithelial Cancer Cells SK-OV-3ip-red.

PubMed

Konovalova, E V; Shulga, A A; Chumakov, S P; Khodarovich, Yu M; Woo, Eui-Jeon; Deev, S M

2017-11-01

Stable red fluorescing line of human ovarian epithelial cancer cells SK-OV-3ip-red was generated expressing gene coding for protein TurboFP635 (Katushka) fluorescing in the far-red spectrum region with excitation and emission peaks at 588 and 635 nm, respectively. Fluorescence of SK-OV-3ip-red line remained high during long-term cell culturing and after cryogenic freezing. The obtained cell line SK-OV-3ip-red can serve a basis for a model of a scattered tumor with numerous/extended metastases and used both for testing anticancer drugs inhibiting metastasis growth and for non-invasive monitoring of the growth dynamics with high precision.

Induced pluripotent stem cells: advances to applications

PubMed Central

Nelson, Timothy J; Martinez-Fernandez, Almudena; Yamada, Satsuki; Ikeda, Yasuhiro; Perez-Terzic, Carmen; Terzic, Andre

2010-01-01

Induced pluripotent stem cell (iPS) technology has enriched the armamentarium of regenerative medicine by introducing autologous pluripotent progenitor pools bioengineered from ordinary somatic tissue. Through nuclear reprogramming, patient-specific iPS cells have been derived and validated. Optimizing iPS-based methodology will ensure robust applications across discovery science, offering opportunities for the development of personalized diagnostics and targeted therapeutics. Here, we highlight the process of nuclear reprogramming of somatic tissues that, when forced to ectopically express stemness factors, are converted into bona fide pluripotent stem cells. Bioengineered stem cells acquire the genuine ability to generate replacement tissues for a wide-spectrum of diseased conditions, and have so far demonstrated therapeutic benefit upon transplantation in model systems of sickle cell anemia, Parkinson’s disease, hemophilia A, and ischemic heart disease. The field of regenerative medicine is therefore primed to adopt and incorporate iPS cell-based advancements as a next generation stem cell platforms. PMID:21165156

Phospholipase C-β1 and β4 Contribute to Non-Genetic Cell-to-Cell Variability in Histamine-Induced Calcium Signals in HeLa Cells

PubMed Central

Ishida, Sachiko; Matsu-ura, Toru; Fukami, Kiyoko; Michikawa, Takayuki; Mikoshiba, Katsuhiko

2014-01-01

A uniform extracellular stimulus triggers cell-specific patterns of Ca2+ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP3) concentration changes using a fluorescent IP3 sensor in single HeLa cells showing different patterns of histamine-induced Ca2+ oscillations in terms of the time constant of Ca2+ spike amplitude decay and the Ca2+ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca2+ signals and we found that there were cell-specific IP3 dynamics depending on the patterns of Ca2+ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP3 increases in HeLa cells. Modulation of IP3 dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca2+ oscillations, such as the time constant of the temporal changes in the Ca2+ spike amplitude and the Ca2+ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP3 production, rather than IP3-induced Ca2+ release, can cause cell-to-cell variability in the patterns of Ca2+ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca2+ signals evoked by G protein-coupled receptor stimulation. PMID:24475116

Highly Expandable Human iPS Cell-Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High-Throughput Screening.

PubMed

Cheng, Chialin; Fass, Daniel M; Folz-Donahue, Kat; MacDonald, Marcy E; Haggarty, Stephen J

2017-01-11

Reprogramming of human somatic cells into induced pluripotent stem (iPS) cells has greatly expanded the set of research tools available to investigate the molecular and cellular mechanisms underlying central nervous system (CNS) disorders. Realizing the promise of iPS cell technology for the identification of novel therapeutic targets and for high-throughput drug screening requires implementation of methods for the large-scale production of defined CNS cell types. Here we describe a protocol for generating stable, highly expandable, iPS cell-derived CNS neural progenitor cells (NPC) using multi-dimensional fluorescence activated cell sorting (FACS) to purify NPC defined by cell surface markers. In addition, we describe a rapid, efficient, and reproducible method for generating excitatory cortical-like neurons from these NPC through inducible expression of the pro-neural transcription factor Neurogenin 2 (iNgn2-NPC). Finally, we describe methodology for the use of iNgn2-NPC for probing human neuroplasticity and mechanisms underlying CNS disorders using high-content, single-cell-level automated microscopy assays. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Human induced pluripotent stem cells labeled with fluorescent magnetic nanoparticles for targeted imaging and hyperthermia therapy for gastric cancer.

PubMed

Li, Chao; Ruan, Jing; Yang, Meng; Pan, Fei; Gao, Guo; Qu, Su; Shen, You-Lan; Dang, Yong-Jun; Wang, Kan; Jin, Wei-Lin; Cui, Da-Xiang

2015-09-01

Human induced pluripotent stem (iPS) cells exhibit great potential for generating functional human cells for medical therapies. In this paper, we report for use of human iPS cells labeled with fluorescent magnetic nanoparticles (FMNPs) for targeted imaging and synergistic therapy of gastric cancer cells in vivo. Human iPS cells were prepared and cultured for 72 h. The culture medium was collected, and then was co-incubated with MGC803 cells. Cell viability was analyzed by the MTT method. FMNP-labeled human iPS cells were prepared and injected into gastric cancer-bearing nude mice. The mouse model was observed using a small-animal imaging system. The nude mice were irradiated under an external alternating magnetic field and evaluated using an infrared thermal mapping instrument. Tumor sizes were measured weekly. iPS cells and the collected culture medium inhibited the growth of MGC803 cells. FMNP-labeled human iPS cells targeted and imaged gastric cancer cells in vivo, as well as inhibited cancer growth in vivo through the external magnetic field. FMNP-labeled human iPS cells exhibit considerable potential in applications such as targeted dual-mode imaging and synergistic therapy for early gastric cancer.

Generation of male differentiated germ cells from various types of stem cells.

PubMed

Hou, Jingmei; Yang, Shi; Yang, Hao; Liu, Yang; Liu, Yun; Hai, Yanan; Chen, Zheng; Guo, Ying; Gong, Yuehua; Gao, Wei-Qiang; Li, Zheng; He, Zuping

2014-06-01

Infertility is a major and largely incurable disease caused by disruption and loss of germ cells. It affects 10-15% of couples, and male factor accounts for half of the cases. To obtain human male germ cells 'especially functional spermatids' is essential for treating male infertility. Currently, much progress has been made on generating male germ cells, including spermatogonia, spermatocytes, and spermatids, from various types of stem cells. These germ cells can also be used in investigation of the pathology of male infertility. In this review, we focused on advances on obtaining male differentiated germ cells from different kinds of stem cells, with an emphasis on the embryonic stem (ES) cells, the induced pluripotent stem (iPS) cells, and spermatogonial stem cells (SSCs). We illustrated the generation of male differentiated germ cells from ES cells, iPS cells and SSCs, and we summarized the phenotype for these stem cells, spermatocytes and spermatids. Moreover, we address the differentiation potentials of ES cells, iPS cells and SSCs. We also highlight the advantages, disadvantages and concerns on derivation of the differentiated male germ cells from several types of stem cells. The ability of generating mature and functional male gametes from stem cells could enable us to understand the precise etiology of male infertility and offer an invaluable source of autologous male gametes for treating male infertility of azoospermia patients. © 2014 Society for Reproduction and Fertility.

Human Induced Pluripotent Stem Cell-Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation.

PubMed

Sharmin, Sazia; Taguchi, Atsuhiro; Kaku, Yusuke; Yoshimura, Yasuhiro; Ohmori, Tomoko; Sakuma, Tetsushi; Mukoyama, Masashi; Yamamoto, Takashi; Kurihara, Hidetake; Nishinakamura, Ryuichi

2016-06-01

Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator-like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm-like structures. Microarray analysis of sorted iPS cell-derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell-derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell-derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases. Copyright © 2016 by the American Society of Nephrology.

MHC-matched induced pluripotent stem cells can attenuate cellular and humoral immune responses but are still susceptible to innate immunity in pigs.

PubMed

Mizukami, Yoshihisa; Abe, Tomoyuki; Shibata, Hiroaki; Makimura, Yukitoshi; Fujishiro, Shuh-hei; Yanase, Kimihide; Hishikawa, Shuji; Kobayashi, Eiji; Hanazono, Yutaka

2014-01-01

Recent studies have revealed negligible immunogenicity of induced pluripotent stem (iPS) cells in syngeneic mice and in autologous monkeys. Therefore, human iPS cells would not elicit immune responses in the autologous setting. However, given that human leukocyte antigen (HLA)-matched allogeneic iPS cells would likely be used for medical applications, a more faithful model system is needed to reflect HLA-matched allogeneic settings. Here we examined whether iPS cells induce immune responses in the swine leukocyte antigen (SLA)-matched setting. iPS cells were generated from the SLA-defined C1 strain of Clawn miniature swine, which were confirmed to develop teratomas in mice, and transplanted into the testes (n = 4) and ovary (n = 1) of C1 pigs. No teratomas were found in pigs on 47 to 125 days after transplantation. A Mixed lymphocyte reaction revealed that T-cell responses to the transplanted MHC-matched (C1) iPS cells were significantly lower compared to allogeneic cells. The humoral immune responses were also attenuated in the C1-to-C1 setting. More importantly, even MHC-matched iPS cells were susceptible to innate immunity, NK cells and serum complement. iPS cells lacked the expression of SLA class I and sialic acids. The in vitro cytotoxic assay showed that C1 iPS cells were targeted by NK cells and serum complement of C1. In vivo, the C1 iPS cells developed larger teratomas in NK-deficient NOG (T-B-NK-) mice (n = 10) than in NK-competent NOD/SCID (T-B-NK+) mice (n = 8) (p<0.01). In addition, C1 iPS cell failed to form teratomas after incubation with the porcine complement-active serum. Taken together, MHC-matched iPS cells can attenuate cellular and humoral immune responses, but still susceptible to innate immunity in pigs.

Induced pluripotent stem cells for regenerative medicine.

PubMed

Hirschi, Karen K; Li, Song; Roy, Krishnendu

2014-07-11

With the discovery of induced pluripotent stem (iPS) cells, it is now possible to convert differentiated somatic cells into multipotent stem cells that have the capacity to generate all cell types of adult tissues. Thus, there is a wide variety of applications for this technology, including regenerative medicine, in vitro disease modeling, and drug screening/discovery. Although biological and biochemical techniques have been well established for cell reprogramming, bioengineering technologies offer novel tools for the reprogramming, expansion, isolation, and differentiation of iPS cells. In this article, we review these bioengineering approaches for the derivation and manipulation of iPS cells and focus on their relevance to regenerative medicine.

Cross talk between metabotropic and ionotropic glutamate receptor-mediated signaling in parallel fiber-induced inositol 1,4,5-trisphosphate production in cerebellar Purkinje cells.

PubMed

Okubo, Yohei; Kakizawa, Sho; Hirose, Kenzo; Iino, Masamitsu

2004-10-27

In many excitatory glutamatergic synapses, both ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs) are closely distributed on the postsynaptic membrane. However, the functional significance of the close distribution of the two types of glutamate receptors has not been fully clarified. In this study, we examined the functional interaction between iGluR and mGluR at parallel fiber (PF)--> Purkinje cell synapses in the generation of inositol 1,4,5-trisphosphate (IP3), a key second messenger that regulates many important cellular functions. We visualized local IP3 dynamics in Purkinje cells using the green fluorescent protein-tagged pleckstrin homology domain (GFP-PHD) as a fluorescent IP3 probe. Purkinje cells were transduced with Sindbis virus encoding GFP-PHD and imaged with a two-photon laser scanning microscope. Translocation of GFP-PHD from the plasma membrane to the cytoplasm attributable to an increase in IP3 concentration was observed on PF stimulation in fine dendrites of Purkinje cells. Surprisingly, this PF-induced IP3 production was blocked not only by the group I mGluR antagonist but also by the AMPA receptor (AMPAR) antagonist. The PF-induced IP3 production was blocked by either the inhibition of G-protein activation by GDP-betaS or intracellular Ca2+ buffering by BAPTA. These results show that IP3 production is mediated cooperatively by group I mGluR and AMPAR through G-protein activation and Ca2+ influx at PF--> Purkinje cell synapses, identifying the robust cross talk between iGluR and mGluR for the generation of IP3 signals.

Induction of pluripotent stem cells from fibroblast cultures.

PubMed

Takahashi, Kazutoshi; Okita, Keisuke; Nakagawa, Masato; Yamanaka, Shinya

2007-01-01

Clinical application of embryonic stem (ES) cells faces difficulties regarding use of embryos, as well as tissue rejection after implantation. One way to circumvent these issues is to generate pluripotent stem cells directly from somatic cells. Somatic cells can be reprogrammed to an embryonic-like state by the injection of a nucleus into an enucleated oocyte or by fusion with ES cells. However, little is known about the mechanisms underlying these processes. We have recently shown that the combination of four transcription factors can generate ES-like pluripotent stem cells directly from mouse fibroblast cultures. The cells, named induced pluripotent stem (iPS) cells, can be differentiated into three germ layers and committed to chimeric mice. Here we describe detailed methods and tips for the generation of iPS cells.

Cerebral organoids derived from Sandhoff disease-induced pluripotent stem cells exhibit impaired neurodifferentiation[S

PubMed Central

Allende, Maria L.; Cook, Emily K.; Larman, Bridget C.; Nugent, Adrienne; Brady, Jacqueline M.; Golebiowski, Diane; Sena-Esteves, Miguel; Tifft, Cynthia J.

2018-01-01

Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of β-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB-corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB-corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB-corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses. PMID:29358305

Cerebral organoids derived from Sandhoff disease-induced pluripotent stem cells exhibit impaired neurodifferentiation.

PubMed

Allende, Maria L; Cook, Emily K; Larman, Bridget C; Nugent, Adrienne; Brady, Jacqueline M; Golebiowski, Diane; Sena-Esteves, Miguel; Tifft, Cynthia J; Proia, Richard L

2018-03-01

Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of β-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB -corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB -corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB -corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses.

Differentiation of Odontoblast-Like Cells From Mouse Induced Pluripotent Stem Cells by Pax9 and Bmp4 Transfection.

PubMed

Seki, Daisuke; Takeshita, Nobuo; Oyanagi, Toshihito; Sasaki, Shutaro; Takano, Ikuko; Hasegawa, Masakazu; Takano-Yamamoto, Teruko

2015-09-01

The field of tooth regeneration has progressed in recent years, and human tooth regeneration could become viable in the future. Because induced pluripotent stem (iPS) cells can differentiate into odontogenic cells given appropriate conditions, iPS cells are a potential cell source for tooth regeneration. However, a definitive method to induce iPS cell-derived odontogenic cells has not been established. We describe a novel method of odontoblast differentiation from iPS cells using gene transfection. We generated mouse iPS cell-derived neural crest-like cells (iNCLCs), which exhibited neural crest markers. Next, we differentiated iNCLCs into odontoblast-like cells by transfection of Pax9 and Bmp4 expression plasmids. Exogenous Pax9 upregulated expression of Msx1 and dentin matrix protein 1 (Dmp1) in iNCLCs but not bone morphogenetic protein 4 (Bmp4) or dentin sialophosphoprotein (Dspp). Exogenous Bmp4 upregulated expression of Msx1, Dmp1, and Dspp in iNCLCs, but not Pax9. Moreover, cotransfection of Pax9 and Bmp4 plasmids in iNCLCs revealed a higher expression of Pax9 than when Pax9 plasmid was used alone. In contrast, exogenous Pax9 downregulated Bmp4 overexpression. Cotransfection of Pax9 and Bmp4 synergistically upregulated Dmp1 expression; however, Pax9 overexpression downregulated exogenous Bmp4-induced Dspp expression. Together, these findings suggest that an interaction between exogenous Pax9- and Bmp4-induced signaling modulated Dmp1 and Dspp expression. In conclusion, transfection of Pax9 and Bmp4 expression plasmids in iNCLCs induced gene expression associated with odontoblast differentiation, suggesting that iNCLCs differentiated into odontoblast-like cells. The iPS cell-derived odontoblast-like cells could be a useful cell source for tooth regeneration. It has been reported that induced pluripotent stem (iPS) cells differentiate into odontogenic cells by administration of recombinant growth factors and coculture with odontogenic cells. Therefore, they can be potential cell sources for tooth regeneration. However, these previous methods still have problems, such as usage of other cell types, heterogeneity of differentiated cells, and tumorigenicity. In the present study, a novel method to differentiate iPS cells into odontoblast-like cells without tumorigenicity using gene transfection was established. It is an important advance in the establishment of efficient methods to generate homogeneous functional odontogenic cells derived from iPS cells. ©AlphaMed Press.

Potential of laryngeal muscle regeneration using induced pluripotent stem cell-derived skeletal muscle cells.

PubMed

Dirja, Bayu Tirta; Yoshie, Susumu; Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Nakamura, Ryosuke; Otsuki, Koshi; Nomoto, Yukio; Wada, Ikuo; Hazama, Akihiro; Omori, Koichi

2016-01-01

Conclusion Induced pluripotent stem (iPS) cells may be a new potential cell source for laryngeal muscle regeneration in the treatment of vocal fold atrophy after recurrent laryngeal nerve paralysis. Objectives Unilateral vocal fold paralysis can lead to degeneration, atrophy, and loss of force of the thyroarytenoid muscle. At present, there are some treatments such as thyroplasty, arytenoid adduction, and vocal fold injection. However, such treatments cannot restore reduced mass of the thyroarytenoid muscle. iPS cells have been recognized as supplying a potential resource for cell transplantation. The aim of this study was to assess the effectiveness of the use of iPS cells for the regeneration of laryngeal muscle through the evaluation of both in vitro and in vivo experiments. Methods Skeletal muscle cells were generated from tdTomato-labeled iPS cells using embryoid body formation. Differentiation into skeletal muscle cells was analyzed by gene expression and immunocytochemistry. The tdTomato-labeled iPS cell-derived skeletal muscle cells were transplanted into the left atrophied thyroarytenoid muscle. To evaluate the engraftment of these cells after transplantation, immunohistochemistry was performed. Results The tdTomato-labeled iPS cells were successfully differentiated into skeletal muscle cells through an in vitro experiment. These cells survived in the atrophied thyroarytenoid muscle after transplantation.

β-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs.

PubMed

Fujita, Atsushi; Uchida, Naoya; Haro-Mora, Juan J; Winkler, Thomas; Tisdale, John

2016-06-01

Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells represent a potential alternative source for red blood cell transfusion. However, when using traditional methods with embryoid bodies, ES cell-derived erythroid cells predominantly express embryonic type ɛ-globin, with lesser fetal type γ-globin and very little adult type β-globin. Furthermore, no β-globin expression is detected in iPS cell-derived erythroid cells. ES cell-derived sacs (ES sacs) have been recently used to generate functional platelets. Due to its unique structure, we hypothesized that ES sacs serve as hemangioblast-like progenitors capable to generate definitive erythroid cells that express β-globin. With our ES sac-derived erythroid differentiation protocol, we obtained ∼120 erythroid cells per single ES cell. Both primitive (ɛ-globin expressing) and definitive (γ- and β-globin expressing) erythroid cells were generated from not only ES cells but also iPS cells. Primitive erythropoiesis is gradually switched to definitive erythropoiesis during prolonged ES sac maturation, concurrent with the emergence of hematopoietic progenitor cells. Primitive and definitive erythroid progenitor cells were selected on the basis of glycophorin A or CD34 expression from cells within the ES sacs before erythroid differentiation. This selection and differentiation strategy represents an important step toward the development of in vitro erythroid cell production systems from pluripotent stem cells. Further optimization to improve expansion should be required for clinical application. Stem Cells 2016;34:1541-1552. © 2016 AlphaMed Press.

Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Ma, Ming-San; Kannan, Vishnu; de Vries, Anneriek E; Czepiel, Marcin; Wesseling, Evelyn M; Balasubramaniyan, Veerakumar; Kuijer, Roel; Vissink, Arjan; Copray, Sjef C V M; Raghoebar, Gerry M

2017-01-01

New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

ChIP-seq and ChIP-exo profiling of Pol II, H2A.Z, and H3K4me3 in human K562 cells.

PubMed

Mchaourab, Zenab F; Perreault, Andrea A; Venters, Bryan J

2018-03-06

The human K562 chronic myeloid leukemia cell line has long served as an experimental paradigm for functional genomic studies. To systematically and functionally annotate the human genome, the ENCODE consortium generated hundreds of functional genomic data sets, such as chromatin immunoprecipitation coupled to sequencing (ChIP-seq). While ChIP-seq analyses have provided tremendous insights into gene regulation, spatiotemporal insights were limited by a resolution of several hundred base pairs. ChIP-exonuclease (ChIP-exo) is a refined version of ChIP-seq that overcomes this limitation by providing higher precision mapping of protein-DNA interactions. To study the interplay of transcription initiation and chromatin, we profiled the genome-wide locations for RNA polymerase II (Pol II), the histone variant H2A.Z, and the histone modification H3K4me3 using ChIP-seq and ChIP-exo. In this Data Descriptor, we present detailed information on parallel experimental design, data generation, quality control analysis, and data validation. We discuss how these data lay the foundation for future analysis to understand the relationship between the occupancy of Pol II and nucleosome positions at near base pair resolution.

IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria.

PubMed

Baumgart, Meike; Luder, Kerstin; Grover, Shipra; Gätgens, Cornelia; Besra, Gurdyal S; Frunzke, Julia

2013-12-30

The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown. Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An ipsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Further studies revealed the absence of inositol-derived lipids in the cell wall and a complete loss of mycothiol biosynthesis. The phenotype of the C. glutamicum ipsA deletion mutant was complemented to different extend by homologs from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. Myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro. This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development.

Reprogramming fibroblasts into induced pluripotent stem cells with Bmi1

PubMed Central

Moon, Jai-Hee; Heo, June Seok; Kim, Jun Sung; Jun, Eun Kyoung; Lee, Jung Han; Kim, Aeree; Kim, Jonggun; Whang, Kwang Youn; Kang, Yong-Kook; Yeo, Seungeun; Lim, Hee-Joung; Han, Dong Wook; Kim, Dong-Wook; Oh, Sejong; Yoon, Byung Sun; Schöler, Hans R; You, Seungkwon

2011-01-01

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4. PMID:21709693

The PLC/IP3R/PKC Pathway is Required for Ethanol-enhanced GABA Release

PubMed Central

Kelm, M. Katherine; Weinberg, Richard J.; Criswell, Hugh E.; Breese, George R.

2010-01-01

Summary Research on the actions of ethanol at the GABAergic synapse has traditionally focused on postsynaptic mechanisms, but recent data demonstrate that ethanol also increases both evoked and spontaneous GABA release in many brain regions. Using whole-cell voltage-clamp recordings, we previously showed that ethanol increases spontaneous GABA release at the rat interneuron-Purkinje cell synapse. This presynaptic ethanol effect is dependent on calcium release from internal stores, possibly through activation of inositol 1,4,5-trisphosphate receptors (IP3Rs). After confirming that ethanol targets vesicular GABA release, in the present study we used electron microscopic immunohistochemistry to demonstrate that IP3Rs are located in presynaptic terminals of cerebellar interneurons. Activation of IP3Rs requires binding of IP3, generated through activation of phospholipase C (PLC). We find that the PLC antagonist edelfosine prevents ethanol from increasing spontaneous GABA release. Diacylglycerol generated by PLC and calcium released by activation of the IP3R activate protein kinase C (PKC). Ethanol-enhanced GABA release was blocked by two PKC antagonists, chelerythrine and calphostin C. When a membrane impermeable PKC antagonist, PKC (19-36), was delivered intracellularly to the postsynaptic neuron, ethanol continued to increase spontaneous GABA release. Overall, these results suggest that activation of the PLC/IP3R/PKC pathway is necessary for ethanol to increase spontaneous GABA release from presynaptic terminals onto Purkinje cells. PMID:20206640

Chicken Induced Pluripotent Stem Cells: Establishment and Characterization.

PubMed

Fuet, Aurelie; Pain, Bertrand

2017-01-01

In mammals, the introduction of the OSKM (Oct4, Sox2, Klf4, and c-Myc) genes into somatic cells has allowed generating induced pluripotent stem (iPS) cells. So far, this process has been only clearly demonstrated in mammals. Here, using chicken as an avian model, we describe a set of protocols allowing the establishment, characterization, maintenance, differentiation, and injection of putative reprogrammed chicken Induced Pluripotent Stem (iPS) cells.

Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation

PubMed Central

Sharmin, Sazia; Taguchi, Atsuhiro; Kaku, Yusuke; Yoshimura, Yasuhiro; Ohmori, Tomoko; Sakuma, Tetsushi; Mukoyama, Masashi; Yamamoto, Takashi; Kurihara, Hidetake

2016-01-01

Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro. These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm–like structures. Microarray analysis of sorted iPS cell–derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo. Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell–derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell–derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases. PMID:26586691

IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria

PubMed Central

2013-01-01

Background The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown. Results Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An ipsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Further studies revealed the absence of inositol-derived lipids in the cell wall and a complete loss of mycothiol biosynthesis. The phenotype of the C. glutamicum ipsA deletion mutant was complemented to different extend by homologs from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. Myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro. Conclusions This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development. PMID:24377418

Efficient biotechnological approach for lentiviral transduction of induced pluripotent stem cells.

PubMed

Zare, Mehrak; Soleimani, Masoud; Mohammadian, Mozhdeh; Akbarzadeh, Abolfazl; Havasi, Parvaneh; Zarghami, Nosratollah

2016-01-01

Induced pluripotent stem (iPS) cells are generated from differentiated adult somatic cells by reprogramming them. Unlimited self-renewal, and the potential to differentiate into any cell type, make iPS cells very promising candidates for basic and clinical research. Furthermore, iPS cells can be genetically manipulated for use as therapeutic tools. DNA can be introduced into iPS cells, using lentiviral vectors, which represent a helpful choice for efficient transduction and stable integration of transgenes. In this study, we compare two methods of lentiviral transduction of iPS cells, namely, the suspension method and the hanging drop method. In contrast to the conventional suspension method, in the hanging drop method, embryoid body (EB) formation and transduction occur concurrently. The iPS cells were cultured to form EBs, and then transduced with lentiviruses, using the conventional suspension method and the hanging drop method, to express miR-128 and green fluorescent protein (GFP). The number of transduced cells were assessed by fluorescent microscopy and flow cytometry. MTT assay and real-time PCR were performed to determine the cell viability and transgene expression, respectively. Morphologically, GFP+ cells were more detectable in the hanging drop method, and this finding was quantified by flow cytometric analysis. According to the results of the MTT assay, cell viability was considerably higher in the hanging drop method, and real-time PCR represented a higher relative expression of miR-128 in the iPS cells introduced with lentiviruses in drops. Altogether, it seems that lentiviral transduction of challenging iPS cells using the hanging drop method offers a suitable and sufficient strategy in their gene transfer, with less toxicity than the conventional suspension method.

Inositol pyrophosphates mediate the DNA-PK/ATM-p53 cell death pathway by regulating CK2 phosphorylation of Tti1/Tel2

PubMed Central

Rao, Feng; Cha, Jiyoung; Xu, Jing; Xu, Risheng; Vandiver, M. Scott; Tyagi, Richa; Tokhunts, Robert; Koldobskiy, Michael A.; Fu, Chenglai; Barrow, Roxanne; Wu, Mingxuan; Fiedler, Dorothea; Barrow, James C.; Snyder, Solomon H.

2014-01-01

The apoptotic actions of p53 require its phosphorylation by a family of phosphoinositide-3-kinase-related-kinases (PIKKs), which include DNA-PKcs and ATM. These kinases are stabilized by the TTT (Tel2, Tti1, Tti2) co-chaperone family, whose actions are mediated by CK2 phosphorylation. The inositol pyrophosphates, such as 5-diphosphoinositol pentakisphosphate (IP7), are generated by a family of inositol hexakisphosphate kinases (IP6Ks) of which IP6K2 has been implicated in p53-associated cell death. In the present study we report a novel apoptotic signaling cascade linking CK2, TTT, the PIKKs, and p53. We demonstrate that IP7, formed by IP6K2, binds CK2 to enhance its phosphorylation of the TTT complex thereby stabilizing DNA-PKcs and ATM. This process stimulates p53 phosphorylation at serine-15 to activate the cell death program in human cancer cells and in murine B cells. PMID:24657168

Investigation of tissue-specific expression and functions of MLF1-IP during development and in the immune system.

PubMed

Wang, Xuehai; Marcinkiewicz, Martin; Gatain, Yaned; Bouchard, Maxime; Mao, Jianning; Tremblay, Michel; Uetani, Noriko; Hanissian, Silva; Qi, Shijie; Wu, Jiangping; Luo, Hongyu

2013-01-01

Myeloid leukemia factor 1-interacting protein (MLF1-IP) has been found to exert functions in mitosis, although studies have been conducted only in cell lines up to now. To understand its roles during ontogeny and immunity, we analyzed its mRNA expression pattern by in situ hybridization and generated MLF1-IP gene knockout (KO) mice. MLF1-IP was expressed at elevated levels in most rudimentary tissues during the mid-gestation stage, between embryonic day 9.5 (e9.5) and e15.5. It declined afterwards in these tissues, but was very high in the testes and ovaries in adulthood. At post-natal day 10 (p10), the retina and cerebellum still expressed moderate MLF1-IP levels, although these tissues do not contain fast-proliferating cells at this stage. MLF1-IP expression in lymphoid organs, such as the thymus, lymph nodes, spleen and bone marrow, was high between e15.5 and p10, and decreased in adulthood. MLF1-IP KO embryos failed to develop beyond e6.5. On the other hand, MLF1-IP(+/-) mice were alive and fertile, with no obvious anomalies. Lymphoid organ size, weight, cellularity and cell sub-populations in MLF1-IP(+/-) mice were in the normal range. The functions of MLF1-IP(+/-) T cells and naïve CD4 cells, in terms of TCR-stimulated proliferation and Th1, Th17 and Treg cell differentiation in vitro, were comparable to those of wild type T cells. Our study demonstrates that MLF1-IP performs unique functions during mouse embryonic development, particularly around e6.5, when there was degeneration of epiblasts. However, the cells could proliferate dozens of rounds without MLF1-IP. MLF1-IP expression at about 50% of its normal level is sufficient to sustain mice life and the development of their immune system without apparent abnormalities. Our results also raise an intriguing question that MLF1-IP might have additional functions unrelated to cell proliferation.

Investigation of Tissue-Specific Expression and Functions of MLF1-IP during Development and in the Immune System

PubMed Central

Wang, Xuehai; Marcinkiewicz, Martin; Gatain, Yaned; Bouchard, Maxime; Mao, Jianning; Tremblay, Michel; Uetani, Noriko; Hanissian, Silva; Qi, Shijie; Wu, Jiangping; Luo, Hongyu

2013-01-01

Myeloid leukemia factor 1-interacting protein (MLF1-IP) has been found to exert functions in mitosis, although studies have been conducted only in cell lines up to now. To understand its roles during ontogeny and immunity, we analyzed its mRNA expression pattern by in situ hybridization and generated MLF1-IP gene knockout (KO) mice. MLF1-IP was expressed at elevated levels in most rudimentary tissues during the mid-gestation stage, between embryonic day 9.5 (e9.5) and e15.5. It declined afterwards in these tissues, but was very high in the testes and ovaries in adulthood. At post-natal day 10 (p10), the retina and cerebellum still expressed moderate MLF1-IP levels, although these tissues do not contain fast-proliferating cells at this stage. MLF1-IP expression in lymphoid organs, such as the thymus, lymph nodes, spleen and bone marrow, was high between e15.5 and p10, and decreased in adulthood. MLF1-IP KO embryos failed to develop beyond e6.5. On the other hand, MLF1-IP+/− mice were alive and fertile, with no obvious anomalies. Lymphoid organ size, weight, cellularity and cell sub-populations in MLF1-IP+/− mice were in the normal range. The functions of MLF1-IP+/− T cells and naïve CD4 cells, in terms of TCR-stimulated proliferation and Th1, Th17 and Treg cell differentiation in vitro, were comparable to those of wild type T cells. Our study demonstrates that MLF1-IP performs unique functions during mouse embryonic development, particularly around e6.5, when there was degeneration of epiblasts. However, the cells could proliferate dozens of rounds without MLF1-IP. MLF1-IP expression at about 50% of its normal level is sufficient to sustain mice life and the development of their immune system without apparent abnormalities. Our results also raise an intriguing question that MLF1-IP might have additional functions unrelated to cell proliferation. PMID:23724000

Multiphoton autofluorescence lifetime imaging of induced pluripotent stem cells

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada

2017-06-01

The multiphoton fluorescence lifetime imaging tomograph MPTflex with its flexible 360-deg scan head, articulated arm, and tunable femtosecond laser source was employed to study induced pluripotent stem cell (iPS) cultures. Autofluorescence (AF) lifetime imaging was performed with 250-ps temporal resolution and submicron spatial resolution using time-correlated single-photon counting. The two-photon excited AF was based on the metabolic coenzymes NAD(P)H and flavin adenine dinucleotide/flavoproteins. iPS cells generated from mouse embryonic fibroblasts (MEFs) and cocultured with growth-arrested MEFs as feeder cells have been studied. Significant differences on AF lifetime signatures were identified between iPS and feeder cells as well as between their differentiating counterparts.

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy.

PubMed

Friedrichs, Stephanie; Malan, Daniela; Voss, Yvonne; Sasse, Philipp

2015-01-08

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening.

Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis

PubMed Central

Son, Aran; Kim, Min Seuk; Jo, Hae; Byun, Hae Mi

2012-01-01

The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca2+ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP3 and evaluated IP3-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca2+ signaling proteins such as IP3 receptors (IP3Rs), plasma membrane Ca2+ ATPase, and sarco/endoplasmic reticulum Ca2+ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP3 was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP3 levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP3Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP3Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP3 levels and the IP3-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis. PMID:22416217

Epigenome analysis of pluripotent stem cells

PubMed Central

Ricupero, Christopher L.; Swerdel, Mavis R.; Hart, Ronald P.

2015-01-01

Summary Mis-regulation of gene expression due to epigenetic abnormalities has been linked with complex genetic disorders, psychiatric illness and cancer. In addition, the dynamic epigenetic changes that occur in pluripotent stem cells are believed to impact regulatory networks essential for proper lineage development. Chromatin immunoprecipitation (ChIP) is a technique used to isolate and enrich chromatin fragments using antibodies against specific chromatin modifications, such as DNA binding proteins or covalent histone modifications. Until recently, many ChIP protocols required millions of cells for each immunoprecipitation. This severely limited analysis of rare cell populations or post-mitotic, differentiated cell lines. Here, we describe a low cell number ChIP protocol with next generation sequencing and analysis, that has the potential to uncover novel epigenetic regulatory pathways that were previously difficult or impossible to obtain. PMID:23546758

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples.

PubMed

Rehimi, Rizwan; Bartusel, Michaela; Solinas, Francesca; Altmüller, Janine; Rada-Iglesias, Alvaro

2017-08-29

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 10 4 - 5 x 10 5 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Modeling Alzheimer’s disease with human induced pluripotent stem (iPS) cells

PubMed Central

Mungenast, Alison E.; Siegert, Sandra; Tsai, Li-Huei

2018-01-01

In the last decade, induced pluripotent stem (iPS) cells have revolutionized the utility of human in vitro models of neurological disease. The iPS-derived and differentiated cells allow researchers to study the impact of a distinct cell type in health and disease as well as performing therapeutic drug screens on a human genetic background. In particular, clinical trials for Alzheimer’s disease (AD) have been often failing. Two of the potential reasons are first, the species gap involved in proceeding from initial discoveries in rodent models to human studies, and second, an unsatisfying patient stratification, meaning subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers. iPS cells overcome this obstacles and will improve our understanding of disease subtypes in AD. They allow researchers conducting in depth characterization of neural cells from both familial and sporadic AD patients as well as preclinical screens on human cells. In this review, we briefly outline the status quo of iPS cell research in neurological diseases along with the general advantages and pitfalls of these models. We summarize how genome-editing techniques such as CRISPR/Cas will allow researchers to reduce the problem of genomic variability inherent to human studies, followed by recent iPS cell studies relevant to AD. We then focus on current techniques for the differentiation of iPS cells into neural cell types that are relevant to AD research. Finally, we discuss how the generation of three-dimensional cell culture systems will be important for understanding AD phenotypes in a complex cellular milieu, and how both two- and three-dimensional iPS cell models can provide platforms for drug discovery and translational studies into the treatment of AD. PMID:26657644

Modeling Alzheimer's disease with human induced pluripotent stem (iPS) cells.

PubMed

Mungenast, Alison E; Siegert, Sandra; Tsai, Li-Huei

2016-06-01

In the last decade, induced pluripotent stem (iPS) cells have revolutionized the utility of human in vitro models of neurological disease. The iPS-derived and differentiated cells allow researchers to study the impact of a distinct cell type in health and disease as well as performing therapeutic drug screens on a human genetic background. In particular, clinical trials for Alzheimer's disease (AD) have been failing. Two of the potential reasons are first, the species gap involved in proceeding from initial discoveries in rodent models to human studies, and second, an unsatisfying patient stratification, meaning subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers. iPS cells overcome this obstacles and will improve our understanding of disease subtypes in AD. They allow researchers conducting in depth characterization of neural cells from both familial and sporadic AD patients as well as preclinical screens on human cells. In this review, we briefly outline the status quo of iPS cell research in neurological diseases along with the general advantages and pitfalls of these models. We summarize how genome-editing techniques such as CRISPR/Cas9 will allow researchers to reduce the problem of genomic variability inherent to human studies, followed by recent iPS cell studies relevant to AD. We then focus on current techniques for the differentiation of iPS cells into neural cell types that are relevant to AD research. Finally, we discuss how the generation of three-dimensional cell culture systems will be important for understanding AD phenotypes in a complex cellular milieu, and how both two- and three-dimensional iPS cell models can provide platforms for drug discovery and translational studies into the treatment of AD. Copyright © 2015 Elsevier Inc. All rights reserved.

MicroRNA‐199b Modulates Vascular Cell Fate During iPS Cell Differentiation by Targeting the Notch Ligand Jagged1 and Enhancing VEGF Signaling

PubMed Central

Chen, Ting; Kelaini, Sophia; Cochrane, Amy; Guha, Shaunta T.; Hu, Yanhua; Stitt, Alan W.; Xu, Qingbo

2015-01-01

Abstract Aims: Recent ability to derive endothelial cells (ECs) from induced pluripotent stem (iPS) cells holds a great therapeutic potential for personalized medicine and stem cell therapy. We aimed that better understanding of the complex molecular signals that are evoked during iPS cell differentiation toward ECs may allow specific targeting of their activities to enhance cell differentiation and promote tissue regeneration. Methods and Results: In this study, we have generated mouse iPS cells from fibroblasts using established protocol. When iPS cells were cultivated on type IV mouse collagen‐coated dishes in differentiation medium, cell differentiation toward vascular lineages were observed. To study the molecular mechanisms of iPS cell differentiation, we found that miR‐199b is involved in EC differentiation. A step‐wise increase in expression of miR‐199 was detected during EC differentiation. Notably, miR‐199b targeted the Notch ligand JAG1, resulting in vascular endothelial growth factor (VEGF) transcriptional activation and secretion through the transcription factor STAT3. Upon shRNA‐mediated knockdown of the Notch ligand JAG1, the regulatory effect of miR‐199b was ablated and there was robust induction of STAT3 and VEGF during EC differentiation. Knockdown of JAG1 also inhibited miR‐199b‐mediated inhibition of iPS cell differentiation toward smooth muscle markers. Using the in vitro tube formation assay and implanted Matrigel plugs, in vivo, miR‐199b also regulated VEGF expression and angiogenesis. Conclusions: This study indicates a novel role for miR‐199b as a regulator of the phenotypic switch during vascular cell differentiation derived from iPS cells by regulating critical signaling angiogenic responses. Stem Cells 2015;33:1405–1418 PMID:25535084

Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model.

PubMed

Yang, Jing; Lam, Dang Hoang; Goh, Sally Sallee; Lee, Esther Xingwei; Zhao, Ying; Tay, Felix Chang; Chen, Can; Du, Shouhui; Balasundaram, Ghayathri; Shahbazi, Mohammad; Tham, Chee Kian; Ng, Wai Hoe; Toh, Han Chong; Wang, Shu

2012-05-01

Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs. Copyright © 2012 AlphaMed Press.

Generation of induced pluripotent stem cells derived from an autosomal dominant polycystic kidney disease patient with a p.Ser1457fs mutation in PKD1.

PubMed

Huang, Ching-Ying; Ho, Ming-Ching; Lee, Jia-Jung; Hwang, Daw-Yang; Ko, Hui-Wen; Cheng, Yu-Che; Hsu, Yu-Hung; Lu, Huai-En; Chen, Hung-Chun; Hsieh, Patrick C H

2017-10-01

Autosomal dominant polycystic kidney disease is one of the most prevalent forms of inherited cystic kidney disease, and can be characterized by kidney cyst formation and enlargement. Here we report the generation of a Type 1 ADPKD disease iPS cell line, IBMS-iPSC-012-12, which retains the conserved deletion of PKD1, normal karyotype and exhibits the properties of pluripotent stem cells such as ES-like morphology, expression of pluripotent markers and capacity to differentiate into all three germ layers. Our results show that we have successfully generated a patient-specific iPS cell line with a mutation in PKD1 for study of renal disease pathophysiology. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Scalable Electrophysiological Investigation of iPS Cell-Derived Cardiomyocytes Obtained by a Lentiviral Purification Strategy

PubMed Central

Friedrichs, Stephanie; Malan, Daniela; Voss, Yvonne; Sasse, Philipp

2015-01-01

Disease-specific induced pluripotent stem (iPS) cells can be generated from patients and differentiated into functional cardiomyocytes for characterization of the disease and for drug screening. In order to obtain pure cardiomyocytes for automated electrophysiological investigation, we here report a novel non-clonal purification strategy by using lentiviral gene transfer of a puromycin resistance gene under the control of a cardiac-specific promoter. We have applied this method to our previous reported wild-type and long QT syndrome 3 (LQTS 3)-specific mouse iPS cells and obtained a pure cardiomyocyte population. These cells were investigated by action potential analysis with manual and automatic planar patch clamp technologies, as well as by recording extracellular field potentials using a microelectrode array system. Action potentials and field potentials showed the characteristic prolongation at low heart rates in LQTS 3-specific, but not in wild-type iPS cell-derived cardiomyocytes. Hence, LQTS 3-specific cardiomyocytes can be purified from iPS cells with a lentiviral strategy, maintain the hallmarks of the LQTS 3 disease and can be used for automated electrophysiological characterization and drug screening. PMID:26237021

Human IP10-scFv and DC-induced CTL synergistically inhibit the growth of glioma in a xenograft model.

PubMed

Wang, Xuan; Zhang, Fang-Cheng; Zhao, Hong-Yang; Lu, Xiao-Ling; Sun, Yun; Xiong, Zhi-Yong; Jiang, Xiao-Bing

2014-08-01

The epidermal growth factor receptor (EGFR) mutant of EGFRvIII is highly expressed in glioma cells, and the EGFRvIII-specific dendritic cell (DC)-induced tumor antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) may hold promise in cancer immunotherapy. Interferon (IFN)-γ-inducible protein (IP)-10 (IP-10) is a potent inhibitor of angiogenesis and can recruit CXCR3(+) T cells, including CD8(+) T cells, which are important for the control of tumor growth. In this study, we assessed if the combination of IP10-EGFRvIIIscFv with DC-induced CTLs would improve the therapeutic antitumor efficacy. IP10-scFv was generated by linking the human IP-10 gene with the DNA fragment for anti-EGFRvIIIscFv with a (Gly4Ser)3 flexible linker, purified by affinity chromatography, and characterized for its anti-EGFRvIII immunoreactivity and chemotactic activity. DCs were isolated from human peripheral blood monocyte cells and pulsed with EGFRvIII-peptide, then co-cultured with autologous CD8(+) T cells. BALB/c-nu mice were inoculated with human glioma U87-EGFRvIII cells in the brain and treated intracranially with IP10-scFv and/or intravenously with DC-induced CTLs for evaluating the therapeutic effect. Treatment with both IP10-scFv and EGFRvIII peptide-pulsed, DC-induced CTL synergistically inhibited the growth of glioma and prolonged the survival of tumor-bearing mice, which was accompanied by the inhibition of tumor angiogenesis and enhancement of cytotoxicity, thereby increasing the numbers of brain-infiltrating lymphocytes (BILs) and prolonging the residence time of CTLs in the tumor.

Modelling IRF8 Deficient Human Hematopoiesis and Dendritic Cell Development with Engineered iPS Cells.

PubMed

Sontag, Stephanie; Förster, Malrun; Qin, Jie; Wanek, Paul; Mitzka, Saskia; Schüler, Herdit M; Koschmieder, Steffen; Rose-John, Stefan; Seré, Kristin; Zenke, Martin

2017-04-01

Human induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers, including hematopoietic stem cells and their progeny. Interferon regulatory factor 8 (IRF8) is a transcription factor, which acts in hematopoiesis as lineage determining factor for myeloid cells, including dendritic cells (DC). Autosomal recessive or dominant IRF8 mutations occurring in patients cause severe monocytic and DC immunodeficiency. To study IRF8 in human hematopoiesis we generated human IRF8-/- iPS cells and IRF8-/- embryonic stem (ES) cells using RNA guided CRISPR/Cas9n genome editing. Upon induction of hematopoietic differentiation, we demonstrate that IRF8 is dispensable for iPS cell and ES cell differentiation into hemogenic endothelium and for endothelial-to-hematopoietic transition, and thus development of hematopoietic progenitors. We differentiated iPS cell and ES cell derived progenitors into CD141+ cross-presenting cDC1 and CD1c+ classical cDC2 and CD303+ plasmacytoid DC (pDC). We found that IRF8 deficiency compromised cDC1 and pDC development, while cDC2 development was largely unaffected. Additionally, in an unrestricted differentiation regimen, IRF8-/- iPS cells and ES cells exhibited a clear bias toward granulocytes at the expense of monocytes. IRF8-/- DC showed reduced MHC class II expression and were impaired in cytokine responses, migration, and antigen presentation. Taken together, we engineered a human IRF8 knockout model that allows studying molecular mechanisms of human immunodeficiencies in vitro, including the pathophysiology of IRF8 deficient DC. Stem Cells 2017;35:898-908. © 2017 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background.

PubMed

Carstea, Ana Claudia; Pirity, Melinda K; Dinnyes, Andras

2009-12-31

In mice, gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics. This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression ("knock-out"), or to introduce mutant or modified alleles at the locus of interest ("knock-in"). The efficacy of producing live, transgenic mice challenges our understanding of this complex process, and of the factors which influence germline competence of embryonic stem cell lines. Increasingly, evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell (ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly strain-dependent and it may also influence the germline transmission capability. Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential. There are a number of options for generating chimeras from ES cells (ES chimera mice); however, each method has its advantages and disadvantages. Recent developments in induced pluripotent stem (iPS) cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera technologies. The aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell lines. The most recent methods for generating specifically ES and iPS chimera mice, including the advantages and disadvantages of each method are also discussed.

The directed differentiation of human iPS cells into kidney podocytes.

PubMed

Song, Bi; Smink, Alexandra M; Jones, Christina V; Callaghan, Judy M; Firth, Stephen D; Bernard, Claude A; Laslett, Andrew L; Kerr, Peter G; Ricardo, Sharon D

2012-01-01

The loss of glomerular podocytes is a key event in the progression of chronic kidney disease resulting in proteinuria and declining function. Podocytes are slow cycling cells that are considered terminally differentiated. Here we provide the first report of the directed differentiation of induced pluripotent stem (iPS) cells to generate kidney cells with podocyte features. The iPS-derived podocytes share a morphological phenotype analogous with cultured human podocytes. Following 10 days of directed differentiation, iPS podocytes had an up-regulated expression of mRNA and protein localization for podocyte markers including synaptopodin, nephrin and Wilm's tumour protein (WT1), combined with a down-regulation of the stem cell marker OCT3/4. In contrast to human podocytes that become quiescent in culture, iPS-derived cells maintain a proliferative capacity suggestive of a more immature phenotype. The transduction of iPS podocytes with fluorescent labeled-talin that were immunostained with podocin showed a cytoplasmic contractile response to angiotensin II (AII). A permeability assay provided functional evidence of albumin uptake in the cytoplasm of iPS podocytes comparable to human podocytes. Moreover, labeled iPS-derived podocytes were found to integrate into reaggregated metanephric kidney explants where they incorporated into developing glomeruli and co-expressed WT1. This study establishes the differentiation of iPS cells to kidney podocytes that will be useful for screening new treatments, understanding podocyte pathogenesis, and offering possibilities for regenerative medicine.

Differential gene expression profiles in neurons generated from lymphoblastoid B-cell line-derived iPS cells from monozygotic twin cases with treatment-resistant schizophrenia and discordant responses to clozapine.

PubMed

Nakazawa, Takanobu; Kikuchi, Masataka; Ishikawa, Mitsuru; Yamamori, Hidenaga; Nagayasu, Kazuki; Matsumoto, Takuya; Fujimoto, Michiko; Yasuda, Yuka; Fujiwara, Mikiya; Okada, Shota; Matsumura, Kensuke; Kasai, Atsushi; Hayata-Takano, Atsuko; Shintani, Norihito; Numata, Shusuke; Takuma, Kazuhiro; Akamatsu, Wado; Okano, Hideyuki; Nakaya, Akihiro; Hashimoto, Hitoshi; Hashimoto, Ryota

2017-03-01

Schizophrenia is a chronic psychiatric disorder with complex genetic and environmental origins. While many antipsychotics have been demonstrated as effective in the treatment of schizophrenia, a substantial number of schizophrenia patients are partially or fully unresponsive to the treatment. Clozapine is the most effective antipsychotic drug for treatment-resistant schizophrenia; however, clozapine has rare but serious side-effects. Furthermore, there is inter-individual variability in the drug response to clozapine treatment. Therefore, the identification of the molecular mechanisms underlying the action of clozapine and drug response predictors is imperative. In the present study, we focused on a pair of monozygotic twin cases with treatment-resistant schizophrenia, in which one twin responded well to clozapine treatment and the other twin did not. Using induced pluripotent stem (iPS) cell-based technology, we generated neurons from iPS cells derived from these patients and subsequently performed RNA-sequencing to compare the transcriptome profiles of the mock or clozapine-treated neurons. Although, these iPS cells similarly differentiated into neurons, several genes encoding homophilic cell adhesion molecules, such as protocadherin genes, showed differential expression patterns between these two patients. These results, which contribute to the current understanding of the molecular mechanisms of clozapine action, establish a new strategy for the use of monozygotic twin studies in schizophrenia research. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Transdifferentiation and reprogramming: Overview of the processes, their similarities and differences.

PubMed

Cieślar-Pobuda, Artur; Knoflach, Viktoria; Ringh, Mikael V; Stark, Joachim; Likus, Wirginia; Siemianowicz, Krzysztof; Ghavami, Saeid; Hudecki, Andrzej; Green, Jason L; Łos, Marek J

2017-07-01

Reprogramming, or generation of induced pluripotent stem (iPS) cells (functionally similar to embryonic stem cells or ES cells) by the use of transcription factors (typically: Oct3/4, Sox2, c-Myc, Klf4) called "Yamanaka factors" (OSKM), has revolutionized regenerative medicine. However, factors used to induce stemness are also overexpressed in cancer. Both, ES cells and iPS cells cause teratoma formation when injected to tissues. This raises a safety concern for therapies based on iPS derivates. Transdifferentiation (lineage reprogramming, or -conversion), is a process in which one mature, specialized cell type changes into another without entering a pluripotent state. This process involves an ectopic expression of transcription factors and/or other stimuli. Unlike in the case of reprogramming, tissues obtained by this method do not carry the risk of subsequent teratomagenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Conference Scene: Induced pluripotent cells: a new path for regenerative medicine. 7 October 2010, BioPark, Welwyn Garden City, Hertfordshire, UK.

PubMed

Crutzen, Hélène S G

2011-01-01

Embryonic stem cells and induced pluripotent stem (iPS) cells, which are embryonic stem-like cells derived from adult tissues, have the broadest differentiation potential. These cells are unique in their ability to self-renew, to be maintained in an undifferentiated state for long periods of culturing and to give rise to many different cell lineages including germ-line cells. They therefore represent an invaluable tool for facilitating research towards the realization of regenerative medicine. The recent developments in embryonic stem cell and iPS cell technology have allowed human cell models to be developed that will hopefully provide novel platforms for disease analysis not only at the basic science level, but also for drug discovery and screening, and other clinical applications. This 1-day conference, chaired by Professor Peter Andrews from the University of Sheffield, UK, and Dr Chris Denning from the University of Nottingham, UK, focused on generation of iPS cells, their differentiation into specific fates and applications to disease modeling. It consisted of 11 talks by UK-based and international researchers, and three posters; Ms Azra Fatima from Cologne University, Germany, won the competition for her poster on the derivation of iPS cells from a patient with arrhythmogenic right ventricular cardiomyopathy.

3D spheroid culture of hESC/hiPSC-derived hepatocyte-like cells for drug toxicity testing.

PubMed

Takayama, Kazuo; Kawabata, Kenji; Nagamoto, Yasuhito; Kishimoto, Keisuke; Tashiro, Katsuhisa; Sakurai, Fuminori; Tachibana, Masashi; Kanda, Katsuhiro; Hayakawa, Takao; Furue, Miho Kusuda; Mizuguchi, Hiroyuki

2013-02-01

Although it is expected that hepatocyte-like cells differentiated from human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells will be utilized in drug toxicity testing, the actual applicability of hepatocyte-like cells in this context has not been well examined so far. To generate mature hepatocyte-like cells that would be applicable for drug toxicity testing, we established a hepatocyte differentiation method that employs not only stage-specific transient overexpression of hepatocyte-related transcription factors but also a three-dimensional spheroid culture system using a Nanopillar Plate. We succeeded in establishing protocol that could generate more matured hepatocyte-like cells than our previous protocol. In addition, our hepatocyte-like cells could sensitively predict drug-induced hepatotoxicity, including reactive metabolite-mediated toxicity. In conclusion, our hepatocyte-like cells differentiated from human ES cells or iPS cells have potential to be applied in drug toxicity testing. Copyright © 2012 Elsevier Ltd. All rights reserved.

Comparison of efficiency of terminal differentiation of oligodendrocytes from induced pluripotent stem cells versus embryonic stem cells in vitro.

PubMed

Tokumoto, Yasuhito; Ogawa, Shinichiro; Nagamune, Teruyuki; Miyake, Jun

2010-06-01

Oligodendrocytes are the myelinating cells of the central nervous system (CNS), and defects in these cells can result in the loss of CNS functions. Although oligodendrocyte progenitor cells transplantation therapy is an effective cure for such symptoms, there is no readily available source of these cells. Recent studies have described the generation of induced pluripotent stem cells (iPS cells) from somatic cells, leading to anticipation of this technique as a novel therapeutic tool in regenerative medicine. In this study, we evaluated the ability of iPS cells derived from mouse embryonic fibroblasts to differentiate into oligodendrocytes and compared this with the differential ability of mouse embryonic stem cells (ES cells). Experiments using an in vitro oligodendrocyte differentiation protocol that was optimized to ES cells demonstrated that 2.3% of iPS cells differentiated into O4(+) oligodendrocytes compared with 24.0% of ES cells. However, the rate of induction of A2B5(+) oligodendrocyte precursor cell (OPC) was similar for both iPS-derived cells and ES-derived cells (14.1% and 12.6%, respectively). These findings suggest that some intracellular factors in iPS cells inhibit the terminal differentiation of oligodendrocytes from the OPC stage. (c) 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

ChIP-PIT: Enhancing the Analysis of ChIP-Seq Data Using Convex-Relaxed Pair-Wise Interaction Tensor Decomposition.

PubMed

Zhu, Lin; Guo, Wei-Li; Deng, Su-Ping; Huang, De-Shuang

2016-01-01

In recent years, thanks to the efforts of individual scientists and research consortiums, a huge amount of chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experimental data have been accumulated. Instead of investigating them independently, several recent studies have convincingly demonstrated that a wealth of scientific insights can be gained by integrative analysis of these ChIP-seq data. However, when used for the purpose of integrative analysis, a serious drawback of current ChIP-seq technique is that it is still expensive and time-consuming to generate ChIP-seq datasets of high standard. Most researchers are therefore unable to obtain complete ChIP-seq data for several TFs in a wide variety of cell lines, which considerably limits the understanding of transcriptional regulation pattern. In this paper, we propose a novel method called ChIP-PIT to overcome the aforementioned limitation. In ChIP-PIT, ChIP-seq data corresponding to a diverse collection of cell types, TFs and genes are fused together using the three-mode pair-wise interaction tensor (PIT) model, and the prediction of unperformed ChIP-seq experimental results is formulated as a tensor completion problem. Computationally, we propose efficient first-order method based on extensions of coordinate descent method to learn the optimal solution of ChIP-PIT, which makes it particularly suitable for the analysis of massive scale ChIP-seq data. Experimental evaluation the ENCODE data illustrate the usefulness of the proposed model.

An In Vitro Expansion System for Generation of Human iPS Cell-Derived Hepatic Progenitor-Like Cells Exhibiting a Bipotent Differentiation Potential

PubMed Central

Yanagida, Ayaka; Ito, Keiichi; Chikada, Hiromi; Nakauchi, Hiromitsu; Kamiya, Akihide

2013-01-01

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13highCD133+ cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development. PMID:23935837

An in vitro expansion system for generation of human iPS cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.

PubMed

Yanagida, Ayaka; Ito, Keiichi; Chikada, Hiromi; Nakauchi, Hiromitsu; Kamiya, Akihide

2013-01-01

Hepatoblasts, hepatic stem/progenitor cells in liver development, have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In regenerative medicine and drug screening for the treatment of severe liver diseases, human induced pluripotent stem (iPS) cell-derived mature functional hepatocytes are considered to be a potentially good cell source. However, induction of proliferation of these cells is difficult ex vivo. To circumvent this problem, we generated hepatic progenitor-like cells from human iPS cells using serial cytokine treatments in vitro. Highly proliferative hepatic progenitor-like cells were purified by fluorescence-activated cell sorting using antibodies against CD13 and CD133 that are known cell surface markers of hepatic stem/progenitor cells in fetal and adult mouse livers. When the purified CD13(high)CD133(+) cells were cultured at a low density with feeder cells in the presence of suitable growth factors and signaling inhibitors (ALK inhibitor A-83-01 and ROCK inhibitor Y-27632), individual cells gave rise to relatively large colonies. These colonies consisted of two types of cells expressing hepatocytic marker genes (hepatocyte nuclear factor 4α and α-fetoprotein) and a cholangiocytic marker gene (cytokeratin 7), and continued to proliferate over long periods of time. In a spheroid formation assay, these cells were found to express genes required for mature liver function, such as cytochrome P450 enzymes, and secrete albumin. When these cells were cultured in a suitable extracellular matrix gel, they eventually formed a cholangiocytic cyst-like structure with epithelial polarity, suggesting that human iPS cell-derived hepatic progenitor-like cells have a bipotent differentiation ability. Collectively these data indicate that this novel procedure using an in vitro expansion system is useful for not only liver regeneration but also for the determination of molecular mechanisms that regulate liver development.

c-MYC independent nuclear reprogramming favors cardiogenic potential of induced pluripotent stem cells

PubMed Central

Martinez-Fernandez, Almudena; Nelson, Timothy J.; Ikeda, Yasuhiro; Terzic, Andre

2010-01-01

Induced pluripotent stem cell (iPS) technology has launched a new platform in regenerative medicine aimed at deriving unlimited replacement tissue from autologous sources through somatic cell reprogramming using stemness factor sets. In this way, authentic cardiomyocytes have been obtained from iPS and recently demonstrated in proof-of-principle studies to repair infarcted heart. Optimizing the cardiogenic potential of iPS progeny would ensure a maximized yield of bioengineered cardiac tissue. Here, we reprogrammed fibroblasts in the presence or absence of c-MYC to determine if the acquired cardiogenicity is sensitive to the method of nuclear reprogramming. Using lentiviral constructs that expressed stemness factors SOX2, OCT4, and KLF4 with or without c-MYC, iPS clones generated through fibroblast reprogramming demonstrated indistinguishable characteristics for 5 days of differentiation with similar cell morphology, growth rates, and chimeric embryo integration. However, 4-factor c-MYC dependent nuclear reprogramming produced iPS progeny that consistently prolonged the expression of pluripotent Oct-4 and Fgf4 genes and repressed cardiac differentiation. In contrast, 3-factor c-MYC-less iPS clones efficiently up-regulated pre-cardiac (CXCR4, Flk-1, and Mesp1/2) and cardiac (Nkx2.5, Mef2c, and Myocardin) gene expression patterns. In fact, 3-factor iPS progeny demonstrated early and robust cardiogenesis during in vitro differentiation with consistent beating activity, sarcomere maturation, and rhythmical intracellular calcium dynamics. Thus, nuclear reprogramming independent of c-MYC enhances production of pluripotent stem cells with innate cardiogenic potential. PMID:20221419

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor expression and function.

PubMed

Bill, Anke; Rosethorne, Elizabeth M; Kent, Toby C; Fawcett, Lindsay; Burchell, Lynn; van Diepen, Michiel T; Marelli, Anthony; Batalov, Sergey; Miraglia, Loren; Orth, Anthony P; Renaud, Nicole A; Charlton, Steven J; Gosling, Martin; Gaither, L Alex; Groot-Kormelink, Paul J

2014-01-01

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

High Throughput Mutagenesis for Identification of Residues Regulating Human Prostacyclin (hIP) Receptor Expression and Function

PubMed Central

Kent, Toby C.; Fawcett, Lindsay; Burchell, Lynn; van Diepen, Michiel T.; Marelli, Anthony; Batalov, Sergey; Miraglia, Loren; Orth, Anthony P.; Renaud, Nicole A.; Charlton, Steven J.; Gosling, Martin; Gaither, L. Alex; Groot-Kormelink, Paul J.

2014-01-01

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs. PMID:24886841

The fused in sarcoma protein forms cytoplasmic aggregates in motor neurons derived from integration-free induced pluripotent stem cells generated from a patient with familial amyotrophic lateral sclerosis carrying the FUS-P525L mutation.

PubMed

Liu, Xinxiu; Chen, Jiayu; Liu, Wenchao; Li, Xiaogang; Chen, Qi; Liu, Tao; Gao, Shaorong; Deng, Min

2015-07-01

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motor neurons (MNs) and has no effective treatment. Mutations in the fused in sarcoma (FUS) gene and abnormal aggregation of FUS protein have been reported in ALS. However, the mechanisms involved in ALS are poorly understood. Clinical drug trails have failed due to a lack of appropriate disease models, including a lack of access to MNs from ALS patients. Induced pluripotent stem (iPS) cells derived from patients with ALS provide an indispensable resource for in vitro mechanistic studies and for future patient-specific cell-based therapies. Previous reports demonstrated that viral-based ALS-iPS cells generated from fibroblasts harvested from Caucasian populations are ideal for basic research; however, ALS-iPS cells are precluded from cell-based therapeutic applications because of the risks associated with the integration of viral sequences into the genome and inconvenience associated with dermal biopsies. To establish a model for use in clinical applications, using episomal vectors, we generated an integration-free iPS cell line from peripheral blood mononuclear cells (PBMCs) harvested from a familial ALS (FALS) patient carrying the FUS-P525L mutation and a healthy control. Furthermore, we successfully differentiated ALS patient-specific iPS cells into MNs and subsequently detected cytoplasmic mislocalization and formation of FUS protein aggregates in MNs due to the FUS-P525L mutation. Our findings offer a cell-based disease model for use in further elucidating ALS pathogenesis and provide a tool for exploring gene repair coupled with cell replacement therapy.

A Phase I Study of iPS Cell Generation From Patients With COPD

ClinicalTrials.gov

2018-03-20

Thoracic Diseases; Respiratory Tract Diseases; Cancer of Lung; Cancer of the Lung; Lung Cancer; Lung Diseases, Obstructive; COPD; Pulmonary Emphysema; Neoplasms, Lung; Neoplasms, Pulmonary; Pulmonary Cancer; Pulmonary Neoplasms; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell

Inducing pluripotency in somatic cells from the snow leopard (Panthera uncia), an endangered felid.

PubMed

Verma, R; Holland, M K; Temple-Smith, P; Verma, P J

2012-01-01

Induced pluripotency is a new approach to produce embryonic stem-like cells from somatic cells that provides a unique means to understand both pluripotency and lineage assignment. To investigate whether this technology could be applied to endangered species, where the limited availability of gametes makes production and research on embryonic stem cells difficult, we attempted generation of induced pluripotent stem (iPS) cells from snow leopard (Panthera uncia) fibroblasts by retroviral transfection with Moloney-based retroviral vectors (pMXs) encoding four factors (OCT4, SOX2, KLF4 and cMYC). This resulted in the formation of small colonies of cells, which could not be maintained beyond four passages (P4). However, addition of NANOG, to the transfection cocktail produced stable iPS cell colonies, which formed as early as D3. Colonies of cells were selected at D5 and expanded in vitro. The resulting cell line was positive for alkaline phosphatase (AP), OCT4, NANOG, and Stage-Specific embryonic Antigen-4 (SSEA-4) at P14. RT-PCR also confirmed that endogenous OCT4 and NANOG were expressed by snow leopard iPS cells from P4. All five human transgenes were transcribed at P4, but OCT4, SOX2 and NANOG transgenes were silenced as early as P14; therefore, reprogramming of the endogenous pluripotent genes had occurred. When injected into immune-deficient mice, snow leopard iPS cells formed teratomas containing tissues representative of the three germ layers. In conclusion, this was apparently the first derivation of iPS cells from the endangered snow leopard and the first report on induced pluripotency in felid species. Addition of NANOG to the reprogramming cocktail was essential for derivation of iPS lines in this felid. The iPS cells provided a unique source of pluripotent cells with utility in conservation through cryopreservation of genetics, as a source of reprogrammed donor cells for nuclear transfer or for directed differentiation to gametes in the future. Copyright © 2012 Elsevier Inc. All rights reserved.

Stimulation of generation of inositol phosphates by carbamoylcholine and its inhibition by phorbol esters and iodide in dog thyroid cells.

PubMed Central

Laurent, E; Mockel, J; Takazawa, K; Erneux, C; Dumont, J E

1989-01-01

The action of carbamoylcholine (Cchol), NaF and other agonists on the generation of inositol phosphates (IPs) was studied in dog thyroid slices prelabelled with myo-[2-3H]inositol. The stimulation by Cchol (0.1 microM-0.1 mM) of IPs accumulation through activation of a muscarinic receptor [Graff, Mockel, Laurent, Erneux & Dumont (1987) FEBS Lett. 210, 204-210] was pertussis- and cholera-toxin insensitive. Ins(1,4,5)P3, Ins(1,3,4)P3 and InsP4 were generated. NaF (5-20 mM) also increased IPs generation (Graff et al., 1987); this effect was potentiated by AlCl3 (10 microM) and unaffected by pertussis toxin. Although phorbol dibutyrate (5 microM) abolished the cholinergic stimulation of IPs generation (Graff et al., 1987), it did not affect the fluoride-induced response. Cchol and NaF did not require extracellular Ca2+ to exert their effect, and neither KCl-induced membrane depolarization nor ionophore A23187 (10 microM) had any influence on basal IPs levels, or on cholinergic stimulation. However, more stringent Ca2+ depletion with EGTA (0.1 or 1 mM) decreased basal IPs levels as well as the amplitude of the stimulation by Cchol without abolishing it. Dibutyryl cyclic AMP, forskolin, cholera toxin and prostaglandin E1 had no effect on basal IPs levels and did not decrease the response to Cchol. Iodide (4 or 40 microM) also strongly decreased the cholinergic action on IPs, this inhibition being relieved by methimazole (1 mM). Our data suggest that Cchol activates a phospholipase C hydrolysing PtdIns(4,5)P2 in the dog thyroid cell in a cyclic AMP-independent manner. This activation requires no extracellular Ca2+ and depends on a GTP-binding protein insensitive to both cholera toxin and requires no extracellular Ca2+ and depends on a GTP-binding protein insensitive to both cholera toxin and pertussis toxin. The data are consistent with a rapid metabolism of Ins(1,4,5)P3 to Ins(1,3,4)P3 via the Ins(1,4,5)P3 3-kinase pathway, followed by dephosphorylation by a 5-phosphomonoesterase. Indeed, a Ca2+-sensitive InsP3 3-kinase activity was demonstrated in tissue homogenate. Stimulation of protein kinase C and an organified form of iodine inhibit the Cchol-induced IPs generation. The negative feedback of activated protein kinase C could be exerted at the level of the receptor or of the receptor-G-protein interaction. PMID:2557011

Generation of hyaline cartilaginous tissue from mouse adult dermal fibroblast culture by defined factors

PubMed Central

Hiramatsu, Kunihiko; Sasagawa, Satoru; Outani, Hidetatsu; Nakagawa, Kanako; Yoshikawa, Hideki; Tsumaki, Noriyuki

2011-01-01

Repair of cartilage injury with hyaline cartilage continues to be a challenging clinical problem. Because of the limited number of chondrocytes in vivo, coupled with in vitro de-differentiation of chondrocytes into fibrochondrocytes, which secrete type I collagen and have an altered matrix architecture and mechanical function, there is a need for a novel cell source that produces hyaline cartilage. The generation of induced pluripotent stem (iPS) cells has provided a tool for reprogramming dermal fibroblasts to an undifferentiated state by ectopic expression of reprogramming factors. Here, we show that retroviral expression of two reprogramming factors (c-Myc and Klf4) and one chondrogenic factor (SOX9) induces polygonal chondrogenic cells directly from adult dermal fibroblast cultures. Induced cells expressed marker genes for chondrocytes but not fibroblasts, i.e., the promoters of type I collagen genes were extensively methylated. Although some induced cell lines formed tumors when subcutaneously injected into nude mice, other induced cell lines generated stable homogenous hyaline cartilage–like tissue. Further, the doxycycline-inducible induction system demonstrated that induced cells are able to respond to chondrogenic medium by expressing endogenous Sox9 and maintain chondrogenic potential after substantial reduction of transgene expression. Thus, this approach could lead to the preparation of hyaline cartilage directly from skin, without generating iPS cells. PMID:21293062

78 FR 37554 - Government-Owned Inventions; Availability for Licensing

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-21

... that utilize cord blood as a stem cell source. Potential Commercial Applications: Drug delivery to... Stem Cells by Blocking CD47 Receptor Signaling Description of Technology: NIH researchers have... generation of self-renewing cells with a high proliferative capacity. Induced pluripotent stem cells (iPS...

Metabolic rescue in pluripotent cells from patients with mtDNA disease.

PubMed

Ma, Hong; Folmes, Clifford D L; Wu, Jun; Morey, Robert; Mora-Castilla, Sergio; Ocampo, Alejandro; Ma, Li; Poulton, Joanna; Wang, Xinjian; Ahmed, Riffat; Kang, Eunju; Lee, Yeonmi; Hayama, Tomonari; Li, Ying; Van Dyken, Crystal; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Koski, Amy; Mitalipov, Nargiz; Amato, Paula; Wolf, Don P; Huang, Taosheng; Terzic, Andre; Laurent, Louise C; Izpisua Belmonte, Juan Carlos; Mitalipov, Shoukhrat

2015-08-13

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

IP-FCM measures physiologic protein-protein interactions modulated by signal transduction and small-molecule drug inhibition.

PubMed

Smith, Stephen E P; Bida, Anya T; Davis, Tessa R; Sicotte, Hugues; Patterson, Steven E; Gil, Diana; Schrum, Adam G

2012-01-01

Protein-protein interactions (PPI) mediate the formation of intermolecular networks that control biological signaling. For this reason, PPIs are of outstanding interest in pharmacology, as they display high specificity and may represent a vast pool of potentially druggable targets. However, the study of physiologic PPIs can be limited by conventional assays that often have large sample requirements and relatively low sensitivity. Here, we build on a novel method, immunoprecipitation detected by flow cytometry (IP-FCM), to assess PPI modulation during either signal transduction or pharmacologic inhibition by two different classes of small-molecule compounds. First, we showed that IP-FCM can detect statistically significant differences in samples possessing a defined PPI change as low as 10%. This sensitivity allowed IP-FCM to detect a PPI that increases transiently during T cell signaling, the antigen-inducible interaction between ZAP70 and the T cell antigen receptor (TCR)/CD3 complex. In contrast, IP-FCM detected no ZAP70 recruitment when T cells were stimulated with antigen in the presence of the src-family kinase inhibitor, PP2. Further, we tested whether IP-FCM possessed sufficient sensitivity to detect the effect of a second, rare class of compounds called SMIPPI (small-molecule inhibitor of PPI). We found that the first-generation non-optimized SMIPPI, Ro-26-4550, inhibited the IL-2:CD25 interaction detected by IP-FCM. This inhibition was detectable using either a recombinant CD25-Fc chimera or physiologic full-length CD25 captured from T cell lysates. Thus, we demonstrate that IP-FCM is a sensitive tool for measuring physiologic PPIs that are modulated by signal transduction and pharmacologic inhibition.

Induced pluripotent stem cells from a spinal muscular atrophy patient

PubMed Central

Ebert, Allison D.; Yu, Junying; Rose, Ferrill F.; Mattis, Virginia B.; Lorson, Christian L.; Thomson, James A.; Svendsen, Clive N.

2009-01-01

Spinal muscular atrophy (SMA) is one of the most common inherited forms of neurological disease leading to infant mortality. Patients exhibit selective loss of lower motor neurons resulting in muscle weakness, paralysis, and often death. Although patient fibroblasts have been used extensively to study SMA, motor neurons have a unique anatomy and physiology which may underlie their vulnerability to the disease process. Here we report the generation of induced pluripotent stem (iPS) cells from skin fibroblast samples taken from a child with SMA. These cells expanded robustly in culture, maintained the disease genotype, and generated motor neurons that showed selective deficits compared to those derived from the child's unaffected mother. This is the first study to show human iPS cells can be used to model the specific pathology seen in a genetically inherited disease. As such, it represents a promising resource to study disease mechanisms, screen novel drug compounds, and develop new therapies. PMID:19098894

The Bcl-2 homolog Nrz inhibits binding of IP3 to its receptor to control calcium signaling during zebrafish epiboly.

PubMed

Bonneau, Benjamin; Nougarède, Adrien; Prudent, Julien; Popgeorgiev, Nikolay; Peyriéras, Nadine; Rimokh, Ruth; Gillet, Germain

2014-02-11

Members of the Bcl-2 protein family regulate mitochondrial membrane permeability and also localize to the endoplasmic reticulum where they control Ca(2+) homeostasis by interacting with inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs). In zebrafish, Bcl-2-like 10 (Nrz) is required for Ca(2+) signaling during epiboly and gastrulation. We characterized the mechanism by which Nrz controls IP3-mediated Ca(2+) release during this process. We showed that Nrz was phosphorylated during early epiboly, and that in embryos in which Nrz was knocked down, reconstitution with Nrz bearing mutations designed to prevent its phosphorylation disrupted cyclic Ca(2+) transients and the assembly of the actin-myosin ring and led to epiboly arrest. In cultured cells, wild-type Nrz, but not Nrz with phosphomimetic mutations, interacted with the IP3 binding domain of IP3R1, inhibited binding of IP3 to IP3R1, and prevented histamine-induced increases in cytosolic Ca(2+). Collectively, these data suggest that Nrz phosphorylation is necessary for the generation of IP3-mediated Ca(2+) transients and the formation of circumferential actin-myosin cables required for epiboly. Thus, in addition to their role in apoptosis, by tightly regulating Ca(2+) signaling, Bcl-2 family members participate in the cellular events associated with early vertebrate development, including cytoskeletal dynamics and cell movement.

Brain-specific enhancers for cell-based therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Visel, Axel; Rubenstein, John L.R.; Chen, Ying-Jiun

Herein are described a set of novel specific human enhancers for specific forebrain cell types used to study and select for human neural progenitor cells. This approach enables the ability to generate interneurons from human ES, iPS and iN cells, making them available for human transplantation and for molecular/cellular analyzes. These approaches are also directly applicable to generating other neuronal cell types, such as cortical and striatal projection neurons, which have implications for many human diseases.

Altered hematopoiesis in trisomy 21 as revealed through in vitro differentiation of isogenic human pluripotent cells

PubMed Central

MacLean, Glenn A.; Menne, Tobias F.; Guo, Guoji; Sanchez, Danielle J.; Park, In-Hyun; Daley, George Q.; Orkin, Stuart H.

2012-01-01

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver, a preleukemic condition termed transient myeloproliferative disorder, and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins, human embryionic stem (hES), and induced pluripotent stem (iPS) cells, were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation, we isolated disomic and trisomic subclones from the same parental iPS line, thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like, γ-globin expressing, definitive hematopoiesis, we found that trisomic cells of hES, iPS, or isogenic origins exhibited a two- to fivefold increase in a population of CD43+(Leukosialin)/CD235+(Glycophorin A) hematopoietic cells, accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage. PMID:23045682

Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishizuka, Toshiaki, E-mail: tishizu@ndmc.ac.jp; Goshima, Hazuki; Ozawa, Ayako

2012-03-30

Highlights: Black-Right-Pointing-Pointer Treatment with angiotensin II enhanced LIF-induced DNA synthesis of mouse iPS cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the DNA synthesis via induction of superoxide. Black-Right-Pointing-Pointer Treatment with angiotensin II significantly increased JAK/STAT3 phosphorylation. Black-Right-Pointing-Pointer Angiotensin II enhanced differentiation into mesodermal progenitor cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the differentiation via activation of p38 MAPK. -- Abstract: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stemmore » (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT{sub 1}R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5 days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression. Treatment with Ang II enhanced the phosphorylation of p38 MAPK in Col IV- exposed iPS cells. These results suggest that the stimulation of mouse iPS cells with AT{sub 1}R may enhance LIF-induced DNA synthesis, by augmenting the generation of superoxide and activating JAK/STAT3, and that AT{sub 1}R stimulation may enhance Col IV-induced differentiation into mesodermal progenitor cells via p38 MAPK activation.« less

[Cellular mechanism of the generation of spontaneous activity in gastric muscle].

PubMed

Nakamura, Eri; Kito, Yoshihiko; Fukuta, Hiroyasu; Yanai, Yoshimasa; Hashitani, Hikaru; Yamamoto, Yoshimichi; Suzuki, Hikaru

2004-03-01

In gastric smooth muscles, interstitial cells of Cajal (ICC) might be the pacemaker cells of spontaneous activities since ICC are rich in mitochondria and are connected with smooth muscle cells via gap junctions. Several types of ICC are distributed widely in the stomach wall. A group of ICC distributed in the myenteric layer (ICC-MY) were the pacemaker cells of gastrointestinal smooth muscles. Pacemaker potentials were generated in ICC-MY, and the potentials were conducted to circular smooth muscles to trigger slow waves and also conducted to longitudinal muscles to form follower potentials. In circular muscle preparations, interstitial cells distributed within muscle bundles (ICC-IM) produced unitary potentials, which were conducted to circular muscles to form slow potentials by summation. In mutant mice lacking inositol trisphosphate (IP(3)) receptor, slow waves were absent in gastric smooth muscles. The generation of spontaneous activity was impaired by the inhibition of Ca(2+)-release from internal stores through IP(3) receptors, inhibition of mitochondrial Ca(2+)-handling with proton pump inhibitors, and inhibition of ATP-sensitive K(+)-channels at the mitochondrial inner membrane. These results suggested that mitochondrial Ca(2+)-handling causes the generation of spontaneous activity in pacemaker cells. Possible involvement of protein kinase C (PKC) in the Ca(2+) signaling system was also suggested.

Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents.

PubMed

Ding, K H; Husain, S; Akhtar, R A; Isales, C M; Abdel-Latif, A A

1997-09-01

The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP3) production and intracellular calcium ([Ca2+]i) mobilization, and their regulation by cAMP-elevating agents were investigated in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. CCh produced time- and dose-dependent increases in IP3 production; the t1/2 and EC50 values were 68 s and 0.5 microM, respectively. The muscarinic agonist provoked a transient increase in [Ca2+]i which reached maximum within 77 s, and increased [Ca2+]i mobilization in a concentration-dependent manner with an EC50 of 1.4 microM. Thapsigargin, a Ca(2+)-pump inhibitor, caused a rapid rise in [Ca2+]i and subsequent addition of CCh was without effect. Both CCh-induced IP3 production and CCh-induced [Ca2+]i mobilization were more potently antagonized by 4-DAMP, an M3 muscarinic receptor antagonist, than by pirenzepine, an M1 receptor antagonist, suggesting that both responses are mediated through the M3 receptor subtype. Treatment of the cells with U73122, a phospholipase C (PLC) inhibitor, resulted in a concentration-dependent decrease in both CCh-stimulated IP3 production and [Ca2+]i mobilization. These data indicate close correlation between enhanced IP3 production and [Ca2+]i mobilization in these smooth muscle cells and suggest that the CCh-stimulated increase in [Ca2+]i could be mediated through increased IP3 production. Isoproterenol (ISO) inhibited CCh-induced IP3 production (IC50 = 80 nM) and [Ca2+]i mobilization (IC50 = 0.17 microM) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 microM) and GTP gamma S (0.1 microM). Pretreatment of the cells with ISO or forskolin, 5 microM each, produced membrane fractions in which CCh-stimulated PLC activity was significantly attenuated. Furthermore, when microsomal fractions isolated from SV-CISM-2 cells were phosphorylated with Protein kinase A (PKA), the CCh- and GTP gamma S-stimulated IP3 production were significantly inhibited. It can be concluded from these studies that in SV-CISM-2 cells, activation of M3 muscarinic receptors results in stimulation of PLC-mediated PIP2 hydrolysis, generating IP3 which mobilizes [Ca2+]i. Furthermore, elevation of cAMP may inhibit IP3 production and [Ca2+]i mobilization through mechanisms involving PKA-dependent phosphorylation of PLC, G-proteins, IP3 receptor and/or IP3 metabolizing enzymes.

Active Generation and Propagation of Ca2+ Signals within Tunneling Membrane Nanotubes

PubMed Central

Smith, Ian F.; Shuai, Jianwei; Parker, Ian

2011-01-01

A new mechanism of cell-cell communication was recently proposed after the discovery of tunneling nanotubes (TNTs) between cells. TNTs are membrane protrusions with lengths of tens of microns and diameters of a few hundred nanometers that permit the exchange of membrane and cytoplasmic constituents between neighboring cells. TNTs have been reported to mediate intercellular Ca2+ signaling; however, our simulations indicate that passive diffusion of Ca2+ ions alone would be inadequate for efficient transmission between cells. Instead, we observed spontaneous and inositol trisphosphate (IP3)-evoked Ca2+ signals within TNTs between cultured mammalian cells, which sometimes remained localized and in other instances propagated as saltatory waves to evoke Ca2+ signals in a connected cell. Consistent with this, immunostaining showed the presence of both endoplasmic reticulum and IP3 receptors along the TNT. We propose that IP3 receptors may actively propagate intercellular Ca2+ signals along TNTs via Ca2+-induced Ca2+ release, acting as amplification sites to overcome the limitations of passive diffusion in a chemical analog of electrical transmission of action potentials. PMID:21504718

Legionella phosphatase hydrolyzes phosphatidylinositol 4,5-bisphosphate and inosital triphosphate in human neutrophils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dowling, J.N.; Saha, A.K.; Glew, R.H.

1987-05-01

Legionella are facultative intracellular bacterial pathogens which multiply in host phagocytes. L. micdadei cells contain an acid phosphatase (ACP) that blocks superoxide anion production by human neutrophils stimulated with the formylated peptide, fMLP. The possibility that ACP acts by interefering with polyphosphoinositide metabolism and the production of the intracellular second messenger, inositol triphosphate (IP3) was explored. When neutrophil phosphoinositides were labeled with TSP, incubation of the cells with ACP caused an 85% loss of the labeled phosphatidylinositol-4,5-bisphosphate (PIP2) over 2 h. Treatment of (TH)inositol-labeled neutrophils with ACP for 30 min resulted in a 20% decrease of labeled PIP2. Following fMLPmore » stimulation, the fractional reduction in PIP2 and the fractional increase in IP3 was the same in ACP-treated and untreated neutrophils, but the total quantity of IP3 was reduced by ACP pre-treatment. The reduction in IP3 generated following fMLP stimulation seems to be due primarily to the decreased amount of PIP2 available for hydrolysis. However, some loss of IP3 due to direct hydrolysis by ACP cannot be ruled out. The Legionella phosphatase may compromise neutrophil response to the bacteria by hydrolyzing PIP2, the prognitor of IP3, and by hydrolyzing IP3 itself.« less

Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4–signalosome complex to regulate DNA repair and cell death

PubMed Central

Rao, Feng; Xu, Jing; Khan, A. Basit; Gadalla, Moataz M.; Cha, Jiyoung Y.; Xu, Risheng; Tyagi, Richa; Dang, Yongjun; Chakraborty, Anutosh; Snyder, Solomon H.

2014-01-01

Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1–3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN–CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4–CSN complex, thereby regulating nucleotide excision repair and cell death. PMID:25349427

Substance P receptor desensitization requires receptor activation but not phospholipase C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sugiya, Hiroshi; Putney, J.W. Jr.

1988-08-01

Previous studies have shown that exposure of parotid acinar cells to substance P at 37{degree}C results in activation of phospholipase C, formation of ({sup 3}H)inositol 1,4,5-trisphosphate (IP{sub 3}), and persistent desensitization of the substance P response. In cells treated with antimycin in medium containing glucose, ATP was decreased to {approximately}20% of control values, IP{sub 3} formation was completely inhibited, but desensitization was unaffected. When cells were treated with antimycin in the absence of glucose, cellular ATP was decreased to {approximately}5% of control values, and both IP{sub 3} formation and desensitization were blocked. A series of substance P-related peptides increased themore » formation of ({sup 3}H)IP{sub 3} and induced desensitization of the substance P response with a similar rank order of potencies. The substance P antagonist, (D-Pro{sup 2}, D-Try{sup 7,9})-substance P, inhibited substance P-induced IP{sub 3} formation and desensitization but did not induce desensitization. These results suggest that the desensitization of substance P-induced IP{sub 3} formation requires agonist activation of a P-type substance P receptor, and that one or more cellular ATP-dependent processes are required for this reaction. However, activation of phospholipase C and the generation of inositol phosphates does not seem to be a prerequisite for desensitization.« less

Unanticipated region- and cell-specific downregulation of individual KChIP auxiliary subunit isotypes in Kv4.2 knock-out mouse brain.

PubMed

Menegola, Milena; Trimmer, James S

2006-11-22

Kv4 family voltage-gated potassium channel alpha subunits and Kv channel-interacting protein (KChIP) and dipeptidyl aminopeptidase-like protein subunits comprise somatodendritic A-type channels in mammalian neurons. Recently, a mouse was generated with a targeted deletion of Kv4.2, a Kv4 alpha subunit expressed in many but not all mammalian brain neurons. Kv4.2-/- mice are grossly indistinguishable from wild-type (WT) littermates. Here we used immunohistochemistry to analyze expression of component Kv4 and KChIP subunits of A-type channels in WT and Kv4.2-/- brains. We found that the expression level, and cellular and subcellular distribution of the other prominent brain Kv4 family member Kv4.3, was indistinguishable between WT and Kv4.2-/- samples. However, we found unanticipated regional and cell-specific decreases in expression of KChIPs. The degree of altered expression of individual KChIP isoforms in different regions and neurons precisely follows the level of Kv4.2 normally found at those sites and presumably their extent of association of these KChIPs with Kv4.2. The dramatic effects of Kv4.2 deletion on KChIP expression suggest that, in addition to previously characterized effects of KChIPs on the functional properties, trafficking, and turnover rate of Kv4 channels, Kv4:KChIP association may confer reciprocal Kv4.2-dependent effects on KChIPs. The impact of Kv4.2 deletion on KChIP expression also supports the major role of KChIPs as auxiliary subunits of Kv4 channels.

Genome-wide ChIP-seq mapping and analysis of butyrate-induced H3K9 and H3K27 acetylation and epigenomic landscape alteration in bovine cells

USDA-ARS?s Scientific Manuscript database

Utilizing next-generation sequencing technology, combined with ChIP (Chromatin Immunoprecipitation) technology, we analyzed histone modification (acetylation) induced by butyrate and the large-scale mapping of the epigenomic landscape of normal histone H3 and acetylated histone H3K9 and H3K27. To d...

Interleukin-18 enhances IL-18R/Nox1 binding, and mediates TRAF3IP2-dependent smooth muscle cell migration. Inhibition by simvastatin

PubMed Central

Valente, Anthony J.; Yoshida, Tadashi; Izadpanah, Reza; Delafontaine, Patrice; Siebenlist, Ulrich; Chandrasekar, Bysani

2013-01-01

We investigated the role of TRAF3 interacting protein 2 (TRAF3IP2), a redox-sensitive adapter protein and an upstream regulator of IKK and JNK in interleukin (IL)-18 induced smooth muscle cell migration, and the mechanism of its inhibition by simvastatin. The pleiotropic cytokine IL-18 induced human coronary artery SMC migration through the induction of TRAF3IP2. IL-18 induced Nox1-dependent ROS generation, TRAF3IP2 expression, and IKK/NF-κB and JNK/AP-1 activation. IL-18 induced its own expression and that of its receptor subunit IL-18Rα. Using co-IP/IB and GST pull-down assays, we show for the first time that the subunits of the IL-18R heterodimer physically associate with Nox1 under basal conditions, and IL-18 appears to enhance their binding. Importantly, the HMG-coA reductase inhibitor simvastatin attenuated IL-18-induced TRAF3IP2 induction. These inhibitory effects were reversed by mevalonate and geranylgeranylpyrophosphate (GGPP), but not by farnesylpyrophosphate (FPP). Interestingly, simvastatin, GGPP, FPP, or Rac1 inhibition did not modulate ectopically expressed TRAF3IP2. The promigratory effects of IL-18 are mediated through TRAF3IP2 in a redox-sensitive manner, and this may involve IL-18R/Nox1 physical association. Further, Simvastatin inhibits inducible, but not ectopically-expressed TRAF3IP2. Targeting TRAF3IP2 may blunt progression of hyperplastic vascular diseases in vivo. PMID:23541442

Mitochondrial calcium handling within the interstitial cells of Cajal

PubMed Central

Cheng, Leo K.

2014-01-01

The interstitial cells of Cajal (ICC) drive rhythmic pacemaking contractions in the gastrointestinal system. The ICC generate pacemaking signals by membrane depolarizations associated with the release of intracellular calcium (Ca2+) in the endoplasmic reticulum (ER) through inositol-trisphosphate (IP3) receptors (IP3R) and uptake by mitochondria (MT). This Ca2+ dynamic is hypothesized to generate pacemaking signals by calibrating ER Ca2+ store depletions and membrane depolarization with ER store-operated Ca2+ entry mechanisms. Using a biophysically based spatio-temporal model of integrated Ca2+ transport in the ICC, we determined the feasibility of ER depletion timescale correspondence with experimentally observed pacemaking frequencies while considering the impact of IP3R Ca2+ release and MT uptake on bulk cytosolic Ca2+ levels because persistent elevations of free intracellular Ca2+ are toxic to the cell. MT densities and distributions are varied in the model geometry to observe MT influence on free cytosolic Ca2+ and the resulting frequencies of ER Ca2+ store depletions, as well as the sarco-endoplasmic reticulum Ca2+ ATP-ase (SERCA) and IP3 agonist concentrations. Our simulations show that high MT densities observed in the ICC are more relevant to ER establishing Ca2+ depletion frequencies than protection of the cytosol from elevated free Ca2+, whereas the SERCA pump is more relevant to containing cytosolic Ca2+ elevations. Our results further suggest that the level of IP3 agonist stimulating ER Ca2+ release, subsequent MT uptake, and eventual activation of ER store-operated Ca2+ entry may determine frequencies of rhythmic pacemaking exhibited by the ICC across species and tissue types. PMID:24789203

Current advances in the generation of human iPS cells: implications in cell-based regenerative medicine.

PubMed

Revilla, Ana; González, Clara; Iriondo, Amaia; Fernández, Bárbara; Prieto, Cristina; Marín, Carlos; Liste, Isabel

2016-11-01

Over the last few years, the generation of induced pluripotent stem cells (iPSCs) from human somatic cells has proved to be one of the most potentially useful discoveries in regenerative medicine. iPSCs are becoming an invaluable tool to study the pathology of different diseases and for drug screening. However, several limitations still affect the possibility of applying iPS cell-based technology in therapeutic prospects. Most strategies for iPSCs generation are based on gene delivery via retroviral or lentiviral vectors, which integrate into the host's cell genome, causing a remarkable risk of insertional mutagenesis and oncogenic transformation. To avoid such risks, significant advances have been made with non-integrative reprogramming strategies. On the other hand, although many different kinds of somatic cells have been employed to generate iPSCs, there is still no consensus about the ideal type of cell to be reprogrammed. In this review we present the recent advances in the generation of human iPSCs, discussing their advantages and limitations in terms of safety and efficiency. We also present a selection of somatic cell sources, considering their capability to be reprogrammed and tissue accessibility. From a translational medicine perspective, these two topics will provide evidence to elucidate the most suitable combination of reprogramming strategy and cell source to be applied in each human iPSC-based therapy. The wide variety of diseases this technology could treat opens a hopeful future for regenerative medicine. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Generating Porcine Chimeras Using Inner Cell Mass Cells and Parthenogenetic Preimplantation Embryos

PubMed Central

Nakano, Kazuaki; Watanabe, Masahito; Matsunari, Hitomi; Matsuda, Taisuke; Honda, Kasumi; Maehara, Miki; Kanai, Takahiro; Hayashida, Gota; Kobayashi, Mirina; Kuramoto, Momoko; Arai, Yoshikazu; Umeyama, Kazuhiro; Fujishiro, Shuh-hei; Mizukami, Yoshihisa; Nagaya, Masaki; Hanazono, Yutaka; Nagashima, Hiroshi

2013-01-01

Background The development and validation of stem cell therapies using induced pluripotent stem (iPS) cells can be optimized through translational research using pigs as large animal models, because pigs have the closest characteristics to humans among non-primate animals. As the recent investigations have been heading for establishment of the human iPS cells with naïve type characteristics, it is an indispensable challenge to develop naïve type porcine iPS cells. The pluripotency of the porcine iPS cells can be evaluated using their abilities to form chimeras. Here, we describe a simple aggregation method using parthenogenetic host embryos that offers a reliable and effective means of determining the chimera formation ability of pluripotent porcine cells. Methodology/Significant Principal Findings In this study, we show that a high yield of chimeric blastocysts can be achieved by aggregating the inner cell mass (ICM) from porcine blastocysts with parthenogenetic porcine embryos. ICMs cultured with morulae or 4–8 cell-stage parthenogenetic embryos derived from in vitro-matured (IVM) oocytes can aggregate to form chimeric blastocysts that can develop into chimeric fetuses after transfer. The rate of production of chimeric blastocysts after aggregation with host morulae (20/24, 83.3%) was similar to that after the injection of ICMs into morulae (24/29, 82.8%). We also found that 4–8 cell-stage embryos could be used; chimeric blastocysts were produced with a similar efficiency (17/26, 65.4%). After transfer into recipients, these blastocysts yielded chimeric fetuses at frequencies of 36.0% and 13.6%, respectively. Conclusion/Significance Our findings indicate that the aggregation method using parthenogenetic morulae or 4–8 cell-stage embryos offers a highly reproducible approach for producing chimeric fetuses from porcine pluripotent cells. This method provides a practical and highly accurate system for evaluating pluripotency of undifferentiated cells, such as iPS cells, based on their ability to form chimeras. PMID:23626746

Generation of an iPS cell line from bone marrow derived mesenchymal stromal cells from an elderly patient.

PubMed

Megges, Matthias; Geissler, Sven; Duda, Georg N; Adjaye, James

2015-11-01

An induced pluripotent stem cell line was generated from primary human bone marrow derived mesenchymal stromal cells of a 74 year old donor using retroviruses harboring OCT4, SOX2, KLF4 and c-MYC in combination with the following inhibitors TGFβ receptor-SB 431542, MEK-PD325901, and p53-Pifithrin α. Pluripotency was confirmed both in vitro and in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

Blocking adenylyl cyclase inhibits olfactory generator currents induced by "IP(3)-odors".

PubMed

Chen, S; Lane, A P; Bock, R; Leinders-Zufall, T; Zufall, F

2000-07-01

Vertebrate olfactory receptor neurons (ORNs) transduce odor stimuli into electrical signals by means of an adenylyl cyclase/cAMP second messenger cascade, but it remains widely debated whether this cAMP cascade mediates transduction for all odorants or only certain odor classes. To address this problem, we have analyzed the generator currents induced by odors that failed to produce cAMP in previous biochemical assays but instead produced IP(3) ("IP(3)-odors"). We show that in single salamander ORNs, sensory responses to "cAMP-odors" and IP(3)-odors are not mutually exclusive but coexist in the same cells. The currents induced by IP(3)-odors exhibit identical biophysical properties as those induced by cAMP odors or direct activation of the cAMP cascade. By disrupting adenylyl cyclase to block cAMP formation using two potent antagonists of adenylyl cyclase, SQ22536 and MDL12330A, we show that this molecular step is necessary for the transduction of both odor classes. To assess whether these results are also applicable to mammals, we examine the electrophysiological responses to IP(3)-odors in intact mouse main olfactory epithelium (MOE) by recording field potentials. The results show that inhibition of adenylyl cyclase prevents EOG responses to both odor classes in mouse MOE, even when "hot spots" with heightened sensitivity to IP(3)-odors are examined.

Immunohistochemical localization of DPP10 in rat brain supports the existence of a Kv4/KChIP/DPPL ternary complex in neurons.

PubMed

Wang, Wan-Chen; Cheng, Chau-Fu; Tsaur, Meei-Ling

2015-03-01

Subthreshold A-type K(+) currents (ISA s) have been recorded from the cell bodies of hippocampal and neocortical interneurons as well as neocortical pyramidal neurons. Kv4 channels are responsible for the somatodendritic ISA s. It has been proposed that neuronal Kv4 channels are ternary complexes including pore-forming Kv4 subunits, K(+) channel-interacting proteins (KChIPs), and dipeptidyl peptidase-like proteins (DPPLs). However, colocalization evidence was still lacking. The distribution of DPP10 mRNA in rodent brain has been reported but its protein localization remains unknown. In this study, we generated a DPP10 antibody to label DPP10 protein in adult rat brain by immunohistochemistry. Absent from glia, DPP10 proteins appear mainly in the cell bodies of DPP10(+) neurons, not only at the plasma membrane but also in the cytoplasm. At least 6.4% of inhibitory interneurons in the hippocampus coexpressed Kv4.3, KChIP1, and DPP10, with the highest density in the CA1 strata alveus/oriens/pyramidale and the dentate hilus. Colocalization of Kv4.3/KChIP1/DPP10 was also detected in at least 6.9% of inhibitory interneurons scattered throughout the neocortex. Both hippocampal and neocortical Kv4.3/KChIP1/DPP10(+) inhibitory interneurons expressed parvalbumin or somatostatin, but not calbindin or calretinin. Furthermore, we found colocalization of Kv4.2/Kv4.3/KChIP3/DPP10 in neocortical layer 5 pyramidal neurons and olfactory bulb mitral cells. Together, although DPP10 is also expressed in some brain neurons lacking Kv4 (such as parvalbumin- and somatostatin-positive Golgi cells in the cerebellum), colocalization of DPP10 with Kv4 and KChIP at the plasma membrane of ISA -expressing neuron somata supports the existence of Kv4/KChIP/DPPL ternary complex in vivo. © 2014 Wiley Periodicals, Inc.

Screening of drugs to treat 8p11 myeloproliferative syndrome using patient-derived induced pluripotent stem cells with fusion gene CEP110-FGFR1.

PubMed

Yamamoto, Shohei; Otsu, Makoto; Matsuzaka, Emiko; Konishi, Chieko; Takagi, Haruna; Hanada, Sachiyo; Mochizuki, Shinji; Nakauchi, Hiromitsu; Imai, Kohzoh; Tsuji, Kohichiro; Ebihara, Yasuhiro

2015-01-01

Induced pluripotent stem (iPS) cells provide powerful tools for studying disease mechanisms and developing therapies for diseases. The 8p11 myeloproliferative syndrome (EMS) is an aggressive chronic myeloproliferative disorder (MPD) that is caused by constitutive activation of fibroblast growth factor receptor 1. EMS is rare and, consequently, effective treatment for this disease has not been established. Here, iPS cells were generated from an EMS patient (EMS-iPS cells) to assist the development of effective therapies for EMS. When iPS cells were co-cultured with murine embryonic stromal cells, EMS-iPS cells produced more hematopoietic progenitor and hematopoietic cells, and CD34+ cells derived from EMS-iPS cells exhibited 3.2-7.2-fold more macrophage and erythroid colony forming units (CFUs) than those derived from control iPS cells. These data indicate that EMS-iPS cells have an increased hematopoietic differentiation capacity, which is characteristic of MPDs. To determine whether a tyrosine kinase inhibitor (TKI) could suppress the increased number of CFUs formed by EMS-iPS-induced CD34+ cells, cells were treated with one of four TKIs (CHIR258, PKC 412, ponatinib, and imatinib). CHIR258, PKC 412, and ponatinib reduced the number of CFUs formed by EMS-iPS-induced CD34+ cells in a dose-dependent manner, whereas imatinib did not. Similar effects were observed on primary peripheral blood cells (more than 90% of which were blasts) isolated from the patient. This study provides evidence that the EMS-iPS cell line is a useful tool for the screening of drugs to treat EMS and to investigate the mechanism underlying this disease.

GnT1IP-L specifically inhibits MGAT1 in the Golgi via its luminal domain.

PubMed

Huang, Hung-Hsiang; Hassinen, Antti; Sundaram, Subha; Spiess, Andrej-Nikolai; Kellokumpu, Sakari; Stanley, Pamela

2015-09-15

Mouse GnT1IP-L, and membrane-bound GnT1IP-S (MGAT4D) expressed in cultured cells inhibit MGAT1, the N-acetylglucosaminyltransferase that initiates the synthesis of hybrid and complex N-glycans. However, it is not known where in the secretory pathway GnT1IP-L inhibits MGAT1, nor whether GnT1IP-L inhibits other N-glycan branching N-acetylglucosaminyltransferases of the medial Golgi. We show here that the luminal domain of GnT1IP-L contains its inhibitory activity. Retention of GnT1IP-L in the endoplasmic reticulum (ER) via the N-terminal region of human invariant chain p33, with or without C-terminal KDEL, markedly reduced inhibitory activity. Dynamic fluorescent resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) assays revealed homomeric interactions for GnT1IP-L in the ER, and heteromeric interactions with MGAT1 in the Golgi. GnT1IP-L did not generate a FRET signal with MGAT2, MGAT3, MGAT4B or MGAT5 medial Golgi GlcNAc-tranferases. GnT1IP/Mgat4d transcripts are expressed predominantly in spermatocytes and spermatids in mouse, and are reduced in men with impaired spermatogenesis.

Active generation and propagation of Ca2+ signals within tunneling membrane nanotubes.

PubMed

Smith, Ian F; Shuai, Jianwei; Parker, Ian

2011-04-20

A new mechanism of cell-cell communication was recently proposed after the discovery of tunneling nanotubes (TNTs) between cells. TNTs are membrane protrusions with lengths of tens of microns and diameters of a few hundred nanometers that permit the exchange of membrane and cytoplasmic constituents between neighboring cells. TNTs have been reported to mediate intercellular Ca(2+) signaling; however, our simulations indicate that passive diffusion of Ca(2+) ions alone would be inadequate for efficient transmission between cells. Instead, we observed spontaneous and inositol trisphosphate (IP(3))-evoked Ca(2+) signals within TNTs between cultured mammalian cells, which sometimes remained localized and in other instances propagated as saltatory waves to evoke Ca(2+) signals in a connected cell. Consistent with this, immunostaining showed the presence of both endoplasmic reticulum and IP(3) receptors along the TNT. We propose that IP(3) receptors may actively propagate intercellular Ca(2+) signals along TNTs via Ca(2+)-induced Ca(2+) release, acting as amplification sites to overcome the limitations of passive diffusion in a chemical analog of electrical transmission of action potentials. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Human Biomarker Discovery and Predictive Models for Disease Progression for Idiopathic Pneumonia Syndrome Following Allogeneic Stem Cell Transplantation*

PubMed Central

Schlatzer, Daniela M.; Dazard, Jean-Eudes; Ewing, Rob M.; Ilchenko, Serguei; Tomcheko, Sara E.; Eid, Saada; Ho, Vincent; Yanik, Greg; Chance, Mark R.; Cooke, Kenneth R.

2012-01-01

Allogeneic hematopoietic stem cell transplantation (SCT) is the only curative therapy for many malignant and nonmalignant conditions. Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication that limits successful outcomes. Preclinical models suggest that IPS represents an immune mediated attack on the lung involving elements of both the adaptive and the innate immune system. However, the etiology of IPS in humans is less well understood. To explore the disease pathway and uncover potential biomarkers of disease, we performed two separate label-free, proteomics experiments defining the plasma protein profiles of allogeneic SCT patients with IPS. Samples obtained from SCT recipients without complications served as controls. The initial discovery study, intended to explore the disease pathway in humans, identified a set of 81 IPS-associated proteins. These data revealed similarities between the known IPS pathways in mice and the condition in humans, in particular in the acute phase response. In addition, pattern recognition pathways were judged to be significant as a function of development of IPS, and from this pathway we chose the lipopolysaccaharide-binding protein (LBP) protein as a candidate molecular diagnostic for IPS, and verified its increase as a function of disease using an ELISA assay. In a separately designed study, we identified protein-based classifiers that could predict, at day 0 of SCT, patients who: 1) progress to IPS and 2) respond to cytokine neutralization therapy. Using cross-validation strategies, we built highly predictive classifier models of both disease progression and therapeutic response. In sum, data generated in this report confirm previous clinical and experimental findings, provide new insights into the pathophysiology of IPS, identify potential molecular classifiers of the condition, and uncover a set of markers potentially of interest for patient stratification as a basis for individualized therapy. PMID:22337588

Upregulation of neurovascular communication through filamin abrogation promotes ectopic periventricular neurogenesis.

PubMed

Houlihan, Shauna L; Lanctot, Alison A; Guo, Yan; Feng, Yuanyi

2016-09-24

Neuronal fate-restricted intermediate progenitors (IPs) are derived from the multipotent radial glia (RGs) and serve as the direct precursors for cerebral cortical neurons, but factors that control their neurogenic plasticity remain elusive. Here we report that IPs' neuron production is enhanced by abrogating filamin function, leading to the generation of periventricular neurons independent of normal neocortical neurogenesis and neuronal migration. Loss of Flna in neural progenitor cells (NPCs) led RGs to undergo changes resembling epithelial-mesenchymal transition (EMT) along with exuberant angiogenesis that together changed the microenvironment and increased neurogenesis of IPs. We show that by collaborating with β-arrestin, Flna maintains the homeostatic signaling between the vasculature and NPCs, and loss of this function results in escalated Vegfa and Igf2 signaling, which exacerbates both EMT and angiogenesis to further potentiate IPs' neurogenesis. These results suggest that the neurogenic potential of IPs may be boosted in vivo by manipulating Flna-mediated neurovascular communication.

Normalization, bias correction, and peak calling for ChIP-seq

PubMed Central

Diaz, Aaron; Park, Kiyoub; Lim, Daniel A.; Song, Jun S.

2012-01-01

Next-generation sequencing is rapidly transforming our ability to profile the transcriptional, genetic, and epigenetic states of a cell. In particular, sequencing DNA from the immunoprecipitation of protein-DNA complexes (ChIP-seq) and methylated DNA (MeDIP-seq) can reveal the locations of protein binding sites and epigenetic modifications. These approaches contain numerous biases which may significantly influence the interpretation of the resulting data. Rigorous computational methods for detecting and removing such biases are still lacking. Also, multi-sample normalization still remains an important open problem. This theoretical paper systematically characterizes the biases and properties of ChIP-seq data by comparing 62 separate publicly available datasets, using rigorous statistical models and signal processing techniques. Statistical methods for separating ChIP-seq signal from background noise, as well as correcting enrichment test statistics for sequence-dependent and sonication biases, are presented. Our method effectively separates reads into signal and background components prior to normalization, improving the signal-to-noise ratio. Moreover, most peak callers currently use a generic null model which suffers from low specificity at the sensitivity level requisite for detecting subtle, but true, ChIP enrichment. The proposed method of determining a cell type-specific null model, which accounts for cell type-specific biases, is shown to be capable of achieving a lower false discovery rate at a given significance threshold than current methods. PMID:22499706

Improvement of mouse cloning using nuclear transfer-derived embryonic stem cells and/or histone deacetylase inhibitor.

PubMed

Wakayama, Sayaka; Wakayama, Teruhiko

2010-01-01

Nuclear transfer-derived ES (ntES) cell lines can be established from somatic cell nuclei with a relatively high success rate. Although ntES cells have been shown to be equivalent to ES cells, there are ethical objections concerning human cells, such as the use of fresh oocyte donation from young healthy woman. In contrast, the use of induced pluripotent stem (iPS) cells for cloning poses few ethical problems and is a relatively easy technique compared with nuclear transfer. Therefore, although there are several reports proposing the use of ntES cells as a model of regenerative medicine, the use of these cells in preliminary medical research is waning. However, in theory, 5 to 10 donor cells can establish one ntES cell line and, once established, these cells will propagate indefinitely. These cells can be used to generate cloned animals from ntES cell lines using a second round of NT. Even in infertile and "unclonable" strains of mice, we can generate offspring from somatic cells by combining cloning with ntES technology. Moreover, cloned offspring can be generated potentially even from the nuclei of dead bodies or freeze-dried cells via ntES cells, such as from an extinct frozen animal. Currently, only the ntES technology is available for this purpose, because all other techniques, including iPS cell derivation, require significant numbers of living donor cells. This review describes how to improve the efficiency of cloning, the establishment of clone-derived embryonic stem cells and further applications.

Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes.

PubMed

Kodama, Nao; Iwao, Takahiro; Kabeya, Tomoki; Horikawa, Takashi; Niwa, Takuro; Kondo, Yuki; Nakamura, Katsunori; Matsunaga, Tamihide

2016-06-01

We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

Comparison of gating dynamics of different IP3R channels with immune algorithm searching for channel parameter distributions

NASA Astrophysics Data System (ADS)

Cai, Xiuhong; Li, Xiang; Qi, Hong; Wei, Fang; Chen, Jianyong; Shuai, Jianwei

2016-10-01

The gating properties of the inositol 1, 4, 5-trisphosphate (IP3) receptor (IP3R) are determined by the binding and unbinding capability of Ca2+ ions and IP3 messengers. With the patch clamp experiments, the stationary properties have been discussed for Xenopus oocyte type-1 IP3R (Oo-IP3R1), type-3 IP3R (Oo-IP3R3) and Spodoptera frugiperda IP3R (Sf-IP3R). In this paper, in order to provide insights about the relation between the observed gating characteristics and the gating parameters in different IP3Rs, we apply the immune algorithm to fit the parameters of a modified DeYoung-Keizer model. By comparing the fitting parameter distributions of three IP3Rs, we suggest that the three types of IP3Rs have the similar open sensitivity in responding to IP3. The Oo-IP3R3 channel is easy to open in responding to low Ca2+ concentration, while Sf-IP3R channel is easily inhibited in responding to high Ca2+ concentration. We also show that the IP3 binding rate is not a sensitive parameter for stationary gating dynamics for three IP3Rs, but the inhibitory Ca2+ binding/unbinding rates are sensitive parameters for gating dynamics for both Oo-IP3R1 and Oo-IP3R3 channels. Such differences may be important in generating the spatially and temporally complex Ca2+ oscillations in cells. Our study also demonstrates that the immune algorithm can be applied for model parameter searching in biological systems.

iPS Cell Cultures from a Gerstmann-Sträussler-Scheinker Patient with the Y218N PRNP Mutation Recapitulate tau Pathology.

PubMed

Matamoros-Angles, Andreu; Gayosso, Lucía Mayela; Richaud-Patin, Yvonne; di Domenico, Angelique; Vergara, Cristina; Hervera, Arnau; Sousa, Amaya; Fernández-Borges, Natalia; Consiglio, Antonella; Gavín, Rosalina; López de Maturana, Rakel; Ferrer, Isidro; López de Munain, Adolfo; Raya, Ángel; Castilla, Joaquín; Sánchez-Pernaute, Rosario; Del Río, José Antonio

2018-04-01

Gerstmann-Sträussler-Scheinker (GSS) syndrome is a fatal autosomal dominant neurodegenerative prionopathy clinically characterized by ataxia, spastic paraparesis, extrapyramidal signs and dementia. In some GSS familiar cases carrying point mutations in the PRNP gene, patients also showed comorbid tauopathy leading to mixed pathologies. In this study we developed an induced pluripotent stem (iPS) cell model derived from fibroblasts of a GSS patient harboring the Y218N PRNP mutation, as well as an age-matched healthy control. This particular PRNP mutation is unique with very few described cases. One of the cases presented neurofibrillary degeneration with relevant Tau hyperphosphorylation. Y218N iPS-derived cultures showed relevant astrogliosis, increased phospho-Tau, altered microtubule-associated transport and cell death. However, they failed to generate proteinase K-resistant prion. In this study we set out to test, for the first time, whether iPS cell-derived neurons could be used to investigate the appearance of disease-related phenotypes (i.e, tauopathy) identified in the GSS patient.

Lipid-derived free radical production in superantigen-induced interstitial pneumonia

PubMed Central

Miyakawa, Hisako; Mason, Ronald P.; Jiang, JinJie; Kadiiska, Maria B.

2009-01-01

We studied the free radical generation involved in the development of interstitial pneumonia (IP) in an animal model of autoimmune disease. We observed an electron spin resonance (ESR) spectrum of α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) radical adducts detected in the lipid extract of lungs in autoimmune-prone mice after intratracheal instillation of staphylococcal enterotoxin B. The POBN adducts detected by ESR were paralleled by infiltration of macrophages and neutrophils in the bronchoalveolar lavage fluid. To further investigate the mechanism of free radical generation, mice were pretreated with the macrophage toxicant gadolinium chloride, which significantly suppressed the radical generation. Free radical generation was also decreased by pretreatment with the xanthine oxidase (XO) inhibitor allopurinol, the iron chelator Desferal, and the inducible nitric oxide synthase (iNOS) inhibitor 1400W. Histopathologically, these drugs significantly reduced both the cell infiltration to alveolar septal walls and the synthesis of pulmonary collagen fibers. Experiments with NADPH oxidase knockout mice showed that NADPH oxidase did not contribute to lipid radical generation. These results suggest that lipid-derived carbon-centered free radical production is important in the manifestation of IP and that a macrophage toxicant, an XO inhibitor, an iron chelator, and an iNOS inhibitor protect against both radical generation and the manifestation of IP. PMID:19376221

Optical reprogramming with ultrashort femtosecond laser pulses

NASA Astrophysics Data System (ADS)

Uchugonova, Aisada; Breunig, Hans G.; Batista, Ana; König, Karsten

2015-03-01

The use of sub-15 femtosecond laser pulses in stem cell research is explored with particular emphasis on the optical reprogramming of somatic cells. The reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be evoked through the ectopic expression of defined transcription factors. Conventional approaches utilize retro/lenti-viruses to deliver genes/transcription factors as well as to facilitate the integration of transcription factors into that of the host genome. However, the use of viruses may result in insertional mutations caused by the random integration of genes and as a result, this may limit the use within clinical applications due to the risk of the formation of cancer. In this study, a new approach is demonstrated in realizing non-viral reprogramming through the use of ultrashort laser pulses, to introduce transcription factors into the cell so as to generate iPS cells.

MLF1 interacting protein: a potential gene therapy target for human prostate cancer?

PubMed

Zhang, Lei; Ji, Guoqing; Shao, Yuzhang; Qiao, Shaoyi; Jing, Yuming; Qin, Rongliang; Sun, Huiming; Shao, Chen

2015-02-01

Here, we investigated the role of one gene that has been previously associated with human prostate carcinoma cells-myelodysplasia/myeloid leukemia factor 1 interacting protein (MLF1IP)-in order to better ascertain its role in human prostate carcinogenesis. The prostate cancer cell line PC-3 was lentivirally transfected to silence endogenous MLF1IP gene expression, which was confirmed by real-time quantitative PCR (RT-qPCR). Cellomics ArrayScan VTI imaging and MTT assays were conducted to assess cell proliferation. Cell cycle phase arrest and apoptosis were assayed by flow cytometry. Colony formation was assessed by fluorescence microscopy. MLF1IP gene expression was also analyzed by RT-qPCR in sixteen prostate cancer tissue samples and six healthy control prostate tissue samples from human patients. Cell proliferation was significantly inhibited in MLF1IP-silenced cells relative to control cells. G1 phase, S and G2/M phase cell counts were not significantly changed in MLF1IP-silenced cells relative to control cells. Apoptosis was significantly increased in MLF1IP-silenced cells, while MLF1IP-silenced cells displayed a significantly reduced number of cell colonies, compared to control cells. The 16 human prostate cancer tissue samples revealed no clear upregulation or downregulation in MLF1IP gene expression. MLF1IP significantly promotes prostate cancer cell proliferation and colony formation and significantly inhibits apoptosis without affecting cell cycle phase arrest. Further study is required to conclusively determine whether MLF1IP is upregulated in human prostate cancer tumors and to determine the precise cellular mechanism(s) for MLF1IP in prostate carcinogenesis.

Human Decidua-Derived Mesenchymal Cells Are a Promising Source for the Generation and Cell Banking of Human Induced Pluripotent Stem Cells

PubMed Central

Shofuda, Tomoko; Kanematsu, Daisuke; Fukusumi, Hayato; Yamamoto, Atsuyo; Bamba, Yohei; Yoshitatsu, Sumiko; Suemizu, Hiroshi; Nakamura, Masato; Sugimoto, Yoshikazu; Furue, Miho Kusuda; Kohara, Arihiro; Akamatsu, Wado; Okada, Yohei; Okano, Hideyuki; Yamasaki, Mami; Kanemura, Yonehiro

2013-01-01

Placental tissue is a biomaterial with remarkable potential for use in regenerative medicine. It has a three-layer structure derived from the fetus (amnion and chorion) and the mother (decidua), and it contains huge numbers of cells. Moreover, placental tissue can be collected without any physical danger to the donor and can be matched with a variety of HLA types. The decidua-derived mesenchymal cells (DMCs) are highly proliferative fibroblast-like cells that express a similar pattern of CD antigens as bone marrow-derived mesenchymal cells (BM-MSCs). Here we demonstrated that induced pluripotent stem (iPS) cells could be efficiently generated from DMCs by retroviral transfer of reprogramming factor genes. DMC-hiPS cells showed equivalent characteristics to human embryonic stem cells (hESCs) in colony morphology, global gene expression profile (including human pluripotent stem cell markers), DNA methylation status of the OCT3/4 and NANOG promoters, and ability to differentiate into components of the three germ layers in vitro and in vivo. The RNA expression of XIST and the methylation status of its promoter region suggested that DMC-iPSCs, when maintained undifferentiated and pluripotent, had three distinct states: (1) complete X-chromosome reactivation, (2) one inactive X-chromosome, or (3) an epigenetic aberration. Because DMCs are derived from the maternal portion of the placenta, they can be collected with the full consent of the adult donor and have considerable ethical advantages for cell banking and the subsequent generation of human iPS cells for regenerative applications. PMID:26858858

Morphological Analysis of Live Undifferentiated Cells Derived from Induced Pluripotent Stem Cells.

PubMed

Osawa, Yukihiko; Miyamoto, Tomoyuki; Ohno, Setsuyo; Ohno, Eiji

2018-01-01

Induced pluripotent stem (iPS) cells possess pluripotency and self-renewal ability. Therefore, iPS cells are expected to be useful in regenerative medicine. However, iPS cells form malignant immature teratomas after transplantation into animals, even after differentiation induction. It has been suggested that undifferentiated cells expressing Nanog that remain after differentiation induction are responsible for teratoma formation. Various methods of removing these undifferentiated cells have therefore been investigated, but few methods involve morphological approaches, which may induce less cell damage. In addition, for cells derived from iPS cells to be applied in regenerative medicine, they must be alive. However, detailed morphological analysis of live undifferentiated cells has not been performed. For the above reasons, we assessed the morphological features of live undifferentiated cells remaining after differentiation induction as a basic investigation into the clinical application of iPS cells. As a result, live undifferentiated cells remaining after differentiation induction exhibited a round or oval cytoplasm about 12 μm in diameter and a nucleus. They exhibited nucleo-cytoplasmic (N/C) ratio of about 60% and eccentric nuclei, and they possessed partially granule-like structures in the cytoplasm and prominent nucleoli. Although they were similar to iPS cells, they were smaller than live iPS cells. Furthermore, very small cells were present among undifferentiated cells after differentiation induction. These results suggest that the removal of undifferentiated cells may be possible using the morphological features of live iPS cells and undifferentiated cells after differentiation induction. In addition, this study supports safe regenerative medicine using iPS cells.

Regulation of 1, 4, 5-triphosphate receptor channel gating dynamics by mutant presenilin in Alzheimer's disease cells

NASA Astrophysics Data System (ADS)

Wei, Fang; Li, Xiang; Cai, Meichun; Liu, Yanping; Jung, Peter; Shuai, Jianwei

2017-06-01

In neurons of patients with Alzheimer's disease, the intracellular Ca2+ concentration is increased by its release from the endoplasmic reticulum via the inositol 1, 4, 5-triphosphate receptor (IP3R). In this paper, we discuss the IP3R gating dynamics in familial Alzheimer's disease (FAD) cells induced with presenilin mutation PS1. By fitting the parameters of an IP3R channel model to experimental data of the open probability, the mean open time and the mean closed time of IP3R channels, in control cells and FAD mutant cells, we suggest that the interaction of presenilin mutation PS1 with IP3R channels leads the decrease in the unbinding rates of IP3 and the activating Ca2+ from IP3Rs. As a result, the increased affinities of IP3 and activating Ca2+ for IP3R channels induce the increase in the Ca2+ signal in FAD mutant cells. Specifically, the PS1 mutation decreases the IP3 dissociation rate of IP3R channels significantly in FAD mutant cells. Our results suggest possible novel targets for FAD therapeutic intervention.

ERP44 inhibits human lung cancer cell migration mainly via IP3R2.

PubMed

Huang, Xue; Jin, Meng; Chen, Ying-Xiao; Wang, Jun; Zhai, Kui; Chang, Yan; Yuan, Qi; Yao, Kai-Tai; Ji, Guangju

2016-06-01

Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway.

ERP44 inhibits human lung cancer cell migration mainly via IP3R2

PubMed Central

Zhai, Kui; Chang, Yan; Yuan, Qi; Yao, Kai-Tai; Ji, Guangju

2016-01-01

Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway. PMID:27347718

Chick derived induced pluripotent stem cells by the poly-cistronic transposon with enhanced transcriptional activity.

PubMed

Katayama, Masafumi; Hirayama, Takashi; Tani, Tetsuya; Nishimori, Katsuhiko; Onuma, Manabu; Fukuda, Tomokazu

2018-02-01

Induced pluripotent stem (iPS) cell technology lead terminally differentiated cells into the pluripotent stem cells through the expression of defined reprogramming factors. Although, iPS cells have been established in a number of mammalian species, including mouse, human, and monkey, studies on iPS cells in avian species are still very limited. To establish chick iPS cells, six factors were used within the poly-cistronic reprogramming vector (PB-R6F), containing M3O (MyoD derived transactivation domain fused with Oct3/4), Sox2, Klf4, c-Myc, Lin28, and Nanog. The PB-R6F derived iPS cells were alkaline-phosphatase and SSEA-1 positive, which are markers of pluripotency. Elevated levels of endogenous Oct3/4 and Nanog genes were detected in the established iPS cells, suggesting the activation of the FGF signaling pathway is critical for the pluripotent status. Histological analysis of teratoma revealed that the established chick iPS cells have differentiation ability into three-germ-layer derived tissues. This is the first report of establishment of avian derived iPS cells with a single poly-cistronic transposon based expression system. The establishment of avian derived iPS cells could contribute to the genetic conservation and modification of avian species. © 2017 Wiley Periodicals, Inc.

Wnt3a stimulates maturation of impaired neutrophils developed from severe congenital neutropenia patient-derived pluripotent stem cells.

PubMed

Hiramoto, Takafumi; Ebihara, Yasuhiro; Mizoguchi, Yoko; Nakamura, Kazuhiro; Yamaguchi, Kiyoshi; Ueno, Kazuko; Nariai, Naoki; Mochizuki, Shinji; Yamamoto, Shohei; Nagasaki, Masao; Furukawa, Yoichi; Tani, Kenzaburo; Nakauchi, Hiromitsu; Kobayashi, Masao; Tsuji, Kohichiro

2013-02-19

The derivation of induced pluripotent stem (iPS) cells from individuals of genetic disorders offers new opportunities for basic research into these diseases and the development of therapeutic compounds. Severe congenital neutropenia (SCN) is a serious disorder characterized by severe neutropenia at birth. SCN is associated with heterozygous mutations in the neutrophil elastase [elastase, neutrophil-expressed (ELANE)] gene, but the mechanisms that disrupt neutrophil development have not yet been clarified because of the current lack of an appropriate disease model. Here, we generated iPS cells from an individual with SCN (SCN-iPS cells). Granulopoiesis from SCN-iPS cells revealed neutrophil maturation arrest and little sensitivity to granulocyte-colony stimulating factor, reflecting a disease status of SCN. Molecular analysis of the granulopoiesis from the SCN-iPS cells vs. control iPS cells showed reduced expression of genes related to the wingless-type mmtv integration site family, member 3a (Wnt3a)/β-catenin pathway [e.g., lymphoid enhancer-binding factor 1], whereas Wnt3a administration induced elevation lymphoid enhancer-binding factor 1-expression and the maturation of SCN-iPS cell-derived neutrophils. These results indicate that SCN-iPS cells provide a useful disease model for SCN, and the activation of the Wnt3a/β-catenin pathway may offer a novel therapy for SCN with ELANE mutation.

TOPICAL REVIEW: Stem cell technology using bioceramics: hard tissue regeneration towards clinical application

NASA Astrophysics Data System (ADS)

Ohnishi, Hiroe; Oda, Yasuaki; Ohgushi, Hajime

2010-02-01

Mesenchymal stem cells (MSCs) are adult stem cells which show differentiation capabilities toward various cell lineages. We have already used MSCs for treatments of osteoarthritis, bone necrosis and bone tumor. For this purpose, culture expanded MSCs were combined with various ceramics and then implanted. Because of rejection response to allogeneic MSC implantation, we have utilized patients' own MSCs for the treatment. Bone marrow is a good cell source of MSCs, although the MSCs also exist in adipose tissue. When comparing osteogenic differentiation of these MSCs, bone marrow MSCs show more extensive bone forming capability than adipose MSCs. Thus, the bone marrow MSCs are useful for bone tissue regeneration. However, the MSCs show limited proliferation and differentiation capabilities that hindered clinical applications in some cases. Recent advances reveal that transduction of plural transcription factors into human adult cells results in generation of new type of stem cells called induced pluripotent stem cells (iPS cells). A drawback of the iPS cells for clinical applications is tumor formation after their in vivo implantation; therefore it is difficult to use iPS cells for the treatment. To circumvent the problem, we transduced a single factor of either SOX2 or NANOG into the MSCs and found high proliferation as well as osteogenic differentiation capabilities of the MSCs. The stem cells could be combined with bioceramics for clinical applications. Here, we summarize our recent technologies using adult stem cells in viewpoints of bone tissue regeneration.

Mechanism of proteasomal degradation of inositol trisphosphate receptors in CHO-K1 cells.

PubMed

Bhanumathy, Cunnigaiper D; Nakao, Steven K; Joseph, Suresh K

2006-02-10

myo-Inositol 1,4,5-trisphosphate receptor (IP3R) degradation occurs in response to carbachol (Cch) stimulation of CHO-K1 cells. The response was mediated by endogenous muscarinic receptors and was blocked by atropine or proteasomal inhibitors. We have used these cells to identify the sites of ubiquitination on IP3Rs and study the role of Ca2+ and substrate recognition properties of the degradation system using exogenously expressed IP3R constructs. Employing caspase-3 for IP3R cleavage, we show that Cch promotes polyubiquitination in the N-terminal domain and monoubiquitination in the C-terminal domain. The addition of extracellular Ca2+ to Ca2+-depleted Chinese hamster ovary (CHO) cells initiates IP3R degradation provided Cch is present. This effect is inhibited by thapsigargin. The data suggest that both a sustained elevation of IP3 and a minimal content of Ca2+ in the endoplasmic reticulum lumen is required to initiate IP3R degradation. Transient transfection of IP3R constructs into CHO cells indicated the selective degradation of only the SI+ splice variant of the type I IP3R. This was also the splice form present endogenously in these cells. A pore-defective, nonfunctional SI+ IP3R mutant (D2550A) was also degraded in Cch-stimulated cells. The Cch-mediated response in CHO cells provides a convenient model system to further analyze the Ca2+ dependence and structural requirements of the IP3R proteasomal degradation pathway.

Characterization of inositol phosphates in carrot (Daucus carota L. ) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rincon, M.; Chen, Q.; Boss, W.F.

1989-01-01

We have shown previously that inositol-1,4,5-trisphosphate (IP{sub 3}) stimulates an efflux of {sup 45}Ca{sup 2+} from fusogenic carrot protoplasts. In light of these results, we suggested that IP{sub 3} might serve as a second messenger for the mobilization of intracellular Ca{sup 2+} in higher plant cells. To determine whether or not IP{sub 3} and other inositol phosphates were present in the carrot cells, the cells were labeled with myo-(2-{sup 3}H)inositol for 18 hours and extracted with ice-cold 10% trichloroacetic acid. The inositol metabolites were separated by anion exchange chromatography and by paper electrophoresis. We found that ({sup 3}H)inositol metabolites coelutedmore » with inositol bisphosphate (IP{sub 2}) and IP{sub 3} when separated by anion exchange chromatography. However, we could not detect IP{sub 2} or IP{sub 3} when the inositol metabolites were analyzed by paper electrophoresis even though the polyphosphoinositides, which are the source of IP{sub 2} and IP{sub 3}, were present in these cells. Thus, ({sup 3}H)inositol metabolites other than IP{sub 2} and IP{sub 3} had coeluted on the anion exchange columns. The data indicate that either IP{sub 3} is rapidly metabolized or that it is not present at a detectable level in the carrot cells.« less

Central and Peripheral Mechanisms of Antipsychotic Medication-Induced Metabolic Dysregulation

DTIC Science & Technology

2016-10-01

generation APD haloperidol or second-generation APD olanzapine (via i.p. administration). We will also measure serum fasting glucose and insulin levels...food consumption in beta cell-specific D2R (and wildtype littermate controls) treated with either with first- generation APD haloperidol or second...effects of D2R agonists (dopamine, quinpirole) versus APD antagonists ( haloperidol and olanzapine). § We will treat pancreatic islets from wildtype

Myostatin acts as an autocrine/paracrine negative regulator in myoblast differentiation from human induced pluripotent stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gao, Fei; Kishida, Tsunao; Ejima, Akika

Highlights: ► iPS-derived cells express myostatin and its receptor upon myoblast differentiation. ► Myostatin inhibits myoblast differentiation by inhibiting MyoD and Myo5a induction. ► Silencing of myostatin promotes differentiation of human iPS cells into myoblasts. -- Abstract: Myostatin, also known as growth differentiation factor (GDF-8), regulates proliferation of muscle satellite cells, and suppresses differentiation of myoblasts into myotubes via down-regulation of key myogenic differentiation factors including MyoD. Recent advances in stem cell biology have enabled generation of myoblasts from pluripotent stem cells, but it remains to be clarified whether myostatin is also involved in regulation of artificial differentiation of myoblastsmore » from pluripotent stem cells. Here we show that the human induced pluripotent stem (iPS) cell-derived cells that were induced to differentiate into myoblasts expressed myostatin and its receptor during the differentiation. An addition of recombinant human myostatin (rhMyostatin) suppressed induction of MyoD and Myo5a, resulting in significant suppression of myoblast differentiation. The rhMyostatin treatment also inhibited proliferation of the cells at a later phase of differentiation. RNAi-mediated silencing of myostatin promoted differentiation of human iPS-derived embryoid body (EB) cells into myoblasts. These results strongly suggest that myostatin plays an important role in regulation of myoblast differentiation from iPS cells of human origin. The present findings also have significant implications for potential regenerative medicine for muscular diseases.« less

A ChIP-Seq Data Analysis Pipeline Based on Bioconductor Packages.

PubMed

Park, Seung-Jin; Kim, Jong-Hwan; Yoon, Byung-Ha; Kim, Seon-Young

2017-03-01

Nowadays, huge volumes of chromatin immunoprecipitation-sequencing (ChIP-Seq) data are generated to increase the knowledge on DNA-protein interactions in the cell, and accordingly, many tools have been developed for ChIP-Seq analysis. Here, we provide an example of a streamlined workflow for ChIP-Seq data analysis composed of only four packages in Bioconductor: dada2, QuasR, mosaics, and ChIPseeker. 'dada2' performs trimming of the high-throughput sequencing data. 'QuasR' and 'mosaics' perform quality control and mapping of the input reads to the reference genome and peak calling, respectively. Finally, 'ChIPseeker' performs annotation and visualization of the called peaks. This workflow runs well independently of operating systems (e.g., Windows, Mac, or Linux) and processes the input fastq files into various results in one run. R code is available at github: https://github.com/ddhb/Workflow_of_Chipseq.git.

A ChIP-Seq Data Analysis Pipeline Based on Bioconductor Packages

PubMed Central

Park, Seung-Jin; Kim, Jong-Hwan; Yoon, Byung-Ha; Kim, Seon-Young

2017-01-01

Nowadays, huge volumes of chromatin immunoprecipitation-sequencing (ChIP-Seq) data are generated to increase the knowledge on DNA-protein interactions in the cell, and accordingly, many tools have been developed for ChIP-Seq analysis. Here, we provide an example of a streamlined workflow for ChIP-Seq data analysis composed of only four packages in Bioconductor: dada2, QuasR, mosaics, and ChIPseeker. ‘dada2’ performs trimming of the high-throughput sequencing data. ‘QuasR’ and ‘mosaics’ perform quality control and mapping of the input reads to the reference genome and peak calling, respectively. Finally, ‘ChIPseeker’ performs annotation and visualization of the called peaks. This workflow runs well independently of operating systems (e.g., Windows, Mac, or Linux) and processes the input fastq files into various results in one run. R code is available at github: https://github.com/ddhb/Workflow_of_Chipseq.git. PMID:28416945

Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation

PubMed Central

Sauer, Karsten; Huang, Yina Hsing; Lin, Hongying; Sandberg, Mark; Mayr, Georg W.

2015-01-01

Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5-trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+ signaling in lymphocytes. IP3 phosphorylation by IP3 3-kinases generates inositol 1,3,4,5-tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3 in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high-performance liquid chromatographic separation methods followed by radiolabeling or non-radioactive metal-dye detection. Finally, less broadly applicable nonchromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+ thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development. PMID:19918943

Arpeggio: harmonic compression of ChIP-seq data reveals protein-chromatin interaction signatures

PubMed Central

Stanton, Kelly Patrick; Parisi, Fabio; Strino, Francesco; Rabin, Neta; Asp, Patrik; Kluger, Yuval

2013-01-01

Researchers generating new genome-wide data in an exploratory sequencing study can gain biological insights by comparing their data with well-annotated data sets possessing similar genomic patterns. Data compression techniques are needed for efficient comparisons of a new genomic experiment with large repositories of publicly available profiles. Furthermore, data representations that allow comparisons of genomic signals from different platforms and across species enhance our ability to leverage these large repositories. Here, we present a signal processing approach that characterizes protein–chromatin interaction patterns at length scales of several kilobases. This allows us to efficiently compare numerous chromatin-immunoprecipitation sequencing (ChIP-seq) data sets consisting of many types of DNA-binding proteins collected from a variety of cells, conditions and organisms. Importantly, these interaction patterns broadly reflect the biological properties of the binding events. To generate these profiles, termed Arpeggio profiles, we applied harmonic deconvolution techniques to the autocorrelation profiles of the ChIP-seq signals. We used 806 publicly available ChIP-seq experiments and showed that Arpeggio profiles with similar spectral densities shared biological properties. Arpeggio profiles of ChIP-seq data sets revealed characteristics that are not easily detected by standard peak finders. They also allowed us to relate sequencing data sets from different genomes, experimental platforms and protocols. Arpeggio is freely available at http://sourceforge.net/p/arpeggio/wiki/Home/. PMID:23873955

Arpeggio: harmonic compression of ChIP-seq data reveals protein-chromatin interaction signatures.

PubMed

Stanton, Kelly Patrick; Parisi, Fabio; Strino, Francesco; Rabin, Neta; Asp, Patrik; Kluger, Yuval

2013-09-01

Researchers generating new genome-wide data in an exploratory sequencing study can gain biological insights by comparing their data with well-annotated data sets possessing similar genomic patterns. Data compression techniques are needed for efficient comparisons of a new genomic experiment with large repositories of publicly available profiles. Furthermore, data representations that allow comparisons of genomic signals from different platforms and across species enhance our ability to leverage these large repositories. Here, we present a signal processing approach that characterizes protein-chromatin interaction patterns at length scales of several kilobases. This allows us to efficiently compare numerous chromatin-immunoprecipitation sequencing (ChIP-seq) data sets consisting of many types of DNA-binding proteins collected from a variety of cells, conditions and organisms. Importantly, these interaction patterns broadly reflect the biological properties of the binding events. To generate these profiles, termed Arpeggio profiles, we applied harmonic deconvolution techniques to the autocorrelation profiles of the ChIP-seq signals. We used 806 publicly available ChIP-seq experiments and showed that Arpeggio profiles with similar spectral densities shared biological properties. Arpeggio profiles of ChIP-seq data sets revealed characteristics that are not easily detected by standard peak finders. They also allowed us to relate sequencing data sets from different genomes, experimental platforms and protocols. Arpeggio is freely available at http://sourceforge.net/p/arpeggio/wiki/Home/.

Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.

PubMed

Li, Hongmei Lisa; Gee, Peter; Ishida, Kentaro; Hotta, Akitsu

2016-05-15

Precise gene correction using the CRISPR-Cas9 system in human iPS cells holds great promise for various applications, such as the study of gene functions, disease modeling, and gene therapy. In this review article, we summarize methods for effective editing of genomic sequences of iPS cells based on our experiences correcting dystrophin gene mutations with the CRISPR-Cas9 system. Designing specific sgRNAs as well as having efficient transfection methods and proper detection assays to assess genomic cleavage activities are critical for successful genome editing in iPS cells. In addition, because iPS cells are fragile by nature when dissociated into single cells, a step-by-step confirmation during the cell recovery process is recommended to obtain an adequate number of genome-edited iPS cell clones. We hope that the techniques described here will be useful for researchers from diverse backgrounds who would like to perform genome editing in iPS cells. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Type I intrinsically photosensitive retinal ganglion cells of early post-natal development correspond to the M4 subtype.

PubMed

Sexton, Timothy J; Bleckert, Adam; Turner, Maxwell H; Van Gelder, Russell N

2015-06-21

Intrinsically photosensitive retinal ganglion cells (ipRGCs) mediate circadian light entrainment and the pupillary light response in adult mice. In early development these cells mediate different processes, including negative phototaxis and the timing of retinal vascular development. To determine if ipRGC physiologic properties also change with development, we measured ipRGC cell density and light responses in wild-type mouse retinas at post-natal days 8, 15 and 30. Melanopsin-positive cell density decreases by 17% between post-natal days 8 and 15 and by 25% between days 8 and 30. This decrease is due specifically to a decrease in cells co-labeled with a SMI-32, a marker for alpha-on ganglion cells (corresponding to adult morphologic type M4 ipRGCs). On multi-electrode array recordings, post-natal day 8 (P8) ipRGC light responses show more robust firing, reduced adaptation and more rapid recovery from short and extended light pulses than do the light responses of P15 and P30 ipRGCs. Three ipRGC subtypes - Types I-III - have been defined in early development based on sensitivity and latency on multielectrode array recordings. We find that Type I cells largely account for the unique physiologic properties of P8 ipRGCs. Type I cells have previously been shown to have relatively short latencies and high sensitivity. We now show that Type I cells show have rapid and robust recovery from long and short bright light exposures compared with Type II and III cells, suggesting differential light adaptation mechanisms between cell types. By P15, Type I ipRGCs are no longer detectable. Loose patch recordings of P8 M4 ipRGCs demonstrate Type I physiology. Type I ipRGCs are found only in early development. In addition to their previously described high sensitivity and rapid kinetics, these cells are uniquely resistant to adaptation and recover quickly and fully to short and prolonged light exposure. Type I ipRGCs correspond to the SMI-32 positive, M4 subtype and largely lose melanopsin expression in development. These cells constitute a unique morphologic and physiologic class of ipRGCs functioning early in postnatal development.

EDD enhances cell survival and cisplatin resistance and is a therapeutic target for epithelial ovarian cancer

PubMed Central

Bradley, Amber; Zheng, Hui; Eblen, Scott T.

2014-01-01

The E3 ubiquitin ligase EDD is overexpressed in recurrent, platinum-resistant ovarian cancers, suggesting a role in tumor survival and/or platinum resistance. EDD knockdown by small interfering RNA (siRNA) induced apoptosis in A2780ip2, OVCAR5 and ES-2 ovarian cancer cells, correlating with loss of the prosurvival protein myeloid cell leukemia sequence 1 (Mcl-1) through a glycogen synthase kinase 3 beta-independent mechanism. SiRNA to EDD or Mcl-1 induced comparable levels of apoptosis in A2780ip2 and ES-2 cells. Stable overexpression of Mcl-1 protected cells from apoptosis following EDD knockdown, accompanied by a loss of endogenous, but not exogenous, Mcl-1 protein, suggesting that EDD regulated Mcl-1 synthesis. Indeed, EDD knockdown induced a 1.87-fold decrease in Mcl-1 messenger RNA and EDD transfection enhanced murine Mcl-1 promoter-driven luciferase expression 5-fold. To separate EDD survival and potential cisplatin resistance functions, we generated EDD shRNA stable cell lines that could survive initial EDD knockdown and showed that these cells were 4- to 21-fold more sensitive to cisplatin. Moreover, transient EDD overexpression in COS-7 cells was sufficient to promote cisplatin resistance 2.4-fold, dependent upon its E3 ligase activity. In vivo, mouse intraperitoneal ES-2 and A2780ip2 xenograft experiments showed that mice treated with EDD siRNA by nanoliposomal delivery [1,2-dioleoyl-sn-glycero-3-phophatidylcholine (DOPC)] and cisplatin had significantly less tumor burden than those treated with control siRNA/DOPC alone (ES-2, 77.9% reduction, P = 0.004; A2780ip2, 75.9% reduction, P = 0.042) or control siRNA/DOPC with cisplatin in ES-2 (64.4% reduction, P = 0.035), with a trend in A2780ip2 (60.3% reduction, P = 0.168). These results identify EDD as a dual regulator of cell survival and cisplatin resistance and suggest that EDD is a therapeutic target for ovarian cancer. PMID:24379240

Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function.

PubMed

Scherer, Paul C; Ding, Yan; Liu, Zhiqing; Xu, Jing; Mao, Haibin; Barrow, James C; Wei, Ning; Zheng, Ning; Snyder, Solomon H; Rao, Feng

2016-03-29

The family of cullin-RING E3 Ligases (CRLs) and the constitutive photomorphogenesis 9 (COP9) signalosome (CSN) form dynamic complexes that mediate ubiquitylation of 20% of the proteome, yet regulation of their assembly/disassembly remains poorly understood. Inositol polyphosphates are highly conserved signaling molecules implicated in diverse cellular processes. We now report that inositol hexakisphosphate (IP6) is a major physiologic determinant of the CRL-CSN interface, which includes a hitherto unidentified electrostatic interaction between the N-terminal acidic tail of CSN subunit 2 (CSN2) and a conserved basic canyon on cullins. IP6, with an EC50 of 20 nM, acts as an intermolecular "glue," increasing cullin-CSN2 binding affinity by 30-fold, thereby promoting assembly of the inactive CRL-CSN complexes. The IP6 synthase, Ins(1,3,4,5,6)P5 2-kinase (IPPK/IP5K) binds to cullins. Depleting IP5K increases the percentage of neddylated, active Cul1 and Cul4A, and decreases levels of the Cul1/4A substrates p27 and p21. Besides dysregulating CRL-mediated cell proliferation and UV-induced apoptosis, IP5K depletion potentiates by 28-fold the cytotoxic effect of the neddylation inhibitor MLN4924. Thus, IP5K and IP6 are evolutionarily conserved components of the CRL-CSN system and are potential targets for cancer therapy in conjunction with MLN4924.

Stem/progenitor cells from inflamed human dental pulp retain tissue regeneration potential

PubMed Central

Alongi, Dominick J; Yamaza, Takayoshi; Song, Yingjie; Fouad, Ashraf F; Romberg, Elaine E; Shi, Songtao; Tuan, Rocky S; Huang, George T-J

2011-01-01

Background Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs). Aims To determine whether DPSCs can be identified and isolated from IPs; and if they can be successfully cultured, whether they retain tissue regeneration potential in vivo. Materials & methods DPSCs from freshly collected normal pulps (NPs) and IPs were characterized in vitro and their tissue regeneration potential tested using an in vivo study model. Results The immunohistochemical analysis showed that IPs expressed higher levels of mesenchymal stem cell markers STRO-1, CD90, CD105 and CD146 compared with NPs (p < 0.05). Flow cytometry analysis showed that DPSCs from both NPs and IPs expressed moderate to high levels of CD146, stage-specific embryonic antigen-4, CD73 and CD166. Total population doubling of DPSCs-IPs (44.6 ± 2.9) was lower than that of DPSCs-NPs (58.9 ± 2.5) (p < 0.05), and DPSCs-IPs appeared to have a decreased osteo/dentinogenic potential compared with DPSCs-NPs based on the mineral deposition in cultures. Nonetheless, DPSCs-IPs formed pulp/dentin complexes similar to DPSCs-NPs when transplanted into immunocompromised mice. Conclusion DPSCs-IPs can be isolated and their mesenchymal stem cell marker profiles are similar to those from NPs. Although some stem cell properties of DPSCs-IPs were altered, cells from some samples remained potent in tissue regeneration in vivo. PMID:20465527

Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences

PubMed Central

Yu, Junying; Hu, Kejin; Smuga-Otto, Kim; Tian, Shulan; Stewart, Ron; Slukvin, Igor I.; Thomson, James A.

2009-01-01

Reprogramming differentiated human cells to induced pluripotent stem (iPS) cells has applications in basic biology, drug development, and transplantation. Human iPS cell derivation previously required vectors that integrate into the genome, which can create mutations and limit the utility of the cells in both research and clinical applications. Here we describe the derivation of human iPS cells using non-integrating episomal vectors. After removal of the episome, iPS cells completely free of vector and transgene sequences are derived that are similar to human embryonic stem (ES) cells in proliferative and developmental potential. These results demonstrate that reprogramming human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors, and removes one obstacle to the clinical application of human iPS cells. PMID:19325077

Induced Pluripotent Stem (iPS) Cells in Dentistry: A Review

PubMed Central

Malhotra, Neeraj

2016-01-01

iPS cells are derived from somatic cells via transduction and expression of selective transcription factors. Both viral-integrating (like retroviral) and non-integrating (like, mRNA or protein-based) techniques are available for the production of iPS cells. In the field of dentistry, iPS cells have been derived from stem cells of apical papilla, dental pulp stem cells, and stem cells from exfoliated deciduous teeth, gingival and periodontal ligament fibroblasts, and buccal mucosa fibroblasts. iPS cells have the potential to differentiate into all derivatives of the 3 primary germ layers i.e. ectoderm, endoderm, and mesoderm. They are autogeneically accessible, and can produce patient-specific or disease-specific cell lines without the issue of ethical controversy. They have been successfully tested to produce mesenchymal stem cells-like cells, neural crest-like cells, ameloblasts-like cells, odontoblasts-like cells, and osteoprogenitor cells. These cells can aid in regeneration of periodontal ligament, alveolar bone, cementum, dentin-pulp complex, as well as possible Biotooth formation. However certain key issues like, epigenetic memory of iPS cells, viral-transduction, tumorgenesis and teratoma formation need to be overcome, before they can be successfully used in clinical practice. The article discusses the sources, pros and cons, and current applications of iPS cells in dentistry with an emphasis on encountered challenges and their solutions. PMID:27572712

Inference of hierarchical regulatory network of estrogen-dependent breast cancer through ChIP-based data

PubMed Central

2010-01-01

Background Global profiling of in vivo protein-DNA interactions using ChIP-based technologies has evolved rapidly in recent years. Although many genome-wide studies have identified thousands of ERα binding sites and have revealed the associated transcription factor (TF) partners, such as AP1, FOXA1 and CEBP, little is known about ERα associated hierarchical transcriptional regulatory networks. Results In this study, we applied computational approaches to analyze three public available ChIP-based datasets: ChIP-seq, ChIP-PET and ChIP-chip, and to investigate the hierarchical regulatory network for ERα and ERα partner TFs regulation in estrogen-dependent breast cancer MCF7 cells. 16 common TFs and two common new TF partners (RORA and PITX2) were found among ChIP-seq, ChIP-chip and ChIP-PET datasets. The regulatory networks were constructed by scanning the ChIP-peak region with TF specific position weight matrix (PWM). A permutation test was performed to test the reliability of each connection of the network. We then used DREM software to perform gene ontology function analysis on the common genes. We found that FOS, PITX2, RORA and FOXA1 were involved in the up-regulated genes. We also conducted the ERα and Pol-II ChIP-seq experiments in tamoxifen resistance MCF7 cells (denoted as MCF7-T in this study) and compared the difference between MCF7 and MCF7-T cells. The result showed very little overlap between these two cells in terms of targeted genes (21.2% of common genes) and targeted TFs (25% of common TFs). The significant dissimilarity may indicate totally different transcriptional regulatory mechanisms between these two cancer cells. Conclusions Our study uncovers new estrogen-mediated regulatory networks by mining three ChIP-based data in MCF7 cells and ChIP-seq data in MCF7-T cells. We compared the different ChIP-based technologies as well as different breast cancer cells. Our computational analytical approach may guide biologists to further study the underlying mechanisms in breast cancer cells or other human diseases. PMID:21167036

Protection against cancer by dietary IP6 and inositol.

PubMed

Vucenik, Ivana; Shamsuddin, AbulKalam M

2006-01-01

Inositol hexaphosphate (IP(6)) is a naturally occurring polyphosphorylated carbohydrate, abundantly present in many plant sources and in certain high-fiber diets, such as cereals and legumes. In addition to being found in plants, IP(6) is contained in almost all mammalian cells, although in much smaller amounts, where it is important in regulating vital cellular functions such as signal transduction, cell proliferation, and differentiation. For a long time IP(6) has been recognized as a natural antioxidant. Recently IP(6) has received much attention for its role in cancer prevention and control of experimental tumor growth, progression, and metastasis. In addition, IP(6) possesses other significant benefits for human health, such as the ability to enhance immune system, prevent pathological calcification and kidney stone formation, lower elevated serum cholesterol, and reduce pathological platelet activity. In this review we show the efficacy and discuss some of the molecular mechanisms that govern the action of this dietary agent. Exogenously administered IP(6) is rapidly taken up into cells and dephosphorylated to lower inositol phosphates, which further affect signal transduction pathways resulting in cell cycle arrest. A striking anticancer action of IP(6) was demonstrated in different experimental models. In addition to reducing cell proliferation, IP(6) also induces differentiation of malignant cells. Enhanced immunity and antioxidant properties also contribute to tumor cell destruction. Preliminary studies in humans show that IP(6) and inositol, the precursor molecule of IP(6), appear to enhance the anticancer effect of conventional chemotherapy, control cancer metastases, and improve quality of life. Because it is abundantly present in regular diet, efficiently absorbed from the gastrointestinal tract, and safe, IP(6) + inositol holds great promise in our strategies for cancer prevention and therapy. There is clearly enough evidence to justify the initiation of full-scale clinical trials in humans.

Parallel Inhibition of Dopamine Amacrine Cells and Intrinsically Photosensitive Retinal Ganglion Cells in a Non-Image-Forming Visual Circuit of the Mouse Retina

PubMed Central

Vuong, Helen E.; Hardi, Claudia N.; Barnes, Steven

2015-01-01

An inner retinal microcircuit composed of dopamine (DA)-containing amacrine cells and melanopsin-containing, intrinsically photosensitive retinal ganglion cells (M1 ipRGCs) process information about the duration and intensity of light exposures, mediating light adaptation, circadian entrainment, pupillary reflexes, and other aspects of non-image-forming vision. The neural interaction is reciprocal: M1 ipRGCs excite DA amacrine cells, and these, in turn, feed inhibition back onto M1 ipRGCs. We found that the neuropeptide somatostatin [somatotropin release inhibiting factor (SRIF)] also inhibits the intrinsic light response of M1 ipRGCs and postulated that, to tune the bidirectional interaction of M1 ipRGCs and DA amacrine cells, SRIF amacrine cells would provide inhibitory modulation to both cell types. SRIF amacrine cells, DA amacrine cells, and M1 ipRGCs form numerous contacts. DA amacrine cells and M1 ipRGCs express the SRIF receptor subtypes sst2A and sst4 respectively. SRIF modulation of the microcircuit was investigated with targeted patch-clamp recordings of DA amacrine cells in TH–RFP mice and M1 ipRGCs in OPN4–EGFP mice. SRIF increases K+ currents, decreases Ca2+ currents, and inhibits spike activity in both cell types, actions reproduced by the selective sst2A agonist L-054,264 (N-[(1R)-2-[[[(1S*,3R*)-3-(aminomethyl)cyclohexyl]methyl]amino]-1-(1H-indol-3-ylmethyl)-2-oxoethyl]spiro[1H-indene-1,4′-piperidine]-1′-carboxamide) in DA amacrine cells and the selective sst4 agonist L-803,087 (N2-[4-(5,7-difluoro-2-phenyl-1H-indol-3-yl)-1-oxobutyl]-l-arginine methyl ester trifluoroacetate) in M1 ipRGCs. These parallel actions of SRIF may serve to counteract the disinhibition of M1 ipRGCs caused by SRIF inhibition of DA amacrine cells. This allows the actions of SRIF on DA amacrine cells to proceed with adjusting retinal DA levels without destabilizing light responses by M1 ipRGCs, which project to non-image-forming targets in the brain. SIGNIFICANCE STATEMENT Amacrine cells form multiple microcircuits in the inner retina to mediate visual processing, although their organization and function remain incompletely understood. The somatostatin [somatotropin release inhibiting factor (SRIF)]- and dopamine (DA)-releasing amacrine cells act globally, and, in this study, they are shown to interact and modulate the light response of intrinsically photosensitive retinal ganglion cells (ipRGCs). SRIF amacrine cells target both DA amacrine cells and M1 ipRGCs for inhibition. The parallel actions of SRIF may serve to compensate for the loss of DA-mediated inhibition of M1 ipRGCs. This inhibitory tuning is of particular importance because the DA system mediates a broad range of light adaptational actions in the retina and M1 ipRGCs project to brain areas that influence sleep, mood, cognition, circadian entrainment, and pupillary reflexes. PMID:26631476

TRAF3IP2 mediates atherosclerotic plaque development and vulnerability in ApoE−/− mice

PubMed Central

Prasad, Sakamuri Siva Sankara Vara; Higashi, Yusuke; Sukhanov, Sergiy; Siddesha, Jalahalli M; Delafontaine, Patrice; Siebenlist, Ulrich; Chandrasekar, Bysani

2016-01-01

Background and aims Atherosclerosis is a major cause of heart attack and stroke. Inflammation plays a critical role in the development of atherosclerosis. Since the cytoplasmic adaptor molecule TRAF3IP2 (TRAF3-Interacting Protein 2) plays a causal role in various autoimmune and inflammatory diseases, we hypothesized that TRAF3IP2 mediates atherosclerotic plaque development. Methods TRAF3IP2/ApoE double knockout (DKO) mice were generated by crossing TRAF3IP2−/− and ApoE−/− mice. ApoE−/− mice served as controls. Both DKO and control mice were fed a high-fat diet for 12 weeks. Plasma lipids were measured by ELISA, atherosclerosis by en face analysis of aorta and plaque cross-section measurements at the aortic valve region, plaque necrotic core area, collagen and smooth muscle cell content by histomorphometry, and aortic gene expression by RT-qPCR. Results The plasma lipoprotein profile was not altered by TRAF3IP2 gene deletion in ApoE−/− mice. While total aortic plaque area was decreased in DKO female, but not male mice, the plaque necrotic area was significantly decreased in DKO mice of both genders. Plaque collagen and smooth muscle cell contents were increased significantly in both female and male DKO mice compared to respective controls. Aortic expression of proinflammatory cytokine (Tumor necrosis factor α, TNFα), chemokine (Chemokine (C-X-C motif) Ligand 1, CXCL1) and adhesion molecule (Vascular cell adhesion molecule 1, VCAM1; and Intercellular adhesion molecule 1, ICAM1) gene expression were decreased in both male and female DKO mice. In addition, the male DKO mice showed a markedly reduced expression of extracellular matrix (ECM)-related genes, including TIMP1 (Tissue inhibitor of metalloproteinase 1), RECK (Reversion-Inducing- Cysteine-Rich Protein with Kazal Motifs) and ADAM17 (A Disintegrin And Metalloproteinase 17). Conclusions TRAF3IP2 plays a causal role in atherosclerotic plaque development and vulnerability, possibly by inducing the expression of multiple proinflammatory mediators. TRAF3IP2 could be a potential therapeutic target in atherosclerotic vascular diseases. PMID:27237075

Naive-like Conversion Overcomes the Limited Differentiation Capacity of Induced Pluripotent Stem Cells*

PubMed Central

Honda, Arata; Hatori, Masanori; Hirose, Michiko; Honda, Chizumi; Izu, Haruna; Inoue, Kimiko; Hirasawa, Ryutaro; Matoba, Shogo; Togayachi, Sumie; Miyoshi, Hiroyuki; Ogura, Atsuo

2013-01-01

Although induced pluripotent stem (iPS) cells are indistinguishable from ES cells in their expression of pluripotent markers, their differentiation into targeted cells is often limited. Here, we examined whether the limited capacity of iPS cells to differentiate into neural lineage cells could be mitigated by improving their base-line level of pluripotency, i.e. by converting them into the so-called “naive” state. In this study, we used rabbit iPS and ES cells because of the easy availability of both cell types and their typical primed state characters. Repeated passages of the iPS cells permitted their differentiation into early neural cell types (neural stem cells, neurons, and glial astrocytes) with efficiencies similar to ES cells. However, unlike ES cells, their ability to differentiate later into neural cells (oligodendrocytes) was severely compromised. In contrast, after these iPS cells had been converted to a naive-like state, they readily differentiated into mature oligodendrocytes developing characteristic ramified branches, which could not be attained even with ES cells. These results suggest that the naive-like conversion of iPS cells might endow them with a higher differentiation capacity. PMID:23880763

Chromatin immunoprecipitation (ChIP) method for non-model fruit flies (Diptera: Tephritidae) and evidence of histone modifications.

PubMed

Nagalingam, Kumaran; Lorenc, Michał T; Manoli, Sahana; Cameron, Stephen L; Clarke, Anthony R; Dudley, Kevin J

2018-01-01

Interactions between DNA and proteins located in the cell nucleus play an important role in controlling physiological processes by specifying, augmenting and regulating context-specific transcription events. Chromatin immunoprecipitation (ChIP) is a widely used methodology to study DNA-protein interactions and has been successfully used in various cell types for over three decades. More recently, by combining ChIP with genomic screening technologies and Next Generation Sequencing (e.g. ChIP-seq), it has become possible to profile DNA-protein interactions (including covalent histone modifications) across entire genomes. However, the applicability of ChIP-chip and ChIP-seq has rarely been extended to non-model species because of a number of technical challenges. Here we report a method that can be used to identify genome wide covalent histone modifications in a group of non-model fruit fly species (Diptera: Tephritidae). The method was developed by testing and refining protocols that have been used in model organisms, including Drosophila melanogaster. We demonstrate that this method is suitable for a group of economically important pest fruit fly species, viz., Bactrocera dorsalis, Ceratitis capitata, Zeugodacus cucurbitae and Bactrocera tryoni. We also report an example ChIP-seq dataset for B. tryoni, providing evidence for histone modifications in the genome of a tephritid fruit fly for the first time. Since tephritids are major agricultural pests globally, this methodology will be a valuable resource to study taxa-specific evolutionary questions and to assist with pest management. It also provides a basis for researchers working with other non-model species to undertake genome wide DNA-protein interaction studies.

Elimination of remaining undifferentiated induced pluripotent stem cells in the process of human cardiac cell sheet fabrication using a methionine-free culture condition.

PubMed

Matsuura, Katsuhisa; Kodama, Fumiko; Sugiyama, Kasumi; Shimizu, Tatsuya; Hagiwara, Nobuhisa; Okano, Teruo

2015-03-01

Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture, the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study, we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells, including cardiomyocytes and fibroblasts, using a methionine-free culture condition. When cultured in the methionine-free condition, human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition, gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore, in fabricated cardiac cell sheets, spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination.

Mechanisms underlying activation of transient BK current in rabbit urethral smooth muscle cells and its modulation by IP3-generating agonists

PubMed Central

Kyle, Barry D.; Bradley, Eamonn; Large, Roddy; Sergeant, Gerard P.; McHale, Noel G.; Thornbury, Keith D.

2013-01-01

We used the perforated patch-clamp technique at 37°C to investigate the mechanisms underlying the activation of a transient large-conductance K+ (tBK) current in rabbit urethral smooth muscle cells. The tBK current required an elevation of intracellular Ca2+, resulting from ryanodine receptor (RyR) activation via Ca2+-induced Ca2+ release, triggered by Ca2+ influx through L-type Ca2+ (CaV) channels. Carbachol inhibited tBK current by reducing Ca2+ influx and Ca2+ release and altered the shape of spike complexes recorded under current-clamp conditions. The tBK currents were blocked by iberiotoxin and penitrem A (300 and 100 nM, respectively) and were also inhibited when external Ca2+ was removed or the CaV channel inhibitors nifedipine (10 μM) and Cd2+ (100 μM) were applied. The tBK current was inhibited by caffeine (10 mM), ryanodine (30 μM), and tetracaine (100 μM), suggesting that RyR-mediated Ca2+ release contributed to the activation of the tBK current. When IP3 receptors (IP3Rs) were blocked with 2-aminoethoxydiphenyl borate (2-APB, 100 μM), the amplitude of the tBK current was not reduced. However, when Ca2+ release via IP3Rs was evoked with phenylephrine (1 μM) or carbachol (1 μM), the tBK current was inhibited. The effect of carbachol was abolished when IP3Rs were blocked with 2-APB or by inhibition of muscarinic receptors with the M3 receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide (1 μM). Under current-clamp conditions, bursts of action potentials could be evoked with depolarizing current injection. Carbachol reduced the number and amplitude of spikes in each burst, and these effects were reduced in the presence of 2-APB. In the presence of ryanodine, the number and amplitude of spikes were also reduced, and carbachol was without further effect. These data suggest that IP3-generating agonists can modulate the electrical activity of rabbit urethral smooth muscle cells and may contribute to the effects of neurotransmitters on urethral tone. PMID:23804200

Global Analysis of Transcription Factor-Binding Sites in Yeast Using ChIP-Seq

PubMed Central

Lefrançois, Philippe; Gallagher, Jennifer E. G.; Snyder, Michael

2016-01-01

Transcription factors influence gene expression through their ability to bind DNA at specific regulatory elements. Specific DNA-protein interactions can be isolated through the chromatin immunoprecipitation (ChIP) procedure, in which DNA fragments bound by the protein of interest are recovered. ChIP is followed by high-throughput DNA sequencing (Seq) to determine the genomic provenance of ChIP DNA fragments and their relative abundance in the sample. This chapter describes a ChIP-Seq strategy adapted for budding yeast to enable the genome-wide characterization of binding sites of transcription factors (TFs) and other DNA-binding proteins in an efficient and cost-effective way. Yeast strains with epitope-tagged TFs are most commonly used for ChIP-Seq, along with their matching untagged control strains. The initial step of ChIP involves the cross-linking of DNA and proteins. Next, yeast cells are lysed and sonicated to shear chromatin into smaller fragments. An antibody against an epitope-tagged TF is used to pull down chromatin complexes containing DNA and the TF of interest. DNA is then purified and proteins degraded. Specific barcoded adapters for multiplex DNA sequencing are ligated to ChIP DNA. Short DNA sequence reads (28–36 base pairs) are parsed according to the barcode and aligned against the yeast reference genome, thus generating a nucleotide-resolution map of transcription factor-binding sites and their occupancy. PMID:25213249

Modeling human neurological disorders with induced pluripotent stem cells.

PubMed

Imaizumi, Yoichi; Okano, Hideyuki

2014-05-01

Human induced pluripotent stem (iPS) cells obtained by reprogramming technology are a source of great hope, not only in terms of applications in regenerative medicine, such as cell transplantation therapy, but also for modeling human diseases and new drug development. In particular, the production of iPS cells from the somatic cells of patients with intractable diseases and their subsequent differentiation into cells at affected sites (e.g., neurons, cardiomyocytes, hepatocytes, and myocytes) has permitted the in vitro construction of disease models that contain patient-specific genetic information. For example, disease-specific iPS cells have been established from patients with neuropsychiatric disorders, including schizophrenia and autism, as well as from those with neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. A multi-omics analysis of neural cells originating from patient-derived iPS cells may thus enable investigators to elucidate the pathogenic mechanisms of neurological diseases that have heretofore been unknown. In addition, large-scale screening of chemical libraries with disease-specific iPS cells is currently underway and is expected to lead to new drug discovery. Accordingly, this review outlines the progress made via the use of patient-derived iPS cells toward the modeling of neurological disorders, the testing of existing drugs, and the discovery of new drugs. The production of human induced pluripotent stem (iPS) cells from the patients' somatic cells and their subsequent differentiation into specific cells have permitted the in vitro construction of disease models that contain patient-specific genetic information. Furthermore, innovations of gene-editing technologies on iPS cells are enabling new approaches for illuminating the pathogenic mechanisms of human diseases. In this review article, we outlined the current status of neurological diseases-specific iPS cell research and described recently obtained knowledge in the form of actual examples. © 2013 International Society for Neurochemistry.

Generation of the SCN1A epilepsy mutation in hiPS cells using the TALEN technique

NASA Astrophysics Data System (ADS)

Chen, Wanjuan; Liu, Jingxin; Zhang, Longmei; Xu, Huijuan; Guo, Xiaogang; Deng, Sihao; Liu, Lipeng; Yu, Daiguan; Chen, Yonglong; Li, Zhiyuan

2014-06-01

Human induced pluripotent stem cells (iPSC) can be used to understand the pathological mechanisms of human disease. These cells are a promising source for cell-replacement therapy. However, such studies require genetically defined conditions. Such genetic manipulations can be performed using the novel Transcription Activator-Like Effector Nucleases (TALENs), which generate site-specific double-strand DNA breaks (DSBs) with high efficiency and precision. Combining the TALEN and iPSC methods, we developed two iPS cell lines by generating the point mutation A5768G in the SCN1A gene, which encodes the voltage-gated sodium channel Nav1.1 α subunit. The engineered iPSC maintained pluripotency and successfully differentiated into neurons with normal functional characteristics. The two cell lines differ exclusively at the epilepsy-susceptibility variant. The ability to robustly introduce disease-causing point mutations in normal hiPS cell lines can be used to generate a human cell model for studying epileptic mechanisms and for drug screening.

Monocyte-derived dendritic cells exposed to Der p 1 allergen enhance the recruitment of Th2 cells: major involvement of the chemokines TARC/CCL17 and MDC/CCL22.

PubMed

Hammad, Hamida; Smits, Hermelijn H; Ratajczak, Céline; Nithiananthan, Asokananthan; Wierenga, Eddy A; Stewart, Geoffrey A; Jacquet, Alain; Tonnel, Andre-Bernard; Pestel, Joël

2003-01-01

Dendritic cells (DC) are potent antigen - presenting cells that can orientate the immune response towards a Th1 or a Th2 type. DC produce chemokines that are involved in the recruitment of either Th1 cells, such as IP10 (CXCL10), Th2 cells such as TARC (CCL17) and MDC (CCL22), or non-polarized T cells such as RANTES (CCL5) and MIP-lalpha (CCL3). We investigated whether monocyte-derived DC (MD-DC) generated from healthy donors or from patients sensitive to Dermatophagoides pteronyssinus (Dpt) and exposed to the cysteine-protease Der p 1(allergen of Dpt), could upregulate the expression of chemokines involved in type 1 or type 2 T cell recruitment. MD-DC were pulsed with either Der p 1 or with LPS as the control and the chemokines produced were evaluated using ELISA and chemotaxis assays. Der p 1-pulsed DC from allergic patients showed increased TARC (CCL17) and MDC (CCL22) production without modifying IP-10 (CXCL10) release. Der p 1-pulsed DC from healthy donors showed only increased IP-10 (CXCL10) secretion. RANTES (CCL5) and MIP-lalpha (CCL3) production were similarly increased when DC were from healthy or allergic donors. The selective Th2 clone recruitment activity of supernatants from Der p 1-pulsed DC of allergic patients was inhibited by anti-TARC (CCL17) and anti-MDC (CCL22) neutralizing Abs. By using anti-IP10 (CXCL10) blocking Abs, supernatants of Der p 1-pulsed DC from healthy donors were shown to be involved in the recruitment of Th1 cells. These results suggest that in allergic patients exposed to house dust mites, DC may favour the exacerbation of the Th2 response via the increase in type 2 chemokine production. Copyright John Libbey Eurotext 2003.

[Progress and application prospect of pig induced pluripotent stem cells].

PubMed

Yan, Yi-Bo; Zhang, Yan-Li; Qi, Wei-Wei; Wan, Yong-Jie; Fan, Yi-Xuan; Wang, Feng

2011-04-01

Pig has always been the focus of establishing a big ungulate animal ES cell lines because of its convenient source, genetic similarity with humans, and their importance in animal husbandry, but little development is achieved. Induced pluripotent stem cells technology creates a new method of reprogramming somatic cells to pluripotent state. As the pig iPS cells is established and perfected, pig ES cells will be established in the coming years. The pig iPS cells will give a hint on other livestock ES cells. On the other hand, pig iPS cells can be used to improve the efficiency of transgenic cloning pigs to conduct effective breeding and conservation of breeds. It is particularly important that the pig iPS cells can provide new model for human medical research, a new donor cells for human tissue and organ engineering, and have extensive and far-reaching impact on the biomedical field. Here, we briefly review the major progress of iPS cells, and emphasize current state of pig iPS cells and its application prospect in biomedicine and animal husbandry in order to provide a useful reference for researchers working in this area.

Inositol Hexakisphosphate Inhibits Osteoclastogenesis on RAW 264.7 Cells and Human Primary Osteoclasts

PubMed Central

Arriero, María del Mar; Ramis, Joana M.; Perelló, Joan; Monjo, Marta

2012-01-01

Background Inoxitol hexakisphosphate (IP6) has been found to have an important role in biomineralization and a direct effect inhibiting mineralization of osteoblasts in vitro without impairing extracellular matrix production and expression of alkaline phosphatase. IP6 has been proposed to exhibit similar effects to those of bisphosphonates on bone resorption, however, its direct effect on osteoclasts (OCL) is presently unknown. Methodology/Principal Findings The aim of the present study was to investigate the effect of IP6 on the RAW 264.7 monocyte/macrophage mouse cell line and on human primary osteoclasts. On one hand, we show that IP6 decreases the osteoclastogenesis in RAW 264.7 cells induced by RANKL, without affecting cell proliferation or cell viability. The number of TRAP positive cells and mRNA levels of osteoclast markers such as TRAP, calcitonin receptor, cathepsin K and MMP-9 was decreased by IP6 on RANKL-treated cells. On the contrary, when giving IP6 to mature osteoclasts after RANKL treatment, a significant increase of bone resorption activity and TRAP mRNA levels was found. On the other hand, we show that 1 µM of IP6 inhibits osteoclastogenesis of human peripheral blood mononuclear cells (PBMNC) and their resorption activity both, when given to undifferentiated and to mature osteoclasts. Conclusions/Significance Our results demonstrate that IP6 inhibits osteoclastogenesis on human PBMNC and on the RAW264.7 cell line. Thus, IP6 may represent a novel type of selective inhibitor of osteoclasts and prove useful for the treatment of osteoporosis. PMID:22905230

Generation of integration-free induced pluripotent stem cells (GZHMUi001-A) by reprogramming peripheral blood mononuclear cells from a 47, XXX syndrome patient.

PubMed

Chen, Yuchang; Ou, Zhanhui; Song, Bing; Xian, Yexing; Ouyang, Shuming; Xie, Yuhuan; Xue, Yanting; Sun, Xiaofang

2017-08-01

47, XXX syndrome is one of several sex-chromosomal aneuploidies, and it has an incidence of approximately 1/1000 in newborn females. Because of heterogeneity in X-inactivation, these patients may exhibit a variety of clinical symptoms. Here, we report the generation of an integration-free human induced pluripotent stem cell line (GZHMUi001-A) by using Sendai virus to reprogram peripheral blood mononuclear cells from a 47, XXX syndrome patient with premature ovarian failure. This 47, XXX iPS cell line has characteristics of pluripotent stem cells and is a useful tool for the investigation of this X chromosome aneuploid disease. Copyright © 2017. Published by Elsevier B.V.

Investigation of the pathogenesis of autoimmune diseases by iPS cells.

PubMed

Natsumoto, Bunki; Shoda, Hirofumi; Fujio, Keishi; Otsu, Makoto; Yamamoto, Kazuhiko

2017-01-01

The pluripotent stem cells have a self-renewal ability and can be differentiated into theoretically all of cell types. The induced pluripotent stem (iPS) cells overcame the ethical problems of the human embryonic stem (ES) cell, and enable pathologic analysis of intractable diseases and drug discovery. The in vitro disease model using disease-specific iPS cells enables repeated analyses of human cells without influence of environment factors. Even though autoimmune diseases are polygenic diseases, autoimmune disease-specific iPS cells are thought to be a promising tool for analyzing the pathogenesis of the diseases and drug discovery in future.

ChIPBase: a database for decoding the transcriptional regulation of long non-coding RNA and microRNA genes from ChIP-Seq data.

PubMed

Yang, Jian-Hua; Li, Jun-Hao; Jiang, Shan; Zhou, Hui; Qu, Liang-Hu

2013-01-01

Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) represent two classes of important non-coding RNAs in eukaryotes. Although these non-coding RNAs have been implicated in organismal development and in various human diseases, surprisingly little is known about their transcriptional regulation. Recent advances in chromatin immunoprecipitation with next-generation DNA sequencing (ChIP-Seq) have provided methods of detecting transcription factor binding sites (TFBSs) with unprecedented sensitivity. In this study, we describe ChIPBase (http://deepbase.sysu.edu.cn/chipbase/), a novel database that we have developed to facilitate the comprehensive annotation and discovery of transcription factor binding maps and transcriptional regulatory relationships of lncRNAs and miRNAs from ChIP-Seq data. The current release of ChIPBase includes high-throughput sequencing data that were generated by 543 ChIP-Seq experiments in diverse tissues and cell lines from six organisms. By analysing millions of TFBSs, we identified tens of thousands of TF-lncRNA and TF-miRNA regulatory relationships. Furthermore, two web-based servers were developed to annotate and discover transcriptional regulatory relationships of lncRNAs and miRNAs from ChIP-Seq data. In addition, we developed two genome browsers, deepView and genomeView, to provide integrated views of multidimensional data. Moreover, our web implementation supports diverse query types and the exploration of TFs, lncRNAs, miRNAs, gene ontologies and pathways.

The contribution of inositol 1,4,5-trisphosphate and ryanodine receptors to agonist-induced Ca(2+) signaling of airway smooth muscle cells.

PubMed

Bai, Yan; Edelmann, Martin; Sanderson, Michael J

2009-08-01

The relative contribution of inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) and ryanodine receptors (RyRs) to agonist-induced Ca(2+) signaling in mouse airway smooth muscle cells (SMCs) was investigated in lung slices with phase-contrast or laser scanning microscopy. At room temperature (RT), methacholine (MCh) or 5-hydroxytryptamine (5-HT) induced Ca(2+) oscillations and an associated contraction in small airway SMCs. The subsequent exposure to an IP(3)R antagonist, 2-aminoethoxydiphenyl borate (2-APB), inhibited the Ca(2+) oscillations and induced airway relaxation in a concentration-dependent manner. 2-APB also inhibited Ca(2+) waves generated by the photolytic release of IP(3). However, the RyR antagonist ryanodine had no significant effect, at any concentration, on airway contraction or agonist- or IP(3)-induced Ca(2+) oscillations or Ca(2+) wave propagation. By contrast, a second RyR antagonist, tetracaine, relaxed agonist-contracted airways and inhibited agonist-induced Ca(2+) oscillations in a concentration-dependent manner. However, tetracaine did not affect IP(3)-induced Ca(2+) release or wave propagation nor the Ca(2+) content of SMC Ca(2+) stores as evaluated by Ca(2+)-release induced by caffeine. Conversely, both ryanodine and tetracaine completely blocked agonist-independent slow Ca(2+) oscillations induced by KCl. The inhibitory effects of 2-APB and absence of an effect of ryanodine on MCh-induced airway contraction or Ca(2+) oscillations of SMCs were also observed at 37 degrees C. In Ca(2+)-permeable SMCs, tetracaine inhibited agonist-induced contraction without affecting intracellular Ca(2+) levels indicating that relaxation also resulted from a reduction in Ca(2+) sensitivity. These results indicate that agonist-induced Ca(2+) oscillations in mouse small airway SMCs are primary mediated via IP(3)Rs and that tetracaine induces relaxation by both decreasing Ca(2+) sensitivity and inhibiting agonist-induced Ca(2+) oscillations via an IP(3)-dependent mechanism.

Vector-Free and Transgene-Free Human iPS Cells Differentiate into Functional Neurons and Enhance Functional Recovery after Ischemic Stroke in Mice

PubMed Central

Mohamad, Osama; Faulkner, Ben; Chen, Dongdong; Yu, Shan Ping; Wei, Ling

2013-01-01

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited, stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently, the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition, the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However, the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation, we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice, hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling, increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice. PMID:23717557

Pancreatic β-Cell-Derived IP-10/CXCL10 Isletokine Mediates Early Loss of Graft Function in Islet Cell Transplantation.

PubMed

Yoshimatsu, Gumpei; Kunnathodi, Faisal; Saravanan, Prathab Balaji; Shahbazov, Rauf; Chang, Charles; Darden, Carly M; Zurawski, Sandra; Boyuk, Gulbahar; Kanak, Mazhar A; Levy, Marlon F; Naziruddin, Bashoo; Lawrence, Michael C

2017-11-01

Pancreatic islets produce and secrete cytokines and chemokines in response to inflammatory and metabolic stress. The physiological role of these "isletokines" in health and disease is largely unknown. We observed that islets release multiple inflammatory mediators in patients undergoing islet transplants within hours of infusion. The proinflammatory cytokine interferon-γ-induced protein 10 (IP-10/CXCL10) was among the highest released, and high levels correlated with poor islet transplant outcomes. Transgenic mouse studies confirmed that donor islet-specific expression of IP-10 contributed to islet inflammation and loss of β-cell function in islet grafts. The effects of islet-derived IP-10 could be blocked by treatment of donor islets and recipient mice with anti-IP-10 neutralizing monoclonal antibody. In vitro studies showed induction of the IP-10 gene was mediated by calcineurin-dependent NFAT signaling in pancreatic β-cells in response to oxidative or inflammatory stress. Sustained association of NFAT and p300 histone acetyltransferase with the IP-10 gene required p38 and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) activity, which differentially regulated IP-10 expression and subsequent protein release. Overall, these findings elucidate an NFAT-MAPK signaling paradigm for induction of isletokine expression in β-cells and reveal IP-10 as a primary therapeutic target to prevent β-cell-induced inflammatory loss of graft function after islet cell transplantation. © 2017 by the American Diabetes Association.

Enhancer connectome in primary human cells identifies target genes of disease-associated DNA elements

PubMed Central

Mumbach, Maxwell R; Satpathy, Ansuman T; Boyle, Evan A; Dai, Chao; Gowen, Benjamin G; Cho, Seung Woo; Nguyen, Michelle L; Rubin, Adam J; Granja, Jeffrey M; Kazane, Katelynn R; Wei, Yuning; Nguyen, Trieu; Greenside, Peyton G; Corces, M Ryan; Tycko, Josh; Simeonov, Dimitre R; Suliman, Nabeela; Li, Rui; Xu, Jin; Flynn, Ryan A; Kundaje, Anshul; Khavari, Paul A; Marson, Alexander; Corn, Jacob E; Quertermous, Thomas; Greenleaf, William J; Chang, Howard Y

2018-01-01

The challenge of linking intergenic mutations to target genes has limited molecular understanding of human diseases. Here we show that H3K27ac HiChIP generates high-resolution contact maps of active enhancers and target genes in rare primary human T cell subtypes and coronary artery smooth muscle cells. Differentiation of naive T cells into T helper 17 cells or regulatory T cells creates subtype-specific enhancer–promoter interactions, specifically at regions of shared DNA accessibility. These data provide a principled means of assigning molecular functions to autoimmune and cardiovascular disease risk variants, linking hundreds of noncoding variants to putative gene targets. Target genes identified with HiChIP are further supported by CRISPR interference and activation at linked enhancers, by the presence of expression quantitative trait loci, and by allele-specific enhancer loops in patient-derived primary cells. The majority of disease-associated enhancers contact genes beyond the nearest gene in the linear genome, leading to a fourfold increase in the number of potential target genes for autoimmune and cardiovascular diseases. PMID:28945252

Relating genes to function: identifying enriched transcription factors using the ENCODE ChIP-Seq significance tool.

PubMed

Auerbach, Raymond K; Chen, Bin; Butte, Atul J

2013-08-01

Biological analysis has shifted from identifying genes and transcripts to mapping these genes and transcripts to biological functions. The ENCODE Project has generated hundreds of ChIP-Seq experiments spanning multiple transcription factors and cell lines for public use, but tools for a biomedical scientist to analyze these data are either non-existent or tailored to narrow biological questions. We present the ENCODE ChIP-Seq Significance Tool, a flexible web application leveraging public ENCODE data to identify enriched transcription factors in a gene or transcript list for comparative analyses. The ENCODE ChIP-Seq Significance Tool is written in JavaScript on the client side and has been tested on Google Chrome, Apple Safari and Mozilla Firefox browsers. Server-side scripts are written in PHP and leverage R and a MySQL database. The tool is available at http://encodeqt.stanford.edu. abutte@stanford.edu Supplementary material is available at Bioinformatics online.

An interactive environment for agile analysis and visualization of ChIP-sequencing data.

PubMed

Lerdrup, Mads; Johansen, Jens Vilstrup; Agrawal-Singh, Shuchi; Hansen, Klaus

2016-04-01

To empower experimentalists with a means for fast and comprehensive chromatin immunoprecipitation sequencing (ChIP-seq) data analyses, we introduce an integrated computational environment, EaSeq. The software combines the exploratory power of genome browsers with an extensive set of interactive and user-friendly tools for genome-wide abstraction and visualization. It enables experimentalists to easily extract information and generate hypotheses from their own data and public genome-wide datasets. For demonstration purposes, we performed meta-analyses of public Polycomb ChIP-seq data and established a new screening approach to analyze more than 900 datasets from mouse embryonic stem cells for factors potentially associated with Polycomb recruitment. EaSeq, which is freely available and works on a standard personal computer, can substantially increase the throughput of many analysis workflows, facilitate transparency and reproducibility by automatically documenting and organizing analyses, and enable a broader group of scientists to gain insights from ChIP-seq data.

Lateral diffusion of inositol 1,4,5-trisphosphate receptor type 1 in Purkinje cells is regulated by calcium and actin filaments.

PubMed

Fukatsu, Kazumi; Bannai, Hiroko; Inoue, Takafumi; Mikoshiba, Katsuhiko

2010-09-01

Inositol 1,4,5-trisphosphate receptor type 1 (IP(3) R1) is an intracellular Ca(2+) release channel that plays crucial roles in the functions of Purkinje cells. The dynamics of IP(3) R1 on the endoplasmic reticulum membrane and the distribution of IP(3) R1 in neurons are thought to be important for the spatial regulation of Ca(2+) release. In this study, we analyzed the lateral diffusion of IP(3) R1 in Purkinje cells in cerebellar slice cultures using fluorescence recovery after photobleaching. In the dendrites of Purkinje cells, IP(3) R1 showed lateral diffusion with an effective diffusion constant of approximately 0.30 μm(2) /s, and the diffusion of IP(3) R1 was negatively regulated by actin filaments. We found that actin filaments were also involved in the regulation of IP(3) R1 diffusion in the spine of Purkinje cells. Glutamate or quisqualic acid stimulation, which activates glutamate receptors and leads to a Ca(2+) transient in Purkinje cells, decreased the diffusion of IP(3) R1 and increased the density of actin in spines. These findings indicate that the neuronal activity-dependent augmentation of actin contributes to the stabilization of IP(3) R1 in spines. © 2010 The Authors. Journal Compilation © 2010 International Society for Neurochemistry.

ANTIPROLIFERATIVE EFFECT OF INOSITOL HEXAPHOSPHATE ON HUMAN SKIN MELANOMA CELLS IN VITRO.

PubMed

Wawszczyk, Joanna; Kapral, Małgorzata; Lodowska, Jolanta; Jesse, Katarzyna; Hollek, Andrzej; Węglarz, Ludmiła

2015-01-01

Human malignant melanoma is a highly metastatic tumor with poor prognosis. The majority of metastatic melanomas are resistant to diverse chemotherapeutic agents. Consequently, the search for novel antimelanoma agents continues. In recent years, the interest in plants and their biologically active constituents as a source of novel potential drugs significantly increased. Inositol hexaphosphate (IP6) is a naturally occurring compound that has been shown to inhibit the growth of a wide variety of tumor cells in multiple experimental model systems. The aim of this study was to evaluate the antiproliferative and cytotoxic influence of IP6 on melanotic melanoma cells in vitro. The A2058 cells used as a model of human skin melanoma malignum were exposed to different concentrations of IP6 (0.1-5 mM) for a various period of time and their growth was determined by sulforhodamine B assay after 24, 48 and 72 h. The cytotoxicity of IP6 was measured at 24 and 72 h by XTT assay. IP6 has been found to cause dose-dependent growth suppression of A2058 melanoma cells. At low concentrations (0.1 and 0.5 mM) it did not exert any influence on the cell proliferation as compared to control cultures. Higher concentrations of IP6 (from 1 to 5 mM) had a statistically significant, suppressive effect on cell proliferation after 24 h incubation. When the experimental time period was increased up to 72 h, statistically significant inhibition of cell proliferation was monitored at all IP6 concentrations used. Data obtained from XTT assay indicated that IP6 had dose- and time-dependent cytotoxic effect on melanoma cells. The results demonstrate the antiproliferative and cytotoxic properties of IP6 in a wide range of concentrations on human A2058 melanoma cells. Hence, it can be suggested that IP6 could have a promising therapeutic significance in treating cancer.

Uncertain translation, uncertain benefit and uncertain risk: ethical challenges facing first-in-human trials of induced pluripotent stem (ips) cells.

PubMed

Fung, Ronald K F; Kerridge, Ian H

2013-02-01

The discovery of induced pluripotent stem (iPS) cells in 2006 was heralded as a major breakthrough in stem cell research. Since then, progress in iPS cell technology has paved the way towards clinical application, particularly cell replacement therapy, which has refueled debate on the ethics of stem cell research. However, much of the discourse has focused on questions of moral status and potentiality, overlooking the ethical issues which are introduced by the clinical testing of iPS cell replacement therapy. First-in-human trials, in particular, raise a number of ethical concerns including informed consent, subject recruitment and harm minimisation as well as the inherent uncertainty and risks which are involved in testing medical procedures on humans for the first time. These issues, while a feature of any human research, become more complex in the case of iPS cell therapy, given the seriousness of the potential risks, the unreliability of available animal models, the vulnerability of the target patient group, and the high stakes of such an intensely public area of science. Our paper will present a detailed case study of iPS cell replacement therapy for Parkinson's disease to highlight these broader ethical and epistemological concerns. If we accept that iPS cell technology is fraught with challenges which go far beyond merely refuting the potentiality of the stem cell line, we conclude that iPS cell research should not replace, but proceed alongside embryonic and adult somatic stem cell research to promote cross-fertilisation of knowledge and better clinical outcomes. © 2011 Blackwell Publishing Ltd.

Calcium signalling in salivary gland physiology and dysfunction

PubMed Central

2015-01-01

Abstract Studies over the past four decades have established that Ca2+ is a critical factor in control of salivary gland function and have led to identification of the critical components of this process. The major ion transport mechanisms and ion channels that are involved in fluid secretion have also been established. The key event in activation of fluid secretion is an increase in [Ca2+]i triggered by inositol 1,4,5‐trisphosphate (IP3)‐induced release of Ca2+ from ER via the IP3 receptor (IP3R). IP3Rs determine the site of initiation and the pattern of the [Ca2+]i signal in the cell. However, Ca2+ entry into the cell is required to sustain the elevation of [Ca2+]i and fluid secretion and is mediated by the store‐operated Ca2+ entry (SOCE) mechanism. Orai1, TRPC1, TRPC3 and STIM1 have been identified as critical components of SOCE in these cells. Cells finely tune the generation and amplification of [Ca2+]i signals for regulation of cell function. An important emerging area is the concept that unregulated [Ca2+]i signals in cells can directly cause cell damage, dysfunction and disease. Alternatively, aberrant [Ca2+]i signals can also amplify and increase the rates of cell damage. Such defects in Ca2+ signalling have been described in salivary glands in conjunction with radiation‐induced loss of salivary gland function as well as in the salivary defects associated with the autoimmune exocrinopathy Sjögren's syndrome. Such defects have been associated with altered function or expression of key Ca2+ signalling components, such as STIM proteins and TRP channels. These studies offer new avenues for examining the mechanisms underlying the disease and development of novel clinical targets and therapeutic strategies. PMID:26592972

Calcium signalling in salivary gland physiology and dysfunction.

PubMed

Ambudkar, Indu S

2016-06-01

Studies over the past four decades have established that Ca(2+) is a critical factor in control of salivary gland function and have led to identification of the critical components of this process. The major ion transport mechanisms and ion channels that are involved in fluid secretion have also been established. The key event in activation of fluid secretion is an increase in [Ca(2+) ]i triggered by inositol 1,4,5-trisphosphate (IP3 )-induced release of Ca(2+) from ER via the IP3 receptor (IP3 R). IP3 Rs determine the site of initiation and the pattern of the [Ca(2+) ]i signal in the cell. However, Ca(2+) entry into the cell is required to sustain the elevation of [Ca(2+) ]i and fluid secretion and is mediated by the store-operated Ca(2+) entry (SOCE) mechanism. Orai1, TRPC1, TRPC3 and STIM1 have been identified as critical components of SOCE in these cells. Cells finely tune the generation and amplification of [Ca(2+) ]i signals for regulation of cell function. An important emerging area is the concept that unregulated [Ca(2+) ]i signals in cells can directly cause cell damage, dysfunction and disease. Alternatively, aberrant [Ca(2+) ]i signals can also amplify and increase the rates of cell damage. Such defects in Ca(2+) signalling have been described in salivary glands in conjunction with radiation-induced loss of salivary gland function as well as in the salivary defects associated with the autoimmune exocrinopathy Sjögren's syndrome. Such defects have been associated with altered function or expression of key Ca(2+) signalling components, such as STIM proteins and TRP channels. These studies offer new avenues for examining the mechanisms underlying the disease and development of novel clinical targets and therapeutic strategies. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

MOBE-ChIP: Probing Cell Type-Specific Binding Through Large-Scale Chromatin Immunoprecipitation.

PubMed

Wang, Shenqi; Lau, On Sun

2018-01-01

In multicellular organisms, the initiation and maintenance of specific cell types often require the activity of cell type-specific transcriptional regulators. Understanding their roles in gene regulation is crucial but probing their DNA targets in vivo, especially in a genome-wide manner, remains a technical challenge with their limited expression. To improve the sensitivity of chromatin immunoprecipitation (ChIP) for detecting the cell type-specific signals, we have developed the Maximized Objects for Better Enrichment (MOBE)-ChIP, where ChIP is performed at a substantially larger experimental scale and under low background conditions. Here, we describe the procedure in the study of transcription factors in the model plant Arabidopsis. However, with some modifications, the technique should also be implemented in other systems. Besides cell type-specific studies, MOBE-ChIP can also be used as a general strategy to improve ChIP signals.

IP3R-mediated Ca2+ release regulates protein metabolism in Drosophila neuroendocrine cells: implications for development under nutrient stress.

PubMed

Megha; Hasan, Gaiti

2017-04-15

Successful completion of animal development is fundamentally reliant on nutritional cues. Surviving periods of nutritional insufficiency requires adaptations that are coordinated, in part, by neural circuits. As neuropeptides secreted by neuroendocrine (NE) cells modulate neural circuits, we investigated NE cell function during development under nutrient stress. Starved Drosophila larvae exhibited reduced pupariation if either insulin signaling or IP 3 /Ca 2+ signaling were downregulated in NE cells. Moreover, an IP 3 R (inositol 1,4,5-trisphosphate receptor) loss-of-function mutant displayed reduced protein synthesis, which was rescued by overexpression of either InR (insulin receptor) or IP 3 R in NE cells of the mutant, suggesting that the two signaling pathways might be functionally compensatory. Furthermore, cultured IP 3 R mutant NE cells, but not neurons, exhibited reduced protein translation. Thus cell-specific regulation of protein synthesis by IP 3 R in NE cells influences protein metabolism. We propose that this regulation helps developing animals survive in poor nutritional conditions. © 2017. Published by The Company of Biologists Ltd.

Effects of peritoneal fluid from endometriosis patients on interferon-gamma-induced protein-10 (CXCL10) and interleukin-8 (CXCL8) released by neutrophils and CD4+ T cells.

PubMed

Kim, Ji-Yeon; Lee, Dong-Hyung; Joo, Jong-Kil; Jin, Jun-O; Wang, Ji-Won; Hong, Young-Seoub; Kwak, Jong-Young; Lee, Kyu-Sup

2009-09-01

Intraperitoneal immuno-inflammatory changes may be associated with the pathogenesis of endometriosis. We evaluated the effects of peritoneal fluid obtained from patients with endometriosis (ePF) on the release of interferon-gamma-induced protein-10 (IP-10/CXCL10) and interleukin-8 (IL-8/CXCL8) by neutrophils, CD4(+) T cells, and monocytes. Neutrophils, CD4(+) T cells, and monocytes were cultured with ePF and the chemokine levels in the supernatants were then measured using enzyme-linked immunosorbent assay. The addition of ePF to cultures of CD4(+) T cells led to a significant increase in the release of IP-10 when compared with control PF without endometriosis (cPF). There was a positive correlation between the levels of IL-8 and IP-10 in ePF (R = 0.89, P = 0.041), but not between the levels of IP-10 and IL-8 released by neutrophils, CD4(+) T cells, and monocytes. The levels of IP-10 in ePF were positively correlated with the release of IP-10 by ePF-treated neutrophils (R = 0.89, P < 0.001), CD4(+) T cells (R = 0.93, P < 0.001), and monocytes (R = 0.70, P = 0.01). Moreover, the addition of ePF significantly enhanced the interferon-gamma-induced release of IP-10 by nuetrophils and CD4(+) T cells. These findings suggest that neutrophils and T cells release differential levels of IP-10 and IL-8 in response to stimulation with ePF, and that these cells are a major source of IP-10 in the PF of endometriosis patients.

Mission Applicability and Benefits of Thin-Film Integrated Power Generation and Energy Storage

NASA Technical Reports Server (NTRS)

Hoffman, David; Raffaelle, Ryne P.; Landis, Geoffrey A.; Hepp, Aloysius F.

2001-01-01

This paper discusses the space mission applicability and benefits of a thin-film integrated power generation and energy storage device, i.e., an "Integrated Power Source" or IPS. The characteristics of an IPS that combines thin-film photo-voltaic power generation with thin-film energy storage are described. Mission concepts for a thin-film IPS as a spacecraft main electrical power system, as a decentralized or distributed power source and as an uninterruptible power supply are discussed. For two specific missions, preliminary sizing of an IPS as a main power system is performed and benefits are assessed. IPS developmental challenges that need to be overcome in order to realize the benefits of an IPS are examined. Based on this preliminary assessment, it is concluded that the most likely and beneficial application of an IPS will be as the main power system on a very small "nanosatellite," or in specialized applications serving as a decentralized or distributed power source or uninterruptible power supply.

Mission Applicability and Benefits of Thin-Film Integrated Power Generation and Energy Storage

NASA Technical Reports Server (NTRS)

Hoffman, David J.; Raffaelle, Ryne P.; Landis, Geoffrey A.; Hepp, Aloysius F.

2001-01-01

This paper discusses the space mission applicability and benefits of a thin-film integrated power generation and energy storage device, i.e., an "Integrated Power Source" or IPS. The characteristics of an IPS that combines thin-film photovoltaic power generation with thin-film energy storage are described. Mission concepts for a thin-film IPS as a spacecraft main electrical power system, as a decentralized or distributed power source and as an uninterruptible power supply are discussed. For two specific missions, preliminary sizing of an IPS as a main power system is performed and benefits are assessed. IPS developmental challenges that need to be overcome in order to realize the benefits of an IPS are examined. Based on this preliminary assessment, it is concluded that the most likely and beneficial application of an IPS will be as the main power system on a very small "nanosatellite," or in specialized applications serving as a decentralized or distributed power source or uninterruptible power supply.

PKM2 methylation by CARM1 activates aerobic glycolysis to promote tumorigenesis

PubMed Central

Liu, Fabao; Ma, Fengfei; Wang, Yuyuan; Hao, Ling; Zeng, Hao; Jia, Chenxi; Wang, Yidan; Liu, Peng; Ong, Irene M; Li, Baobin; Chen, Guojun; Jiang, Jiaoyang; Gong, Shaoqin; Li, Lingjun; Xu, Wei

2017-01-01

Metabolic reprogramming is a hallmark of cancer. Herein we discovered that the key glycolytic enzyme pyruvate kinase M2 isoform (PKM2), but not the related isoform PKM1, is methylated by co-activator associated arginine methyltransferase 1 (CARM1). PKM2 methylation reversibly shifts the balance of metabolism from oxidative phosphorylation to aerobic glycolysis in breast cancer cells. Oxidative phosphorylation depends on mitochondria calcium concentration, which becomes critical for cancer cell survival when PKM2 methylation is blocked. By interacting with and suppressing the expression of inositol 1, 4, 5-trisphosphate receptors (IP3Rs), methylated PKM2 inhibits the influx of calcium from endoplasmic reticulum (ER) to mitochondria. Inhibiting PKM2 methylation with a competitive peptide delivered by nanoparticle perturbs metabolic energy balance in cancer cells, leading to decrease of cell proliferation, migration, and metastasis. Collectively, the CARM1-PKM2 axis serves as a metabolic reprogramming mechanism in tumorigenesis, and inhibiting PKM2 methylation generates metabolic vulnerability to IP3R-dependent mitochondrial functions. PMID:29058718

From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium

PubMed Central

Reichman, Sacha; Terray, Angélique; Slembrouck, Amélie; Nanteau, Céline; Orieux, Gaël; Habeler, Walter; Nandrot, Emeline F.; Sahel, José-Alain; Monville, Christelle; Goureau, Olivier

2014-01-01

Progress in retinal-cell therapy derived from human pluripotent stem cells currently faces technical challenges that require the development of easy and standardized protocols. Here, we developed a simple retinal differentiation method, based on confluent human induced pluripotent stem cells (hiPSC), bypassing embryoid body formation and the use of exogenous molecules, coating, or Matrigel. In 2 wk, we generated both retinal pigmented epithelial cells and self-forming neural retina (NR)-like structures containing retinal progenitor cells (RPCs). We report sequential differentiation from RPCs to the seven neuroretinal cell types in maturated NR-like structures as floating cultures, thereby revealing the multipotency of RPCs generated from integration-free hiPSCs. Furthermore, Notch pathway inhibition boosted the generation of photoreceptor precursor cells, crucial in establishing cell therapy strategies. This innovative process proposed here provides a readily efficient and scalable approach to produce retinal cells for regenerative medicine and for drug-screening purposes, as well as an in vitro model of human retinal development and disease. PMID:24912154

Induction of pluripotency by defined factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okita, Keisuke, E-mail: okita@cira.kyoto-u.ac.jp; Yamanaka, Shinya; Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507

2010-10-01

Somatic cells can be reprogrammed into pluripotent stem cells by introducing a combination of several transcription factors. The induced pluripotent stem (iPS) cells from a patient's somatic cells could be useful source of cells for drug discovery and cell transplantation therapies. However, most human iPS cells are made by viral vectors, such as retrovirus and lentivirus, which integrate the reprogramming factors into host genomes and may increase the risk of tumor formation. Studies of the mechanisms underlying the reprogramming and establishment of non-integration methods contribute evidence to resolve the safety concerns associated with iPS cells. On the other hand, patient-specificmore » iPS cells have already been established and used for recapitulating disease pathology.« less

IP-10 protects while MIP-2 promotes experimental anesthetic hapten - induced hepatitis

PubMed Central

Njoku, Dolores B.; Li, Zhaoxia; Mellerson, Jenelle L; Sharma, Rajni; Talor, Monica V.; Barat, Nicole; Rose, Noel R.

2009-01-01

MIP-2 and IFN-γ inducible protein-10 (IP-10) and their respective receptors, CXCR2 and CXCR3, modulate tissue inflammation by recruiting neutrophils or T cells from the spleen or bone marrow. Yet, how these chemokines modulate diseases such as immune-mediated drug-induced liver injury (DILI) is essentially unknown. To investigate how chemokines modulate experimental DILI in our model we used susceptible BALB/c (WT) and IL-4−/− (KO) mice that develop significantly reduced hepatitis and splenic T cell priming to anesthetic haptens and self proteins following TFA-S100 immunizations. We detected CXCR2+ splenic granulocytes in all mice two weeks following immunizations; by 3 weeks, MIP-2 levels (p<0.001) and GR1+ cells were elevated in WT livers, suggesting MIP-2-recruited granulocytes. Elevated splenic CXCR3+ CD4+T cells were identified after 2 weeks in KO mice indicating elevated IP-10 levels which were confirmed during T cell priming. This result suggested that IP-10 reduced T cell priming to critical DILI antigens. Increased T cell proliferation following co-culture of TFA-S100-primed WT splenocytes with anti-IP-10 (p<0.05) confirmed that IP-10 reduced T cell priming to CYP2E1 and TFA. We propose that MIP-2 promotes and IP-10 protects against the development of hepatitis and T cell priming in this murine model. PMID:19131211

Inositol bisphosphate and inositol trisphosphate inhibit cell-to-cell passage of carboxyfluorescein in staminal hairs ofSetcreasea purpurea.

PubMed

Tucker, E B

1988-06-01

pH-buffered carboxyfluorescein (Buffered-CF) alone (control), or Buffered-CF solutions containing one of the following: (1)D-myo-inositol (I); (2)D-myo-inositol 2-monophosphate (IP1); (3)D-myo-inositol 1,4-bisphosphate (IP2); (4)D-myo-inositol 1,4,5-trisphosphate (IP3); (5)D-fructose 2,6-diphosphate (F-2,6P2) were microinjected into the terminal cells of staminal hairs ofSetcreasea purpurea Boom. Passage of the CF from this terminal cell along the chain of cells towards the filament was monitored for 5 min using fluorescence microscopy and quantified using computer-assisted fluorescence-intensity video analysis. Cell-to-cell transport of CF in hairs microinjected with Buffered-CF containing either I, IP1 or F-2,6P2 was similar to that in hairs microinjected with Buffered-CF only. On the other hand, cell-to-cell transport of CF in hairs microinjected with Buffered-CF containing either IP2 or IP3 was inhibited. These results indicate that polyphosphoinositols may be involved in the regulation of intercellular transport of low-molecular-weight, hydrophilic molecules in plants.

The role of DAB2IP in androgen receptor activation during prostate cancer progression.

PubMed

Wu, K; Liu, J; Tseng, S-F; Gore, C; Ning, Z; Sharifi, N; Fazli, L; Gleave, M; Kapur, P; Xiao, G; Sun, X; Oz, O K; Min, W; Alexandrakis, G; Yang, C-R; Hsieh, C-L; Wu, H-C; He, D; Xie, D; Hsieh, J-T

2014-04-10

Altered androgen-receptor (AR) expression and/or constitutively active AR are commonly associated with prostate cancer (PCa) progression. Targeting AR remains a focal point for designing new strategy of PCa therapy. Here, we have shown that DAB2IP, a novel tumor suppressor in PCa, can inhibit AR-mediated cell growth and gene activation in PCa cells via distinct mechanisms. DAB2IP inhibits the genomic pathway by preventing AR nuclear translocation or phosphorylation and suppresses the non-genomic pathway via its unique functional domain to inactivate c-Src. Also, DAB2IP is capable of suppressing AR activation in an androgen-independent manner. In addition, DAB2IP can inhibit several AR splice variants showing constitutive activity in PCa cells. In DAB2IP(-/-) mice, the prostate gland exhibits hyperplastic epithelia, in which AR becomes more active. Consistently, DAB2IP expression inversely correlates with AR activation status particularly in recurrent or metastatic PCa patients. Taken together, DAB2IP is a unique intrinsic AR modulator in normal cells, and likely can be further developed into a therapeutic agent for PCa.

Emptying of Intracellular Calcium Pool and Oxidative Stress Imbalance Are Associated with the Glyphosate-Induced Proliferation in Human Skin Keratinocytes HaCaT Cells

PubMed Central

George, Jasmine; Shukla, Yogeshwer

2013-01-01

We demonstrated that glyphosate possesses tumor promoting potential in mouse skin carcinogenesis and SOD 1, calcyclin (S100A6), and calgranulin B (S100A9) have been associated with this potential, although the mechanism is unclear. We aimed to clarify whether imbalance in between [Ca2+]i levels and oxidative stress is associated with glyphosate-induced proliferation in human keratinocytes HaCaT cells. The [Ca2+]i levels, ROS generation, and expressions of G1/S cyclins, IP3R1, S100A6, S100A9, and SOD 1, and apoptosis-related proteins were investigated upon glyphosate exposure in HaCaT cells. Glyphosate (0.1 mM) significantly induced proliferation, decreases [Ca2+]i, and increases ROS generation in HaCaT cells, whereas antioxidant N-acetyl-L-cysteine (NAC) pretreatment reverts these effects which directly indicated that glyphosate induced cell proliferation by lowering [Ca2+]i levels via ROS generation. Glyphosate also enhanced the expression of G1/S cyclins associated with a sharp decrease in G0/G1 and a corresponding increase in S-phases. Additionally, glyphosate also triggers S100A6/S100A9 expression and decreases IP3R1 and SOD 1 expressions in HaCaT cells. Notably, Ca2+ suppression also prevented apoptotic related events including Bax/Bcl-2 ratio and caspases activation. This study highlights that glyphosate promotes proliferation in HaCaT cells probably by disrupting the balance in between [Ca2+]i levels and oxidative stress which in turn facilitated the downregulation of mitochondrial apoptotic signaling pathways. PMID:24073338

Glial cells isolated from dorsal root ganglia express prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors.

PubMed

Ng, Kai Yu; Wong, Yung Hou; Wise, Helen

2011-07-01

Isolated cells from adult rat dorsal root ganglia (DRG) are frequently used as a model system to study responses of primary sensory neurons to nociceptor sensitizing agents such as prostaglandin E(2) and prostacyclin, which are presumed to act only on the neurons in typical mixed cell cultures. In the present study, we evaluated the expression of prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors in cultures of mixed DRG cells and in purified DRG glia. We show here that EP(4) and IP receptor agonists stimulated adenylyl cyclase activity in both mixed DRG cells and in purified DRG glia, and that these responses were specifically inhibited by EP(4) and IP receptor antagonists, respectively. The presence of EP(4) and IP receptors in DRG glia was further confirmed by the expression of EP(4) and IP receptor immunoreactivity and mRNA. With the increasing awareness of neuron-glial interactions within intact DRG and the use of isolated DRG cells in the study of mechanisms underlying nociception, it will be essential to consider the role played by EP(4) and IP receptor-expressing glial cells when evaluating prostanoid-induced sensitization of DRG neurons. Copyright © 2011 Elsevier B.V. All rights reserved.

Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2.

PubMed

Montgomery, Benjamin C S; Mewes, Jacqueline; Davidson, Chelsea; Burshtyn, Deborah N; Stafford, James L

2009-04-01

Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using 'chimeric' expression constructs and an epitope-tagged 'native' IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.

MLF1IP promotes normal erythroid proliferation and is involved in the pathogenesis of polycythemia vera.

PubMed

Feng, Gege; Zhang, Tianjiao; Liu, Jinqin; Ma, Xiaotang; Li, Bing; Yang, Lin; Zhang, Yue; Xu, Zefeng; Qin, Tiejun; Zhou, Jiaxi; Huang, Gang; Shi, Lihong; Xiao, Zhijian

2017-03-01

Myelodysplasia/myeloid leukemia factor 1-interacting protein (MLF1IP) appears to be an erythroid lineage-specific gene in mice; however, its role in normal erythropoiesis and erythropoietic disorders have not yet been elucidated. Here, we found that MLF1IP is abundantly expressed in human erythroid progenitor cells and that MLF1IP-deficiency reduces cell proliferation resulting from cell cycle arrest. Moreover, MLF1IP expression is exclusively elevated in CFU-E cells from polycythemia vera (PV) patients, and MLF1IP transgenic mice develop a PV-like disorder. Further analyses revealed that the erythroid progenitors and early-stage erythroblasts from these transgenic mice expand by up-regulating cyclin D2 and down-regulating p27 and p21. Thus, our data demonstrate that MLF1IP promotes erythroid proliferation and is involved in the pathogenesis of PV, suggesting that it might be a novel molecular target for erythropoietic disorders. © 2017 Federation of European Biochemical Societies.

Role of Caspase-3 Cleaved IP3R1 on Ca2+ Homeostasis and Developmental Competence of Mouse Oocytes and Eggs

PubMed Central

Zhang, Nan; Fissore, Rafael. A.

2014-01-01

Apoptosis in most cell types is accompanied by altered Ca2+ homeostasis. During apoptosis, caspase-3 mediated cleavage of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) generates a 95-kDa C-terminal fragment (C-IP3R1), which represents the channel domain of the receptor. Aged mouse eggs display abnormal Ca2+ homeostasis and express C-IP3R1, although whether or not C-IP3R1 expression contributes to Ca2+ misregulation or a decrease in developmental competency is unknown. We sought to answer these questions by injecting in mouse oocytes and eggs cRNAs encoding CIP3R1. We found that: 1) expression of C-IP3R1 in eggs lowered the Ca2+ content of the endoplasmic reticulum (ER), although, as C-IP3R1 is quickly degraded at this stage, its expression did not impair pre-implantation embryo development; 2) expression of CIP3R1 in eggs enhanced fragmentation associated with aging; 3) endogenous IP3R1 is required for aging associated apoptosis, as its down-regulation prevented fragmentation, and expression of C-IP3R1 in eggs with downregulated IP3R1 partly restored fragmentation; 4) C-IP3R1 expression in GV oocytes resulted in persistent levels of protein, which abolished the increase in the ER releasable Ca2+ pool that occurs during maturation, undermined the Ca2+ oscillatory ability of matured eggs and their activation potential. Collectively, this study supports a role for IP3R1 and C-IP3R1 in regulating Ca2+ homeostasis and the ER Ca2+ content during oocyte maturation. Nevertheless, the role of C-IP3R1 on Ca2+ homeostasis in aged eggs seems minor, as in MII eggs the majority of endogenous IP3R1 remains intact and C-IP3R1 undergoes rapid turnover. PMID:24692207

Perspectives on human clinical trials of therapies using iPS cells in Japan: reaching the forefront of stem-cell therapies.

PubMed

Song, Peipei; Inagaki, Yoshinori; Sugawara, Yasuhiko; Kokudo, Norihiro

2013-06-01

A research project involving sheets of retinal pigment epithelium constructed from iPS cells derived from patients with age-related maculopathy is one step closer to being approved for clinical trials by the Japanese Government. Now is the time to make therapies using iPS cells clinically available.

Human induced pluripotent stem cells: a review of the US patent landscape.

PubMed

Georgieva, Bilyana P; Love, Jane M

2010-07-01

Human induced pluripotent stem (iPS) cells and human embryonic stem cells are cells that have the ability to differentiate into a variety of cell types. Embryonic stem cells are derived from human embryos; however, by contrast, human iPS cells can be obtained from somatic cells that have undergone a process of 'reprogramming' via genetic manipulation such that they develop pluripotency. Since iPS cells are not derived from human embryos, they are a less complicated source of human pluripotent cells and are considered valuable research tools and potentially useful in therapeutic applications in regenerative medicine. Worldwide, there are only three issued patents concerning iPS cells. Therefore, the patent landscape in this field is largely undefined. This article provides an overview of the issued patents as well as the pending published patent applications in the field.

A Multidisciplinary Biospecimen Bank of Renal Cell Carcinomas Compatible with Discovery Platforms at Mayo Clinic, Scottsdale, Arizona

PubMed Central

Ho, Thai H.; Nateras, Rafael Nunez; Yan, Huihuang; Park, Jin G.; Jensen, Sally; Borges, Chad; Lee, Jeong Heon; Champion, Mia D.; Tibes, Raoul; Bryce, Alan H.; Carballido, Estrella M.; Todd, Mark A.; Joseph, Richard W.; Wong, William W.; Parker, Alexander S.; Stanton, Melissa L.; Castle, Erik P.

2015-01-01

To address the need to study frozen clinical specimens using next-generation RNA, DNA, chromatin immunoprecipitation (ChIP) sequencing and protein analyses, we developed a biobank work flow to prospectively collect biospecimens from patients with renal cell carcinoma (RCC). We describe our standard operating procedures and work flow to annotate pathologic results and clinical outcomes. We report quality control outcomes and nucleic acid yields of our RCC submissions (N=16) to The Cancer Genome Atlas (TCGA) project, as well as newer discovery platforms, by describing mass spectrometry analysis of albumin oxidation in plasma and 6 ChIP sequencing libraries generated from nephrectomy specimens after histone H3 lysine 36 trimethylation (H3K36me3) immunoprecipitation. From June 1, 2010, through January 1, 2013, we enrolled 328 patients with RCC. Our mean (SD) TCGA RNA integrity numbers (RINs) were 8.1 (0.8) for papillary RCC, with a 12.5% overall rate of sample disqualification for RIN <7. Banked plasma had significantly less albumin oxidation (by mass spectrometry analysis) than plasma kept at 25°C (P<.001). For ChIP sequencing, the FastQC score for average read quality was at least 30 for 91% to 95% of paired-end reads. In parallel, we analyzed frozen tissue by RNA sequencing; after genome alignment, only 0.2% to 0.4% of total reads failed the default quality check steps of Bowtie2, which was comparable to the disqualification ratio (0.1%) of the 786-O RCC cell line that was prepared under optimal RNA isolation conditions. The overall correlation coefficients for gene expression between Mayo Clinic vs TCGA tissues ranged from 0.75 to 0.82. These data support the generation of high-quality nucleic acids for genomic analyses from banked RCC. Importantly, the protocol does not interfere with routine clinical care. Collections over defined time points during disease treatment further enhance collaborative efforts to integrate genomic information with outcomes. PMID:26181416

Distribution and developmental changes of ghrelin-immunopositive cells in the pancreas of African ostrich chicks (Struthio camelus).

PubMed

Wang, J X; Li, P; Zhang, X T; Ye, L X

2017-09-01

Ghrelin, the endogenous ligand for the growth hormone secretagogue receptor (GHS-R), is produced by multiple cell types and affects feeding behavior, metabolic regulation, and energy balance. In the mammalian pancreas, the types of endocrine cells that are immunoreactive to ghrelin vary. However, little was known about its distribution and developmental changes in the pancreas of African ostrich chicks (Struthio camelus). In the present study, the distribution, morphological characteristics, and developmental changes of ghrelin-immunopositive (ghrelin-ip) cells in the pancreas of African ostrich chicks were investigated using immunohistochemistry. Ghrelin-ip cells were found in both the pancreatic islets and acinar cell regions. The greatest number of ghrelin-ip cells were found in the pancreatic islets, and were primarily observed at the periphery of the islets; some ghrelin-ip cells were also located in the central portion of the pancreatic islets. Interestingly, from postnatal d 1 to d 90, there was a steady decrease in the number of ghrelin-ip cells in the pancreatic islets and acinar cell regions. These results clearly demonstrated that ghrelin-ip cells exist and decreased with age in the African ostrich pancreas from postnatal d 1 to d90. Thus, these findings indicated that ghrelin may be involved in the development of the pancreas in the African ostrich. © 2017 Poultry Science Association Inc.

Concentration- and time-dependent genotoxicity profiles of isoprene monoepoxides and diepoxide, and the cross-linking potential of isoprene diepoxide in cells.

PubMed

Li, Yan; Pelah, Avishay; An, Jing; Yu, Ying-Xin; Zhang, Xin-Yu

2014-01-01

Isoprene, a possible carcinogen, is a petrochemical and a natural product being primarily produced by plants. It is biotransformed to 2-ethenyl-2-methyloxirane (IP-1,2-O) and 2-(1-methylethenyl)oxirane (IP-3,4-O), both of which can be further metabolized to 2-methyl-2,2'-bioxirane (MBO). MBO is mutagenic, but IP-1,2-O and IP-3,4-O are not. While IP-1,2-O has been reported being genotoxic, the genotoxicity of IP-3,4-O and MBO, and the cross-linking potential of MBO have not been examined. In the present study, we used the comet assay to investigate the concentration- and time-dependent genotoxicity profiles of the three metabolites and the cross-linking potential of MBO in human hepatocyte L02 cells. For the incubation time of 1 h, all metabolites showed positive concentration-dependent profiles with a potency rank order of IP-3,4-O > MBO > IP-1,2-O. In human hepatocellular carcinoma (HepG2) and human leukemia (HL60) cells, IP-3,4-O was still more potent in inducing DNA breaks than MBO at high concentrations (>200 μM), although at low concentrations (≤200 μM) IP-3,4-O exhibited slightly lower or similar potency to MBO. Interestingly, their time-dependent genotoxicity profiles (0.5-4 h) in L02 cells were different from each other: IP-1,2-O and MBO (200 μM) exhibited negative and positive profiles, respectively, with IP-3,4-O lying in between, namely, IP-3,4-O-caused DNA breaks did not change over the exposure time. Further experiments demonstrated that hydrolysis of IP-1,2-O contributed to the negative profile and MBO induced cross-links at high concentrations and long incubation times. Collectively, the results suggested that IP-3,4-O might play a significant role in the toxicity of isoprene.

RdgB2 is required for dim-light input into intrinsically photosensitive retinal ganglion cells

PubMed Central

Walker, Marquis T.; Rupp, Alan; Elsaesser, Rebecca; Güler, Ali D.; Sheng, Wenlong; Weng, Shijun; Berson, David M.; Hattar, Samer; Montell, Craig

2015-01-01

A subset of retinal ganglion cells is intrinsically photosensitive (ipRGCs) and contributes directly to the pupillary light reflex and circadian photoentrainment under bright-light conditions. ipRGCs are also indirectly activated by light through cellular circuits initiated in rods and cones. A mammalian homologue (RdgB2) of a phosphoinositide transfer/exchange protein that functions in Drosophila phototransduction is expressed in the retinal ganglion cell layer. This raised the possibility that RdgB2 might function in the intrinsic light response in ipRGCs, which depends on a cascade reminiscent of Drosophila phototransduction. Here we found that under high light intensities, RdgB2−/− mutant mice showed normal pupillary light responses and circadian photoentrainment. Consistent with this behavioral phenotype, the intrinsic light responses of ipRGCs in RdgB2−/− were indistinguishable from wild-type. In contrast, under low-light conditions, RdgB2−/− mutants displayed defects in both circadian photoentrainment and the pupillary light response. The RdgB2 protein was not expressed in ipRGCs but was in GABAergic amacrine cells, which provided inhibitory feedback onto bipolar cells. We propose that RdgB2 is required in a cellular circuit that transduces light input from rods to bipolar cells that are coupled to GABAergic amacrine cells and ultimately to ipRGCs, thereby enabling ipRGCs to respond to dim light. PMID:26269578

ChIP-seq: advantages and challenges of a maturing technology.

PubMed

Park, Peter J

2009-10-01

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique for genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes. Owing to the tremendous progress in next-generation sequencing technology, ChIP-seq offers higher resolution, less noise and greater coverage than its array-based predecessor ChIP-chip. With the decreasing cost of sequencing, ChIP-seq has become an indispensable tool for studying gene regulation and epigenetic mechanisms. In this Review, I describe the benefits and challenges in harnessing this technique with an emphasis on issues related to experimental design and data analysis. ChIP-seq experiments generate large quantities of data, and effective computational analysis will be crucial for uncovering biological mechanisms.

[In vitro generation of blood red cells from stem cells: a sketch of the future].

PubMed

Mazurier, Christelle; Douay, Luc

2016-01-01

Human adult pluripotent stem cells, stem cells of embryonic origin and induced pluripotent stem cells (iPS) provide cellular sources for new promising regenerative medicine approaches. Because these cells can be patient-specific, they allow considering a personalized medicine appropriate to the diagnosis of each. The generation of cultured red blood cells (cRBC) derived from stem cells is emblematic of personalized medicine. Indeed, these cells have the advantage of being selected according to a blood phenotype of interest and they may provide treatments to patients in situation of impossible transfusion (alloimmunized patients, rare phenotypes). Essential progresses have established proof of concept for this approach, still a concept some years ago. From adult stem cells, all steps of upstream research were successfully achieved, including the demonstration of the feasibility of injection into human. This leads us to believe that Red Blood Cells generated in vitro from stem cells will be the future players of blood transfusion. However, although theoretically ideal, these stem cells raise many biological challenges to overcome, although some tracks are identified. © Société de Biologie, 2016.

Chromatin immunoprecipitation with fixed animal tissues and preparation for high-throughput sequencing.

PubMed

Cotney, Justin L; Noonan, James P

2015-02-02

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) is a powerful method used to identify genome-wide binding patterns of transcription factors and distribution of various histone modifications associated with different chromatin states. In most published studies, ChIP-Seq has been performed on cultured cells grown under controlled conditions, allowing generation of large amounts of material in a homogeneous biological state. Although such studies have provided great insight into the dynamic landscapes of animal genomes, they do not allow the examination of transcription factor binding and chromatin states in adult tissues, developing embryonic structures, or tumors. Such knowledge is critical to understanding the information required to create and maintain a complex biological tissue and to identify noncoding regions of the genome directly involved in tissues affected by complex diseases such as autism. Studying these tissue types with ChIP-Seq can be challenging due to the limited availability of tissues and the lack of complex biological states able to be achieved in culture. These inherent differences require alterations of standard cross-linking and chromatin extraction typically used in cell culture. Here we describe a general approach for using small amounts of animal tissue to perform ChIP-Seq directed at histone modifications and transcription factors. Tissue is homogenized before treatment with formaldehyde to ensure proper cross-linking, and a two-step nuclear isolation is performed to increase extraction of soluble chromatin. Small amounts of soluble chromatin are then used for immunoprecipitation (IP) and prepared for multiplexed high-throughput sequencing. © 2015 Cold Spring Harbor Laboratory Press.

Epigenetic rejuvenation.

PubMed

Manukyan, Maria; Singh, Prim B

2012-05-01

Induced pluripotent stem (iPS) cells have provided a rational means of obtaining histo-compatible tissues for 'patient-specific' regenerative therapies (Hanna et al. 2010; Yamanaka & Blau 2010). Despite the obvious potential of iPS cell-based therapies, there are certain problems that must be overcome before these therapies can become safe and routine (Ohi et al. 2011; Pera 2011). As an alternative, we have recently explored the possibility of using 'epigenetic rejuvenation', where the specialized functions of an old cell are rejuvenated in the absence of any change in its differentiated state (Singh & Zacouto 2010). The mechanism(s) that underpin 'epigenetic rejuvenation' are unknown and here we discuss model systems, using key epigenetic modifiers, which might shed light on the processes involved. Epigenetic rejuvenation has advantages over iPS cell techniques that are currently being pursued. First, the genetic and epigenetic abnormalities that arise through the cycle of dedifferentiation of somatic cells to iPS cells followed by redifferentiation of iPS cells into the desired cell type are avoided (Gore et al. 2011; Hussein et al. 2011; Pera 2011): epigenetic rejuvenation does not require passage through the de-/redifferentiation cycle. Second, because the aim of epigenetic rejuvenation is to ensure that the differentiated cell type retains its specialized function it makes redundant the question of transcriptional memory that is inimical to iPS cell-based therapies (Ohi et al. 2011). Third, to produce unrelated cell types using the iPS technology takes a long time, around three weeks, whereas epigenetic rejuvenation of old cells will take only a matter of days. Epigenetic rejuvenation provides the most safe, rapid and cheap route to successful regenerative medicine. © 2012 The Authors. Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

A mathematical model of calcium dynamics in HSY cells

PubMed Central

Han, Jung Min; Tanimura, Akihiko; Kirk, Vivien; Sneyd, James

2017-01-01

Saliva is an essential part of activities such as speaking, masticating and swallowing. Enzymes in salivary fluid protect teeth and gums from infectious diseases, and also initiate the digestion process. Intracellular calcium (Ca2+) plays a critical role in saliva secretion and regulation. Experimental measurements of Ca2+ and inositol trisphosphate (IP3) concentrations in HSY cells, a human salivary duct cell line, show that when the cells are stimulated with adenosine triphosphate (ATP) or carbachol (CCh), they exhibit coupled oscillations with Ca2+ spike peaks preceding IP3 spike peaks. Based on these data, we construct a mathematical model of coupled Ca2+ and IP3 oscillations in HSY cells and perform model simulations of three different experimental settings to forecast Ca2+ responses. The model predicts that when Ca2+ influx from the extracellular space is removed, oscillations gradually slow down until they stop. The model simulation of applying a pulse of IP3 predicts that photolysis of caged IP3 causes a transient increase in the frequency of the Ca2+ oscillations. Lastly, when Ca2+-dependent activation of PLC is inhibited, we see an increase in the oscillation frequency and a decrease in the amplitude. These model predictions are confirmed by experimental data. We conclude that, although concentrations of Ca2+ and IP3 oscillate, Ca2+ oscillations in HSY cells are the result of modulation of the IP3 receptor by intracellular Ca2+, and that the period is modulated by the accompanying IP3 oscillations. PMID:28199326

Micro-chromatin Immunoprecipation (μChIP) Protocol for Real-time PCR Analysis of a Limited Amount of Cells.

PubMed

Gillotin, Sébastien; Guillemot, François

2016-06-20

Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) is an important strategy to study gene regulation. When availability of cells is limited, however, it can be useful to focus on specific genes to investigate in depth the role of transcription factors or histone marks. Unfortunately, performing ChIP experiments to study transcription factors' binding to DNA can be difficult when biological material is restricted. This protocol describes a robust method to perform μChIP for over-expressed or endogenous transcription factors using ~100,000 cells per ChIP experiment (Masserdotti et al ., 2015). We also describe optimization steps, which we think are critical for this protocol to work and which can be used to further reduce the number of cells.

Fenpropathrin, a Widely Used Pesticide, Causes Dopaminergic Degeneration

PubMed Central

Xiong, Jing; Zhang, Xiaowei; Huang, Jinsha; Chen, Chunnuan; Chen, Zhenzhen; Liu, Ling; Zhang, Guoxin; Yang, Jiaolong; Zhang, Zhentao; Zhang, Zhaohui; Lin, Zhicheng

2017-01-01

Fenpropathrin is one of the widely used pyrethroids in agriculture and household and also reported to have neurotoxic effects in rodent models. In our Parkinson’s disease (PD) clinic, there was a unique patient with a history of daily exposure to fenpropathrin for 6 months prior to developing Parkinsonian symptoms progressively. Since whether fenpropathrin is related to any dopaminergic degeneration was unknown, we aimed in this study to evaluate the neurotoxic effects of fenpropathrin on the dopaminergic system and associated mechanisms in vitro and in vivo. In cultured SH-SY5Y cells, fenpropathrin caused cell death, reactive oxygen species generation, Lewy body-associated proteins aggregation, and Lewy body-like intracytoplasmic inclusions formation. In rodent animals, two different injections of fenpropathrin were used for administrations, intraperitoneal (i.p), or stereotaxical (ST). The rats exhibited lower number of pokes 60 days after first i.p injection, while the rats in ST group showed a significant upregulation of apomorphine-evoked rotations 60 days after first injection. Dcreased tyrosine hydroxylase (TH) and vesicular monoamine transporter 2 (VMAT2) immunoreactivity, while increased dopamine transporter (DAT) immunoreactivity were observed in rats of either i.p or ST group 60 days after the last exposure to fenpropathrin. However, the number of TH-positive cells in the substantia nigra was more reduced 120 days after the first i.p injection than those of 60 days. Our data demonstrated that exposure to fenpropathrin could mimic the pathologic and pathogenetic features of PD especially in late onset cases. These results imply fenpropathrin as a DA neurotoxin and a possible environmental risk factor for PD. PMID:25575680

miRNA-556-3p promotes human bladder cancer proliferation, migration and invasion by negatively regulating DAB2IP expression.

PubMed

Feng, Chen; Sun, Ping; Hu, Jing; Feng, Hua; Li, Mingqiu; Liu, Guibo; Pan, Yanming; Feng, Ying; Xu, Yongliang; Feng, Kejian; Feng, Yukuan

2017-06-01

MicroRNAs (miRNAs) play critical roles in tumorigenesis and metastasis by negatively regulating gene expression through complementary binding to the 3'-untranslated region of target mRNAs. The role of miRNAs in expression of the tumor suppressor DAB2IP in bladder cancer (BC) remains unknown. The aim of the present study was to identify miRNAs targeting DAB2IP and determine their expression and function in BC. We predicted candidate miRNAs targeting DAB2IP using TargetScan software. Dual-luciferase reporter assays confirmed that miRNA-556-3p directly regulated DAB2IP expression. Quantitative RT-PCR and RNase protection assays showed that endogenous miRNA-556-3p expression was significantly upregulated in clinical samples of BC patients and BC cell lines and western blot analysis indicated that DAB2IP expression in BC tissues and BC cell lines was concurrently downregulated. Gain or loss of function studies showed that upregulation of miRNA-556-3p promoted proliferation, invasion, migration and colony formation of BC cells, whereas downregulation resulted in opposite effects. Importantly, restoration of DAB2IP expression rescued the effects induced by miRNA-556-3p. Overexpression of miRNA-556-3p in BC cells not only decreased DAB2IP expression, but also markedly increased Ras GTPase activity and ERK1/2 phosphorylation level. These findings suggest that DAB2IP is a direct target of miRNA-556-3p, and endogenous miRNA-556-3p expression shows inverse correlation with simultaneous DAB2IP expression in BC tissues and cells. miRNA-556-3p functions as a tumor promoter in tumorigenesis and metastasis of BC by targeting DAB2IP. Moreover, miRNA-556-3p-mediated DAB2IP suppression plays an oncogenic role by partial activation of the Ras-ERK pathway.

Long-term Culture of Human iPS Cell-derived Telencephalic Neuron Aggregates on Collagen Gel.

PubMed

Oyama, Hiroshi; Takahashi, Koji; Tanaka, Yoshikazu; Takemoto, Hiroshi; Haga, Hisashi

2018-01-01

It takes several months to form the 3-dimensional morphology of the human embryonic brain. Therefore, establishing a long-term culture method for neuronal tissues derived from human induced pluripotent stem (iPS) cells is very important for studying human brain development. However, it is difficult to keep primary neurons alive for more than 3 weeks in culture. Moreover, long-term adherent culture to maintain the morphology of telencephalic neuron aggregates induced from human iPS cells is also difficult. Although collagen gel has been widely used to support long-term culture of cells, it is not clear whether human iPS cell-derived neuron aggregates can be cultured for long periods on this substrate. In the present study, we differentiated human iPS cells to telencephalic neuron aggregates and examined long-term culture of these aggregates on collagen gel. The results indicated that these aggregates could be cultured for over 3 months by adhering tightly onto collagen gel. Furthermore, telencephalic neuronal precursors within these aggregates matured over time and formed layered structures. Thus, long-term culture of telencephalic neuron aggregates derived from human iPS cells on collagen gel would be useful for studying human cerebral cortex development.Key words: Induced pluripotent stem cell, forebrain neuron, collagen gel, long-term culture.

Interferon-gamma improves impaired dentinogenic and immunosuppressive functions of irreversible pulpitis-derived human dental pulp stem cells

PubMed Central

Sonoda, Soichiro; Yamaza, Haruyoshi; Ma, Lan; Tanaka, Yosuke; Tomoda, Erika; Aijima, Reona; Nonaka, Kazuaki; Kukita, Toshio; Shi, Songtao; Nishimura, Fusanori; Yamaza, Takayoshi

2016-01-01

Clinically, irreversible pulpitis is treated by the complete removal of pulp tissue followed by replacement with artificial materials. There is considered to be a high potential for autologous transplantation of human dental pulp stem cells (DPSCs) in endodontic treatment. The usefulness of DPSCs isolated from healthy teeth is limited. However, DPSCs isolated from diseased teeth with irreversible pulpitis (IP-DPSCs) are considered to be suitable for dentin/pulp regeneration. In this study, we examined the stem cell potency of IP-DPSCs. In comparison with healthy DPSCs, IP-DPSCs expressed lower colony-forming capacity, population-doubling rate, cell proliferation, multipotency, in vivo dentin regeneration, and immunosuppressive activity, suggesting that intact IP-DPSCs may be inadequate for dentin/pulp regeneration. Therefore, we attempted to improve the impaired in vivo dentin regeneration and in vitro immunosuppressive functions of IP-DPSCs to enable dentin/pulp regeneration. Interferon gamma (IFN-γ) treatment enhanced in vivo dentin regeneration and in vitro T cell suppression of IP-DPSCs, whereas treatment with tumor necrosis factor alpha did not. Therefore, these findings suggest that IFN-γ may be a feasible modulator to improve the functions of impaired IP-DPSCs, suggesting that autologous transplantation of IFN-γ-accelerated IP-DPSCs might be a promising new therapeutic strategy for dentin/pulp tissue engineering in future endodontic treatment. PMID:26775677

Transcriptional regulation of intermediate progenitor cell generation during hippocampal development

PubMed Central

Harris, Lachlan; Zalucki, Oressia; Gobius, Ilan; McDonald, Hannah; Osinki, Jason; Harvey, Tracey J.; Essebier, Alexandra; Vidovic, Diana; Gladwyn-Ng, Ivan; Burne, Thomas H.; Heng, Julian I.; Richards, Linda J.; Gronostajski, Richard M.

2016-01-01

During forebrain development, radial glia generate neurons through the production of intermediate progenitor cells (IPCs). The production of IPCs is a central tenet underlying the generation of the appropriate number of cortical neurons, but the transcriptional logic underpinning this process remains poorly defined. Here, we examined IPC production using mice lacking the transcription factor nuclear factor I/X (Nfix). We show that Nfix deficiency delays IPC production and prolongs the neurogenic window, resulting in an increased number of neurons in the postnatal forebrain. Loss of additional Nfi alleles (Nfib) resulted in a severe delay in IPC generation while, conversely, overexpression of NFIX led to precocious IPC generation. Mechanistically, analyses of microarray and ChIP-seq datasets, coupled with the investigation of spindle orientation during radial glial cell division, revealed that NFIX promotes the generation of IPCs via the transcriptional upregulation of inscuteable (Insc). These data thereby provide novel insights into the mechanisms controlling the timely transition of radial glia into IPCs during forebrain development. PMID:27965439

Inhibition by salmeterol and cilomilast of fluticasone-enhanced IP-10 release in airway epithelial cells.

PubMed

Reddy, P J; Aksoy, Mark O; Yang, Yi; Li, Xiu Xia; Ji, Rong; Kelsen, Steven G

2008-02-01

The CXC chemokines, IP-10/CXCL10 and IL-8/CXCL8, play a role in obstructive lung disease by attracting Th1/Tc1 lymphocytes and neutrophils, respectively. Inhaled corticosteroids (ICS) and long acting beta 2-agonists (LABA) are widely used. However, their effect(s) on the release of IP-10 and IL-8 by airway epithelial cells are poorly understood. This study examined the effects of fluticasone, salmeterol, and agents which raise intracellular cAMP (cilomilast and db-cAMP) on the expression of IP-10 and IL-8 protein and mRNA. Studies were performed in cultured human airway epithelial cells during cytokine-stimulated IP-10 and IL-8 release. Cytokine treatment (TNF-alpha, IL-1beta and IFN-gamma) increased IP-10 and IL-8 protein and mRNA levels. Fluticasone (0.1 nM to 1 microM) increased IP-10 but reduced IL-8 protein release without changing IP-10 mRNA levels assessed by real time RT-PCR. The combination of salmeterol (1 micro M) and cilomilast (1-10 mu M) reduced IP-10 but had no effect on IL-8 protein. Salmeterol alone (1 micro M) and db-cAMP alone (1 mM) antagonised the effects of fluticasone on IP-10 but not IL-8 protein. In human airway epithelial cells, inhibition by salmeterol of fluticasone-enhanced IP-10 release may be an important therapeutic effect of the LABA/ICS combination not present when the two drugs are used separately.

Podocalyxin Is a Glycoprotein Ligand of the Human Pluripotent Stem Cell-Specific Probe rBC2LCN

PubMed Central

Tateno, Hiroaki; Matsushima, Asako; Hiemori, Keiko; Onuma, Yasuko; Ito, Yuzuru; Hasehira, Kayo; Nishimura, Ken; Ohtaka, Manami; Takayasu, Satoko; Nakanishi, Mahito; Ikehara, Yuzuru; Nakanishi, Mio; Ohnuma, Kiyoshi; Chan, Techuan; Toyoda, Masashi; Akutsu, Hidenori; Umezawa, Akihiro; Asashima, Makoto

2013-01-01

In comprehensive glycome analysis with a high-density lectin microarray, we have previously shown that the recombinant N-terminal domain of the lectin BC2L-C from Burkholderia cenocepacia (rBC2LCN) binds exclusively to undifferentiated human induced pluripotent stem (iPS) cells and embryonic stem (ES) cells but not to differentiated somatic cells. Here we demonstrate that podocalyxin, a heavily glycosylated type 1 transmembrane protein, is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. When analyzed by DNA microarray, podocalyxin was found to be highly expressed in both iPS cells and ES cells. Western and lectin blotting revealed that rBC2LCN binds to podocalyxin with a high molecular weight of more than 240 kDa in undifferentiated iPS cells of six different origins and four ES cell lines, but no binding was observed in either differentiated mouse feeder cells or somatic cells. The specific binding of rBC2LCN to podocalyxin prepared from a large set of iPS cells (138 types) and ES cells (15 types) was also confirmed using a high-throughput antibody-overlay lectin microarray. Alkaline digestion greatly reduced the binding of rBC2LCN to podocalyxin, indicating that the major glycan ligands of rBC2LCN are presented on O-glycans. Furthermore, rBC2LCN was found to exhibit significant affinity to a branched O-glycan comprising an H type 3 structure (Ka, 2.5 × 104 M−1) prepared from human 201B7 iPS cells, indicating that H type 3 is a most probable potential pluripotency marker. We conclude that podocalyxin is a glycoprotein ligand of rBC2LCN on human iPS cells and ES cells. PMID:23526252

Resveratrol-induced autophagy is dependent on IP3Rs and on cytosolic Ca2.

PubMed

Luyten, Tomas; Welkenhuyzen, Kirsten; Roest, Gemma; Kania, Elzbieta; Wang, Liwei; Bittremieux, Mart; Yule, David I; Parys, Jan B; Bultynck, Geert

2017-06-01

Previous work revealed that intracellular Ca 2+ signals and the inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 R) are essential to increase autophagic flux in response to mTOR inhibition, induced by either nutrient starvation or rapamycin treatment. Here, we investigated whether autophagy induced by resveratrol, a polyphenolic phytochemical reported to trigger autophagy in a non-canonical way, also requires IP 3 Rs and Ca 2+ signaling. Resveratrol augmented autophagic flux in a time-dependent manner in HeLa cells. Importantly, autophagy induced by resveratrol (80μM, 2h) was completely abolished in the presence of 10μM BAPTA-AM, an intracellular Ca 2+ -chelating agent. To elucidate the IP 3 R's role in this process, we employed the recently established HEK 3KO cells lacking all three IP 3 R isoforms. In contrast to the HEK293 wt cells and to HEK 3KO cells re-expressing IP 3 R1, autophagic responses in HEK 3KO cells exposed to resveratrol were severely impaired. These altered autophagic responses could not be attributed to alterations in the mTOR/p70S6K pathway, since resveratrol-induced inhibition of S6 phosphorylation was not abrogated by chelating cytosolic Ca 2+ or by knocking out IP 3 Rs. Finally, we investigated whether resveratrol by itself induced Ca 2+ release. In permeabilized HeLa cells, resveratrol neither affected the sarco- and endoplasmic reticulum Ca 2+ ATPase (SERCA) activity nor the IP 3 -induced Ca 2+ release nor the basal Ca 2+ leak from the ER. Also, prolonged (4 h) treatment with 100μM resveratrol did not affect subsequent IP 3 -induced Ca 2+ release. However, in intact HeLa cells, although resveratrol did not elicit cytosolic Ca 2+ signals by itself, it acutely decreased the ER Ca 2+ -store content irrespective of the presence or absence of IP 3 Rs, leading to a dampened agonist-induced Ca 2+ signaling. In conclusion, these results reveal that IP 3 Rs and cytosolic Ca 2+ signaling are fundamentally important for driving autophagic flux, not only in response to mTOR inhibition but also in response to non-canonical autophagy inducers like resveratrol. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech. Copyright © 2017 Elsevier B.V. All rights reserved.

The therapeutic potential of human adipose-derived mesenchymal stem cells producing CXCL10 in a mouse melanoma lung metastasis model.

PubMed

Mirzaei, Hamed; Salehi, Hossein; Oskuee, Reza Kazemi; Mohammadpour, Ali; Mirzaei, Hamid Reza; Sharifi, Mohammad Reza; Salarinia, Reza; Darani, Hossein Yousofi; Mokhtari, Mojgan; Masoudifar, Aria; Sahebkar, Amirhossein; Salehi, Rasoul; Jaafari, Mahmoud Reza

2018-04-10

Interferon γ-induced protein 10 kDa (IP-10) is a potent chemoattractant and has been suggested to enhance antitumor activity and mediate tumor regression through multiple mechanisms of action. Multiple lines of evidence have indicated that genetically-modified adult stem cells represent a potential source for cell-based cancer therapy. In the current study, we assessed therapeutic potential of human adipose derived mesenchymal stem cells (hADSC) genetically-modified to express IP-10 for the treatment of lung metastasis in an immunocompetent mouse model of metastatic melanoma. A Piggybac vector encoding IP-10 was employed to transfect hADSC ex vivo. Expression and bioactivity of the transgenic protein from hADSCs expressing IP-10 were confirmed prior to in vivo studies. Our results indicated that hADSCs expressing IP-10 could inhibit the growth of B16F10 melanoma cells and significantly prolonged survival. Immunohistochemistry analysis, TUNEL assay and western blot analysis indicated that hADSCs expressing IP-10 inhibited tumor cell growth, hindered tumor infiltration of Tregs, restricted angiogenesis and significantly prolonged survival. In conclusion, our results demonstrated that targeting metastatic tumor sites by hADSC expressing IP-10 could reduce melanoma tumor growth and lung metastasis. Copyright © 2018 Elsevier B.V. All rights reserved.

Abnormalities in human pluripotent cells due to reprogramming mechanisms

PubMed Central

Ma, Hong; Morey, Robert; O’Neil, Ryan C.; He, Yupeng; Daughtry, Brittany; Schultz, Matthew D.; Hariharan, Manoj; Nery, Joseph R.; Castanon, Rosa; Sabatini, Karen; Thiagarajan, Rathi D.; Tachibana, Masahito; Kang, Eunju; Tippner-Hedges, Rebecca; Ahmed, Riffat; Gutierrez, Nuria Marti; Van Dyken, Crystal; Polat, Alim; Sugawara, Atsushi; Sparman, Michelle; Gokhale, Sumita; Amato, Paula; Wolf, Don P.; Ecker, Joseph R.; Laurent, Louise C.; Mitalipov, Shoukhrat

2016-01-01

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the ‘gold standard’, they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies. PMID:25008523

Inhibition of P-Glycoprotein Mediated Efflux in Caco-2 Cells by Phytic Acid.

PubMed

Li, Lujia; Fu, Qingxue; Xia, Mengxin; Xin, Lei; Shen, Hongyi; Li, Guowen; Ji, Guang; Meng, Qianchao; Xie, Yan

2018-01-31

Phytic acid (IP6) is a natural phosphorylated inositol, which is abundantly present in most cereal grains and seeds. This study investigated the effects of IP6 regulation on P-glycoprotein (P-gp) and its potential mechanisms using in situ and in vitro models. The effective permeability of the typical P-gp substrate rhodamine 123 (R123) in colon was significantly increased from (1.69 ± 0.22) × 10 -5 cm/s in the control group to (3.39 ± 0.417) × 10 -5 cm/s (p < 0.01) in the 3.5 mM IP6 group. Additionally, IP6 can concentration-dependently decrease the R123 efflux ratio in both Caco-2 and MDCK II-MDR1 cell monolayers and increase intracellular R123 accumulation in Caco-2 cells. Furthermore, IP6 noncompetitively inhibited P-gp by impacting R123 efflux kinetics. The noncompetitive inhibition of P-gp by IP6 was likely due to decreases in P-gp ATPase activity and P-gp molecular conformational changes induced by IP6. In summary, IP6 is a promising P-gp inhibitor candidate.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orellana, S.A.; Trilivas, I.; Brown, J.H.

Carbachol and guanine nucleotides stimulate formation of the (/sup 3/H)inositol phosphates IP, IP2, and IP3 in saponin-permeabilized monolayers labelled with (/sup 3/H) inositol. Carbachol alone has little effect on formation of the (/sup 3/H) inositol phosphates (IPs), but GTP..gamma..S causes synergistic accumulation of (/sup 3/H)IPs to levels similar to those seen in intact cells. GTP, GppNHp, and GTP..gamma..S all support formation of the (/sup 3/H)IPs, with or without hormone, but GTP..gamma..S is the most effective. In the presence of GTP..gamma..S, the effect of carbachol is dose-dependent. Half-maximal and maximal accumulation of the (/sup 3/H)IPs occur at approx. 5 ..mu..M andmore » approx. 100 ..mu..M carbachol, respectively; values close to those seen in intact cells. GTP..gamma..S alone stimulates formation of the (/sup 3/H)IPs after a brief lag time. The combination of GTP..gamma..S and carbachol both increases the rate of, and decreases the lag in, formation of the (/sup 3/H)IPs. LiCl increases (/sup 3/H)IP and IP2, but not IP3, accumulation; while 2,3-diphosphoglycerate substantially increases that of (/sup 3/H)IP3. GTP..gamma..S and carbachol cause formation of (/sup 3/H)IPs in the absence of Ca/sup + +/, but formation induced by GTP..gamma..S with or without carbachol is Ca/sup + +/-sensitive over a range of physiological concentrations. Although carbachol, Ca/sup + +/, and GTP..gamma..S all have effects on formation of (/sup 3/H)IPs, GTP..gamma..S appears to be a primary and obligatory regulator of phosphoinositide hydrolysis in the permeabilized 1321N1 astrocytoma cell.« less

Transplantation of neurons derived from human iPS cells cultured on collagen matrix into guinea-pig cochleae.

PubMed

Ishikawa, Masaaki; Ohnishi, Hiroe; Skerleva, Desislava; Sakamoto, Tatsunori; Yamamoto, Norio; Hotta, Akitsu; Ito, Juichi; Nakagawa, Takayuki

2017-06-01

The present study examined the efficacy of a neural induction method for human induced pluripotent stem (iPS) cells to eliminate undifferentiated cells and to determine the feasibility of transplanting neurally induced cells into guinea-pig cochleae for replacement of spiral ganglion neurons (SGNs). A stepwise method for differentiation of human iPS cells into neurons was used. First, a neural induction method was established on Matrigel-coated plates; characteristics of cell populations at each differentiation step were assessed. Second, neural stem cells were differentiated into neurons on a three-dimensional (3D) collagen matrix, using the same protocol of culture on Matrigel-coated plates; neuron subtypes in differentiated cells on a 3D collagen matrix were examined. Then, human iPS cell-derived neurons cultured on a 3D collagen matrix were transplanted into intact guinea-pig cochleae, followed by histological analysis. In vitro analyses revealed successful induction of neural stem cells from human iPS cells, with no retention of undifferentiated cells expressing OCT3/4. After the neural differentiation of neural stem cells, approximately 70% of cells expressed a neuronal marker, 90% of which were positive for vesicular glutamate transporter 1 (VGLUT1). The expression pattern of neuron subtypes in differentiated cells on a 3D collagen matrix was identical to that of the differentiated cells on Matrigel-coated plates. In addition, the survival of transplant-derived neurons was achieved when inflammatory responses were appropriately controlled. Our preparation method for human iPS cell-derived neurons efficiently eliminated undifferentiated cells and contributed to the settlement of transplant-derived neurons expressing VGLUT1 in guinea-pig cochleae. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Stimulation of Inositol 1,4,5-Trisphosphate (IP3) Receptor Subtypes by Adenophostin A and Its Analogues

PubMed Central

Saleem, Huma; Tovey, Stephen C.; Riley, Andrew M.; Potter, Barry V. L.; Taylor, Colin W.

2013-01-01

Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular Ca2+ channels. Most animal cells express mixtures of the three IP3R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP3R and it shares with IP3 the essential features of all IP3R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP3. The two essential phosphate groups contribute to closure of the clam-like IP3-binding core (IBC), and thereby IP3R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP3R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP3R. We measured Ca2+ release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP3R and stably expressing single subtypes of mammalian IP3R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP3R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP3R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP3. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP3R subtypes. They demonstrate that differences in the Ca2+ signals evoked by AdA analogues are unlikely to be due to selective regulation of IP3R subtypes. PMID:23469136

Multiple cone pathways are involved in photic regulation of retinal dopamine.

PubMed

Qiao, Sheng-Nan; Zhang, Zhijing; Ribelayga, Christophe P; Zhong, Yong-Mei; Zhang, Dao-Qi

2016-06-30

Dopamine is a key neurotransmitter in the retina and plays a central role in the light adaptive processes of the visual system. The sole source of retinal dopamine is dopaminergic amacrine cells (DACs). We and others have previously demonstrated that DACs are activated by rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) upon illumination. However, it is still not clear how each class of photosensitive cells generates light responses in DACs. We genetically isolated cone function in mice to specifically examine the cone-mediated responses of DACs and their neural pathways. In addition to the reported excitatory input to DACs from light-increment (ON) bipolar cells, we found that cones alternatively signal to DACs via a retrograde signalling pathway from ipRGCs. Cones also produce ON and light-decrement (OFF) inhibitory responses in DACs, which are mediated by other amacrine cells, likely driven by type 1 and type 2/3a OFF bipolar cells, respectively. Dye injections indicated that DACs had similar morphological profiles with or without ON/OFF inhibition. Our data demonstrate that cones utilize specific parallel excitatory and inhibitory circuits to modulate DAC activity and efficiently regulate dopamine release and the light-adaptive state of the retina.

Regulation of myeloid leukemia factor-1 interacting protein (MLF1IP) expression in glioblastoma.

PubMed

Hanissian, Silva H; Teng, Bin; Akbar, Umar; Janjetovic, Zorica; Zhou, Qihong; Duntsch, Christopher; Robertson, Jon H

2005-06-14

The myelodysplasia/myeloid leukemia factor 1-interacting protein MLF1IP is a novel gene which encodes for a putative transcriptional repressor. It is localized to human chromosome 4q35.1 and is expressed in both the nuclei and cytoplasm of cells. Northern and Western blot analyses have revealed MLF1IP to be present at very low amounts in normal brain tissues, whereas a number of human and rat glioblastoma (GBM) cell lines demonstrated a high level expression of the MLF1IP protein. Immunohistochemical analysis of rat F98 and C6 GBM tumor models showed that MLF1IP was highly expressed in the tumor core where it was co-localized with MLF1 and nestin. Moreover, MLF1IP expression was elevated in the contralateral brain where no tumor cells were detected. These observations, together with previous data demonstrating a role for MLF1IP in erythroleukemias, suggest a possible function for this protein in glioma pathogenesis and potentially in other types of malignancies.

Unraveling the biology of bipolar disorder using induced pluripotent stem-derived neurons.

PubMed

Miller, Nathaniel D; Kelsoe, John R

2017-11-01

Bipolar disorder has been studied from numerous angles, from pathological studies to large-scale genomic studies, overall making moderate gains toward an understanding of the disorder. With the advancement of induced pluripotent stem (iPS) cell technology, in vitro models based on patient samples are now available that inherently incorporate the complex genetic variants that largely are the basis for this disorder. A number of groups are starting to apply iPS technology to the study of bipolar disorder. We selectively reviewed the literature related to understanding bipolar disorder based on using neurons derived from iPS cells. So far, most work has used the prototypical iPS cells. However, others have been able to transdifferentiate fibroblasts directly to neurons. Others still have utilized olfactory epithelium tissue as a source of neural-like cells that do not need reprogramming. In general, iPS and related cells can be used for studies of disease pathology, drug discovery, or stem cell therapy. Published studies have primarily focused on understanding bipolar disorder pathology, but initial work is also being done to use iPS technology for drug discovery. In terms of disease pathology, some evidence is pointing toward a differentiation defect with more ventral cell types being prominent. Additionally, there is evidence for a calcium signaling defect, a finding that builds on the genome-wide association study results. Continued work with iPS cells will certainly help us understand bipolar disorder and provide a way forward for improved treatments. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Exo1 plays a major role in DNA end resection in humans and influences double-strand break repair and damage signaling decisions

PubMed Central

Tomimatsu, Nozomi; Mukherjee, Bipasha; Deland, Katherine; Kurimasa, Akihiro; Bolderson, Emma; Khanna, Kum Kum; Burma, Sandeep

2012-01-01

The resection of DNA double-strand breaks (DSBs) to generate ssDNA tails is a pivotal event in the cellular response to these breaks. In the two-step model of resection, primarily elucidated in yeast, initial resection by Mre11/CtIP is followed by extensive resection by two distinct pathways involving Exo1 or BLM/WRN/Dna2. However, resection pathways and their exact contributions in humans in vivo are not as clearly worked out as in yeast. Here, we examined the contribution of Exo1 to DNA end resection in humans in vivo in response to ionizing radiation (IR) and its relationship with other resection pathways (Mre11/CtIP or BLM/WRN). We find that Exo1 plays a predominant role in resection in human cells along with an alternate pathway dependent on WRN. While Mre11 and CtIP stimulate resection in human cells, they are not absolutely required for this process and Exo1 can function in resection even in the absence of Mre11/CtIP. Interestingly, the recruitment of Exo1 to DNA breaks appears to be inhibited by the NHEJ protein Ku80, and the higher level of resection that occurs upon siRNA-mediated depletion of Ku80 is dependent on Exo1. In addition, Exo1 may be regulated by 53BP1 and Brca1, and the restoration of resection in BRCA1-deficient cells upon depletion of 53BP1 is dependent on Exo1. Finally, we find that Exo1-mediated resection facilitates a transition from ATM- to ATR-mediated cell cycle checkpoint signaling. Our results identify Exo1 as a key mediator of DNA end resection and DSB repair and damage signaling decisions in human cells. PMID:22326273

Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application.

PubMed

Zhong, Jian; Ye, Zhenqing; Lenz, Samuel W; Clark, Chad R; Bharucha, Adil; Farrugia, Gianrico; Robertson, Keith D; Zhang, Zhiguo; Ordog, Tamas; Lee, Jeong-Heon

2017-12-21

Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored. We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents. This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines

PubMed Central

Bock, Christoph; Kiskinis, Evangelos; Verstappen, Griet; Gu, Hongcang; Boulting, Gabriella; Smith, Zachary D.; Ziller, Michael; Croft, Gist F.; Amoroso, Mackenzie W.; Oakley, Derek H.; Gnirke, Andreas; Eggan, Kevin; Meissner, Alexander

2011-01-01

SUMMARY The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines. PMID:21295703

An HDAC2-TET1 switch at distinct chromatin regions significantly promotes the maturation of pre-iPS to iPS cells

PubMed Central

Wei, Tingyi; Chen, Wen; Wang, Xiukun; Zhang, Man; Chen, Jiayu; Zhu, Songcheng; Chen, Long; Yang, Dandan; Wang, Guiying; Jia, Wenwen; Yu, Yangyang; Duan, Tao; Wu, Minjuan; Liu, Houqi; Gao, Shaorong; Kang, Jiuhong

2015-01-01

The maturation of induced pluripotent stem cells (iPS) is one of the limiting steps of somatic cell reprogramming, but the underlying mechanism is largely unknown. Here, we reported that knockdown of histone deacetylase 2 (HDAC2) specifically promoted the maturation of iPS cells. Further studies showed that HDAC2 knockdown significantly increased histone acetylation, facilitated TET1 binding and DNA demethylation at the promoters of iPS cell maturation-related genes during the transition of pre-iPS cells to a fully reprogrammed state. We also found that HDAC2 competed with TET1 in the binding of the RbAp46 protein at the promoters of maturation genes and knockdown of TET1 markedly prevented the activation of these genes. Collectively, our data not only demonstrated a novel intrinsic mechanism that the HDAC2-TET1 switch critically regulates iPS cell maturation, but also revealed an underlying mechanism of the interplay between histone acetylation and DNA demethylation in gene regulation. PMID:25934799

Identification and characterization of novel IGFBP5 interacting protein: evidence IGFBP5-IP is a potential regulator of osteoblast cell proliferation

PubMed Central

Amaar, Yousef G.; Tapia, Blanca; Chen, Shin-Tai; Baylink, David J.; Mohan, Subburaman

2010-01-01

Insulin-like growth factor binding protein-5 (IGFBP5) is a multifunctional protein, which acts not only as a traditional binding protein, but also functions as a growth factor independent of IGFs to stimulate bone formation. It has been predicted that the intrinsic growth factor action of IGFBP5 involves binding of IGFBP5 to a putative receptor to induce downstream signaling pathways and/or nuclear translocation of IGFBP5 to influence transcription of genes involved in osteoblast cell proliferation/differentiation. Our study indentified proteins that bound to IGFBP5 using IGFBP5 as bait in a yeast two-hybrid screen of the U2 human osteosarcoma cell cDNA library. One of the clones that interacted strongly with the bait under high-stringency conditions corresponded to a novel IGFBP5 interacting protein (IGFBP5-IP) encoded by a gene that resides in mouse chromosome 10. The interaction between IGFBP5-IP and IGFBP5 is confirmed by in vitro coimmunoprecipitation studies that used pFlag and IGFBP5 polyclonal antibody, and cell lysates overexpressing both IGFBP5-IP and IGFBP5. Northern blot and RT-PCR analysis showed that the IGFBP-IP is expressed in both untransformed normal human osteoblasts and in osteosarcoma cell lines, which are known to produce IGFBP5. To determine the roles of IGFBP5-IP, we evaluated the effect of blocking the expression of IGFBP5-IP on osteoblast proliferation. We found that using a IGFBP5-IP-specific small interfering-hairpin plasmid resulted in a decrease in both basal and IGFBP5-induced osteoblast cell proliferation. On the basis of these findings, we predict that IGFBP5-IP may act as intracellular mediator of growth promoting actions of IGFBP5 and perhaps other osteoregulatory agents in bone cells. PMID:16269403

KChIP1 modulation of Kv4.3-mediated A-type K(+) currents and repetitive firing in hippocampal interneurons.

PubMed

Bourdeau, M L; Laplante, I; Laurent, C E; Lacaille, J-C

2011-03-10

Neuronal A-type K(+) channels regulate action potential waveform, back-propagation and firing frequency. In hippocampal CA1 interneurons located at the stratum lacunosum-moleculare/radiatum junction (LM/RAD), Kv4.3 mediates A-type K(+) currents and a Kv4 β-subunit of the Kv channel interacting protein (KChIP) family, KChIP1, appears specifically expressed in these cells. However, the functional role of this accessory subunit in A-type K(+) currents and interneuron excitability remains largely unknown. Thus, first we studied KChIP1 and Kv4.3 channel interactions in human embryonic kidney 293 (HEK293) cells and determined that KChIP1 coexpression modulated the biophysical properties of Kv4.3 A-type currents (faster recovery from inactivation, leftward shift of activation curve, faster rise time and slower decay) and this modulation was selectively prevented by KChIP1 short interfering RNA (siRNA) knockdown. Next, we evaluated the effects of KChIP1 down-regulation by siRNA on A-type K(+) currents in LM/RAD interneurons in slice cultures. Recovery from inactivation of A-type K(+) currents was slower after KChIP1 down-regulation but other properties were unchanged. In addition, down-regulation of KChIP1 levels did not affect action potential waveform and firing, but increased firing frequency during suprathreshold depolarizations, indicating that KChIP1 regulates interneuron excitability. The effects of KChIP1 down-regulation were cell-specific since CA1 pyramidal cells that do not express KChIP1 were unaffected. Overall, our findings suggest that KChIP1 interacts with Kv4.3 in LM/RAD interneurons, enabling faster recovery from inactivation of A-type currents and thus promoting stronger inhibitory control of firing during sustained activity. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Conserved Kv4 N-terminal domain critical for effects of Kv channel-interacting protein 2.2 on channel expression and gating.

PubMed

Bähring, R; Dannenberg, J; Peters, H C; Leicher, T; Pongs, O; Isbrandt, D

2001-06-29

Association of Kv channel-interacting proteins (KChIPs) with Kv4 channels leads to modulation of these A-type potassium channels (An, W. F., Bowlby, M. R., Betty, M., Cao, J., Ling, H. P., Mendoza, G., Hinson, J. W., Mattsson, K. I., Strassle, B. W., Trimmer, J. S., and Rhodes, K. J. (2000) Nature 403, 553-556). We cloned a KChIP2 splice variant (KChIP2.2) from human ventricle. In comparison with KChIP2.1, coexpression of KChIP2.2 with human Kv4 channels in mammalian cells slowed the onset of Kv4 current inactivation (2-3-fold), accelerated the recovery from inactivation (5-7-fold), and shifted Kv4 steady-state inactivation curves by 8-29 mV to more positive potentials. The features of Kv4.2/KChIP2.2 currents closely resemble those of cardiac rapidly inactivating transient outward currents. KChIP2.2 stimulated the Kv4 current density in Chinese hamster ovary cells by approximately 55-fold. This correlated with a redistribution of immunoreactivity from perinuclear areas to the plasma membrane. Increased Kv4 cell-surface expression and current density were also obtained in the absence of KChIP2.2 when the highly conserved proximal Kv4 N terminus was deleted. The same domain is required for association of KChIP2.2 with Kv4 alpha-subunits. We propose that an efficient transport of Kv4 channels to the cell surface depends on KChIP binding to the Kv4 N-terminal domain. Our data suggest that the binding is necessary, but not sufficient, for the functional activity of KChIPs.

Infiltrating T cells promote renal cell carcinoma (RCC) progression via altering the estrogen receptor β-DAB2IP signals.

PubMed

Yeh, Chiuan-Ren; Ou, Zheng-Yu; Xiao, Guang-Qian; Guancial, Elizabeth; Yeh, Shuyuan

2015-12-29

Previous studies indicated the T cells, one of the most common types of immune cells existing in the microenvironment of renal cell carcinoma (RCC), may influence the progression of RCC. The potential linkage of T cells and the estrogen receptor beta (ERβ), a key player to impact RCC progression, however, remains unclear. Our results demonstrate that RCC cells can recruit more T cells than non-malignant kidney cells. Using an in vitro matrigel invasion system, we found infiltrating T cells could promote RCC cells invasion via increasing ERβ expression and transcriptional activity. Mechanism dissection suggested that co-culturing T cells with RCC cells released more T cell attraction factors, including IFN-γ, CCL3 and CCL5, suggesting a positive regulatory feed-back mechanism. Meanwhile, infiltrating T cells may also promote RCC cell invasion via increased ERβ and decreased DAB2IP expressions, and knocking down DAB2IP can then reverse the T cells-promoted RCC cell invasion. Together, our results suggest that infiltrating T cells may promote RCC cell invasion via increasing the RCC cell ERβ expression to inhibit the tumor suppressor DAB2IP signals. Further mechanism dissection showed that co-culturing T cells with RCC cells could produce more IGF-1 and FGF-7, which may enhance the ERβ transcriptional activity. The newly identified relationship between infiltrating T cells/ERβ/DAB2IP signals may provide a novel therapeutic target in the development of agents against RCC.

Preparation of pancreatic β-cells from human iPS cells with small molecules

PubMed Central

2012-01-01

Human induced pluripotent stem (iPS) cells obtained from patients are expected to be a useful source for cell transplantation therapy, because many patients (including those with type 1 diabetes and severe type 2 diabetes) are on waiting lists for transplantation for a long time due to the shortage of donors. At present, many concerns related to clinical application of human iPS cells have been raised, but rapid development of methods for the establishment, culture, and standardization of iPS cells will lead autologous cell therapy to be realistic sooner or later. However, establishment of a method for preparing some of desired cell types is still challenging. Regarding pancreatic β-cells, there have been many reports about differentiation of these cells from human embryonic stem (ES)/iPS cells, but a protocol for clinical application has still not been established. Since there is clear proof that cell transplantation therapy is effective for diabetes based on the results of clinical islet transplantation, pancreatic β-cells prepared from human iPS cells are considered likely to be effective for reducing the burden on patients. In this article, the current status of procedures for preparing pancreatic β-cells from human ES/iPS cells, including effective use of small molecules, is summarized, and some of the problems that still need to be overcome are discussed. PMID:22722666

Running TCP/IP over ATM Networks.

ERIC Educational Resources Information Center

Witt, Michael

1995-01-01

Discusses Internet protocol (IP) and subnets and describes how IP may operate over asynchronous transfer mode (ATM). Topics include TCP (transmission control protocol), ATM cells and adaptation layers, a basic architectural model for IP over ATM, address resolution, mapping IP to a subnet technology, and connection management strategy. (LRW)

Effects of generation time on spray aerosol transport and deposition in models of the mouth-throat geometry.

PubMed

Worth Longest, P; Hindle, Michael; Das Choudhuri, Suparna

2009-06-01

For most newly developed spray aerosol inhalers, the generation time is a potentially important variable that can be fully controlled. The objective of this study was to determine the effects of spray aerosol generation time on transport and deposition in a standard induction port (IP) and more realistic mouth-throat (MT) geometry. Capillary aerosol generation (CAG) was selected as a representative system in which spray momentum was expected to significantly impact deposition. Sectional and total depositions in the IP and MT geometries were assessed at a constant CAG flow rate of 25 mg/sec for aerosol generation times of 1, 2, and 4 sec using both in vitro experiments and a previously developed computational fluid dynamics (CFD) model. Both the in vitro and numerical results indicated that extending the generation time of the spray aerosol, delivered at a constant mass flow rate, significantly reduced deposition in the IP and more realistic MT geometry. Specifically, increasing the generation time of the CAG system from 1 to 4 sec reduced the deposition fraction in the IP and MT geometries by approximately 60 and 33%, respectively. Furthermore, the CFD predictions of deposition fraction were found to be in good agreement with the in vitro results for all times considered in both the IP and MT geometries. The numerical results indicated that the reduction in deposition fraction over time was associated with temporal dissipation of what was termed the spray aerosol "burst effect." Based on these results, increasing the spray aerosol generation time, at a constant mass flow rate, may be an effective strategy for reducing deposition in the standard IP and in more realistic MT geometries.

Specific receptor for inositol-1,4,5-trisphosphate in permeabilized rabbit neutrophils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bradford, P.G.; Spat, A.; Rubin, R.P.

1986-03-05

Neutrophil chemotaxis and degranulation are resultant, in part, from the mobilization of intracellular calcium by inositol-1,4,5-trisphosphate ((1,4,5)IP/sub 3/), one of the products of chemoattractant-stimulated phospholipase C activity. High specific activity (ca. 40 Ci/mmol) (/sup 32/P)(1,4,5)IP/sub 3/ was prepared from (..gamma..-/sup 32/P)ATP-labeled human erythrocyte ghosts and was used in binding assays with saponin-permeabilized rabbit peritoneal neutrophils. At 4/sup 0/C and in the presence of inhibitors of the IP/sub 3/ 5-phosphomonoesterase, (/sup 32/P)(1,4,5)IP/sub 3/ rapidly associated with a specific binding component which saturated within 60s. Nonspecific binding, taken as the residual binding in the presence of 10 ..mu..M (1,4,5)IP/sub 3/, was 15%more » of the total. No specific binding was detected using intact cells. The specific binding to permeable cells was reversible (t/sup 1/2/ approx. 60s) and could be inhibited in a dose-dependent manner by (1,4,5)IP/sub 3/ (EC/sub 50/ = 30 nM) and by other calcium mobilizing inositol phosphates ((2,4,5)IP/sub 3/) but not by inactive analogs ((1,4)IP/sub 2/, (4,5)IP/sub 2/, (1)IP). The dose-responses of (1,4,5)IP/sub 3/ and (2,4,5)IP/sub 3/ in inhibiting (/sup 32/P)(1,4,5)IP/sub 3/ specific binding correlated well with their abilities to release Ca/sup 2 +/ from nonmitochondrial vesicular stores in the same preparation of cells, suggesting that the authors have identified the physiological receptor for (1,4,5)IP/sub 3/.« less

Limited hair cell induction from human induced pluripotent stem cells using a simple stepwise method.

PubMed

Ohnishi, Hiroe; Skerleva, Desislava; Kitajiri, Shin-ichiro; Sakamoto, Tatsunori; Yamamoto, Norio; Ito, Juichi; Nakagawa, Takayuki

2015-07-10

Disease-specific induced pluripotent stem cells (iPS) cells are expected to contribute to exploring useful tools for studying the pathophysiology of inner ear diseases and to drug discovery for treating inner ear diseases. For this purpose, stable induction methods for the differentiation of human iPS cells into inner ear hair cells are required. In the present study, we examined the efficacy of a simple induction method for inducing the differentiation of human iPS cells into hair cells. The induction of inner ear hair cell-like cells was performed using a stepwise method mimicking inner ear development. Human iPS cells were sequentially transformed into the preplacodal ectoderm, otic placode, and hair cell-like cells. As a first step, preplacodal ectoderm induction, human iPS cells were seeded on a Matrigel-coated plate and cultured in a serum free N2/B27 medium for 8 days according to a previous study that demonstrated spontaneous differentiation of human ES cells into the preplacodal ectoderm. As the second step, the cells after preplacodal ectoderm induction were treated with basic fibroblast growth factor (bFGF) for induction of differentiation into otic-placode-like cells for 15 days. As the final step, cultured cells were incubated in a serum free medium containing Matrigel for 48 days. After preplacodal ectoderm induction, over 90% of cultured cells expressed the genes that express in preplacodal ectoderm. By culture with bFGF, otic placode marker-positive cells were obtained, although their number was limited. Further 48-day culture in serum free media resulted in the induction of hair cell-like cells, which expressed a hair cell marker and had stereocilia bundle-like constructions on their apical surface. Our results indicate that hair cell-like cells are induced from human iPS cells using a simple stepwise method with only bFGF, without the use of xenogeneic cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Phorbol 12-myristate 13-acetate down-regulates Na,K-ATPase independent of its protein kinase C site: decrease in basolateral cell surface area.

PubMed Central

Beron, J; Forster, I; Beguin, P; Geering, K; Verrey, F

1997-01-01

The effect of protein kinase C (PKC) stimulation on the pump current (Ip) generated by the Na,K-ATPase was measured in A6 epithelia apically permeabilized with amphotericin B. Phorbol 12-myristate 13-acetate (PMA) produced a decrease in Ip carried by sodium pumps containing the endogenous Xenopus laevis or transfected Bufo marinus alpha 1 subunits (approximately 30% reduction within 25 min, maximum after 40 min) independent of the PKC phosphorylation site (T15A/S16A). In addition to this major effect of PMA, which was independent of the intracellular sodium concentration and was prevented by the PKC inhibitor bisindolylmaleimide GF 109203X (BIM), another BIM-resistant, PKC site-independent decrease was observed when the Ip was measured at low sodium concentrations (total reduction approximately 50% at 5 mM sodium). Using ouabain binding and cell surface biotinylation, stimulation of PKC was shown to reduce surface Na,K-ATPase by 14 to 20% within 25 min. The same treatment stimulated fluid phase endocytosis sevenfold and decreased by 16.5% the basolateral cell surface area measured by transepithelial capacitance measurements. In conclusion, PKC stimulation produces a decrease in sodium pump function which can be attributed, to a large extent, to a withdrawal of sodium pumps from the basolateral cell surface independent of their PKC site. This reduction of the number of sodium pumps is parallel to a decrease in basolateral membrane area. Images PMID:9188092

Red Wine Consumption is inversely associated with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA Adduct Levels in Prostate

PubMed Central

Rybicki, Benjamin A.; Neslund-Dudas, Christine; Bock, Cathryn H.; Nock, Nora L.; Rundle, Andrew; Jankowski, Michelle; Levin, Albert M.; Beebe-Dimmer, Jennifer; Savera, Adnan T.; Takahashi, Satoru; Shirai, Tomoyuki; Tang, Deliang

2011-01-01

In humans, genetic variation and dietary factors may alter the biologic effects of exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of the major heterocyclic amines generated from cooking meats at high temperatures that has carcinogenic potential through the formation of DNA adducts. Previously, we reported grilled red meat consumption associated with PhIP-DNA adduct levels in human prostate. In the present study, we expanded our investigation to estimate the associations between beverage consumption and PhIP-DNA adduct levels in prostate for 391 prostate cancer cases. Of the 15 beverages analyzed, red wine consumption had the strongest association with PhIP-DNA adduct levels showing an inverse correlation in both tumor (p=0.006) and non-tumor (p=0.002) prostate cells. Red wine consumption differed significantly between African-American and white cases, but PhIP-DNA adduct levels in prostate did not vary by race. In African Americans compared with whites, however, associations between red wine consumption and PhIP-DNA adduct levels were not as strong as associations with specific (e.g., SULT1A1 and UGT1A10 genotypes) and non-specific (e.g., African ancestry) genetic variation. In a multivariable model, the covariate for red wine consumption explained a comparable percentage (13-16%) of the variation in PhIP-DNA adduct levels in prostate across the two racial groups, but the aforementioned genetic factors explained 33% of the PhIP-DNA adduct variation in African-American cases, while only 19% of the PhIPDNA adduct variation in whites. We conclude that red wine consumption may counteract biologic effects of PhIP exposure in human prostate, but genetic factors may play an even larger role, particularly in African Americans. PMID:21846795

Cardiac-restricted Overexpression of TRAF3 Interacting Protein 2 (TRAF3IP2) Results in Spontaneous Development of Myocardial Hypertrophy, Fibrosis, and Dysfunction *

PubMed Central

Sakamuri, Siva S. V. P.; Siddesha, Jalahalli M.; Saifudeen, Zubaida; Ma, Lixin; Siebenlist, Ulrich; Gardner, Jason D.; Chandrasekar, Bysani

2016-01-01

TRAF3IP2 (TRAF3 interacting protein 2; previously known as CIKS or Act1) is a key intermediate in the normal inflammatory response and the pathogenesis of various autoimmune and inflammatory diseases. Induction of TRAF3IP2 activates IκB kinase (IKK)/NF-κB, JNK/AP-1, and c/EBPβ and stimulates the expression of various inflammatory mediators with negative myocardial inotropic effects. To investigate the role of TRAF3IP2 in heart disease, we generated a transgenic mouse model with cardiomyocyte-specific TRAF3IP2 overexpression (TRAF3IP2-Tg). Echocardiography, magnetic resonance imaging, and pressure-volume conductance catheterization revealed impaired cardiac function in 2-month-old male transgenic (Tg) mice as evidenced by decreased ejection fraction, stroke volume, cardiac output, and peak ejection rate. Moreover, the male Tg mice spontaneously developed myocardial hypertrophy (increased heart/body weight ratio, cardiomyocyte cross-sectional area, GATA4 induction, and fetal gene re-expression). Furthermore, TRAF3IP2 overexpression resulted in the activation of IKK/NF-κB, JNK/AP-1, c/EBPβ, and p38 MAPK and induction of proinflammatory cytokines, chemokines, and extracellular matrix proteins in the heart. Although myocardial hypertrophy decreased with age, cardiac fibrosis (increased number of myofibroblasts and enhanced expression and deposition of fibrillar collagens) increased progressively. Despite these adverse changes, TRAF3IP2 overexpression did not result in cell death at any time period. Interestingly, despite increased mRNA expression, TRAF3IP2 protein levels and activation of its downstream signaling intermediates remained unchanged in the hearts of female Tg mice. The female Tg mice also failed to develop myocardial hypertrophy. In summary, these results demonstrate that overexpression of TRAF3IP2 in male mice is sufficient to induce myocardial hypertrophy, cardiac fibrosis, and contractile dysfunction. PMID:27466370

Human iPS cell-engineered cardiac tissue sheets with cardiomyocytes and vascular cells for cardiac regeneration

PubMed Central

Masumoto, Hidetoshi; Ikuno, Takeshi; Takeda, Masafumi; Fukushima, Hiroyuki; Marui, Akira; Katayama, Shiori; Shimizu, Tatsuya; Ikeda, Tadashi; Okano, Teruo; Sakata, Ryuzo; Yamashita, Jun K.

2014-01-01

To realize cardiac regeneration using human induced pluripotent stem cells (hiPSCs), strategies for cell preparation, tissue engineering and transplantation must be explored. Here we report a new protocol for the simultaneous induction of cardiomyocytes (CMs) and vascular cells [endothelial cells (ECs)/vascular mural cells (MCs)], and generate entirely hiPSC-engineered cardiovascular cell sheets, which showed advantageous therapeutic effects in infarcted hearts. The protocol adds to a previous differentiation protocol of CMs by using stage-specific supplementation of vascular endothelial cell growth factor for the additional induction of vascular cells. Using this cell sheet technology, we successfully generated physically integrated cardiac tissue sheets (hiPSC-CTSs). HiPSC-CTS transplantation to rat infarcted hearts significantly improved cardiac function. In addition to neovascularization, we confirmed that engrafted human cells mainly consisted of CMs in >40% of transplanted rats four weeks after transplantation. Thus, our HiPSC-CTSs show promise for cardiac regenerative therapy. PMID:25336194

Vectors to Increase Production Efficiency of Inducible Pluripotent Stem Cell (iPSC) | NCI Technology Transfer Center | TTC

Cancer.gov

This invention describes the discovery that specific p53 isoform increase the number of inducible pluripotent stem cells (iPS). It is known that the activity of p53 regulates the self-renewal and pluripotency of normal and cancer stem cells, and also affects re-programming efficiency of iPS cells. This p53 isoform-based technology provides a more natural process of increasing iPS cell production than previous methods of decreasing p53. NCI seeks licensees for this technology.

Polycistronic lentiviral vector for "hit and run" reprogramming of adult skin fibroblasts to induced pluripotent stem cells.

PubMed

Chang, Chia-Wei; Lai, Yi-Shin; Pawlik, Kevin M; Liu, Kaimao; Sun, Chiao-Wang; Li, Chao; Schoeb, Trenton R; Townes, Tim M

2009-05-01

We report the derivation of induced pluripotent stem (iPS) cells from adult skin fibroblasts using a single, polycistronic lentiviral vector encoding the reprogramming factors Oct4, Sox2, and Klf4. Porcine teschovirus-1 2A sequences that trigger ribosome skipping were inserted between human cDNAs for these factors, and the polycistron was subcloned downstream of the elongation factor 1 alpha promoter in a self-inactivating (SIN) lentiviral vector containing a loxP site in the truncated 3' long terminal repeat (LTR). Adult skin fibroblasts from a humanized mouse model of sickle cell disease were transduced with this single lentiviral vector, and iPS cell colonies were picked within 30 days. These cells expressed endogenous Oct4, Sox2, Nanog, alkaline phosphatase, stage-specific embryonic antigen-1, and other markers of pluripotency. The iPS cells produced teratomas containing tissue derived from all three germ layers after injection into immunocompromised mice and formed high-level chimeras after injection into murine blastocysts. iPS cell lines with as few as three lentiviral insertions were obtained. Expression of Cre recombinase in these iPS cells resulted in deletion of the lentiviral vector, and sequencing of insertion sites demonstrated that remnant 291-bp SIN LTRs containing a single loxP site did not interrupt coding sequences, promoters, or known regulatory elements. These results suggest that a single, polycistronic "hit and run" vector can safely and effectively reprogram adult dermal fibroblasts into iPS cells.

p53 isoform Δ133p53 promotes efficiency of induced pluripotent stem cells and ensures genomic integrity during reprogramming.

PubMed

Gong, Lu; Pan, Xiao; Chen, Haide; Rao, Lingjun; Zeng, Yelin; Hang, Honghui; Peng, Jinrong; Xiao, Lei; Chen, Jun

2016-11-22

Human induced pluripotent stem (iPS) cells have great potential in regenerative medicine, but this depends on the integrity of their genomes. iPS cells have been found to contain a large number of de novo genetic alterations due to DNA damage response during reprogramming. Thus, to maintain the genetic stability of iPS cells is an important goal in iPS cell technology. DNA damage response can trigger tumor suppressor p53 activation, which ensures genome integrity of reprogramming cells by inducing apoptosis and senescence. p53 isoform Δ133p53 is a p53 target gene and functions to not only antagonize p53 mediated apoptosis, but also promote DNA double-strand break (DSB) repair. Here we report that Δ133p53 is induced in reprogramming. Knockdown of Δ133p53 results 2-fold decrease in reprogramming efficiency, 4-fold increase in chromosomal aberrations, whereas overexpression of Δ133p53 with 4 Yamanaka factors showes 4-fold increase in reprogamming efficiency and 2-fold decrease in chromosomal aberrations, compared to those in iPS cells induced only with 4 Yamanaka factors. Overexpression of Δ133p53 can inhibit cell apoptosis and promote DNA DSB repair foci formation during reprogramming. Our finding demonstrates that the overexpression of Δ133p53 not only enhances reprogramming efficiency, but also results better genetic quality in iPS cells.

Mastoparan-Induced Intracellular Ca2+ Fluxes May Regulate Cell-to-Cell Communication in Plants.

PubMed

Tucker, E. B.; Boss, W. F.

1996-06-01

The relationship of Ca2+ and plasmodesmatal closure was examined in staminal hairs of Setcreasea purpurea by microinjecting cells with active mastoparan (Mas-7), inactive mastoparan (Mas-17), active inositol-1,4,5-trisphosphate (IP3), or inactive IP3. Calcium green dextran 10,000 was used to study cellular free Ca2+, and carboxyfluorescein was used to monitor plasmodesmatal closure. When Mas-7 was microinjected into the cytoplasm of cell 1 (the tip cell of a chain of cells), a rapid increase in calcium green dextran-10,000 fluorescence was observed in the cytoplasmic areas on both sides of the plasmodesmata connecting cells 1 and 2 during the same time that the diffusion of carboxyfluorescein through them was blocked. The inhibition of cell-to-cell diffusion was transient, and the closed plasmodesmata reopened within 30 s. The elevated Ca2+ level near plasmodesmata was also transient and returned to base level in about 1.5 min. The transient increase in Ca2+, once initiated in cell 1, repeated with an oscillatory period of 3 min. Elevated Ca2+ and oscillations of Ca2+ were also observed near interconnecting cell walls throughout the chain of cells, indicating that the signal had been transmitted. Previously, we reported that IP3 closed plasmodesmata; now we report that it stimulated Ca2+ and oscillations similar to Mas-7. The effect was specific for similar concentrations of Mas-7 over Mas-17 and active IP3 over inactive IP3. It is important that the Ca2+ channel blocker La3+ eliminated the responses from Mas-7 and IP3, indicating that an influx of Ca2+ was required. These results support the contention that plasmodesmata functioning is regulated via Ca2+ and that IP3 may be an intermediary between the stimulus and Ca2+ elevations.

Mastoparan-Induced Intracellular Ca2+ Fluxes May Regulate Cell-to-Cell Communication in Plants.

PubMed Central

Tucker, E. B.; Boss, W. F.

1996-01-01

The relationship of Ca2+ and plasmodesmatal closure was examined in staminal hairs of Setcreasea purpurea by microinjecting cells with active mastoparan (Mas-7), inactive mastoparan (Mas-17), active inositol-1,4,5-trisphosphate (IP3), or inactive IP3. Calcium green dextran 10,000 was used to study cellular free Ca2+, and carboxyfluorescein was used to monitor plasmodesmatal closure. When Mas-7 was microinjected into the cytoplasm of cell 1 (the tip cell of a chain of cells), a rapid increase in calcium green dextran-10,000 fluorescence was observed in the cytoplasmic areas on both sides of the plasmodesmata connecting cells 1 and 2 during the same time that the diffusion of carboxyfluorescein through them was blocked. The inhibition of cell-to-cell diffusion was transient, and the closed plasmodesmata reopened within 30 s. The elevated Ca2+ level near plasmodesmata was also transient and returned to base level in about 1.5 min. The transient increase in Ca2+, once initiated in cell 1, repeated with an oscillatory period of 3 min. Elevated Ca2+ and oscillations of Ca2+ were also observed near interconnecting cell walls throughout the chain of cells, indicating that the signal had been transmitted. Previously, we reported that IP3 closed plasmodesmata; now we report that it stimulated Ca2+ and oscillations similar to Mas-7. The effect was specific for similar concentrations of Mas-7 over Mas-17 and active IP3 over inactive IP3. It is important that the Ca2+ channel blocker La3+ eliminated the responses from Mas-7 and IP3, indicating that an influx of Ca2+ was required. These results support the contention that plasmodesmata functioning is regulated via Ca2+ and that IP3 may be an intermediary between the stimulus and Ca2+ elevations. PMID:12226302

Mechanism of cisplatin resistance in human urothelial carcinoma cells.

PubMed

Yu, Hui-Min; Wang, Tsing-Cheng

2012-05-01

An isogenic pair of cisplatin-susceptible (NTUB1) and -resistant (NTUB1/P) human urothelial carcinoma cell lines was used to elucidate the mechanism of cisplatin resistance. The significantly lower intracellular platinum (IP) concentration, which resulted from the decreased cisplatin uptake, was found in NTUB1/P cells. The enhancement of IP concentration did not increase the susceptibility of NTUB1/P cells to cisplatin treatment. The reduction of IP concentration as well was unable to enhance the cisplatin-resistance in susceptible NTUB1 cells. This indicated that reduction of IP concentration was not the account for the development of cisplatin resistance here. Instead, the over expression of anti-apoptotic Bcl-2, anti-oxidative heme oxygenase-1 (HO-1) and cell cycle regulator p16INK4 seemed to be more important for the gaining of cisplatin in these human urothelial carcinoma cell. Copyright © 2012 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Taguchi, M.; Field, J.B.

Thyrotropin (TSH) and carbachol stimulated in a dose-dependent manner the accumulation of 3H-glycerophosphoinositol (GPI), 3H-inositol monophosphate (IP1), 3H-inositol bisphosphate (IP2) and 3H-inositol trisphosphate (IP3) in primary cultures of dog thyroid cells prelabeled with myo-(2-3H)inositol. TSH, 250 mU/mL, stimulated 3H-IP3 level after a 10-minute incubation while 10 mU/mL TSH increased it during a 60-minute incubation. The effect of carbachol was more rapid and greater than that of TSH. Carbachol, 100 mumol/L, elevated 3H-IP3 after a 2-minute incubation and 3H-IP3 formation was increased by as little as 1 mumol/L carbachol. TSH stimulation was observed only if the cells were deprived of TSHmore » for 5 days before being labeled with 3H-inositol. Prolongation of the labeling period or addition of TSH, (Bu)2cAMP or carbachol during the labeling increased 3H-inositol incorporation into polyphoinositides (PIPs). When the cells were labeled without any other addition, control and TSH-stimulated 3H-IP3 levels increased in parallel with 3H-PIP levels. However, TSH or carbachol-stimulated 3H-IP3 levels did not increase in proportion to 3H-PIPs level when the cells were labeled with TSH or (Bu)2cAMP. Thus, the ratio of 3H-IP3/3H-PIPs (both control and TSH or carbachol-stimulated) decreased in the cells labeled with TSH or (Bu)2cAMP, which might reflect TSH stimulation of 3H-inositol incorporation into PIPs pool(s) that do not participate in hormone-induced hydrolysis of PIPs.« less

An Impact Assessment Model for Distributed Adaptive Security Situation Assessment

DTIC Science & Technology

2005-01-01

the cargo manifest can be either a 56K modem-based TCP/IP connection (the oval labeled internet) or a 40K wireless modem connection ( cell phone ) that...via a UDP connection on the 40K wireless modem ( cell phone ). For each resource, either alternative may be used to achieve the same goal, but some...Manifests Comm-in Comp- power Comm- out JTF Internet (TCP-IP) Cell phone (TCP-IP) Internet (UDP) Cell phone (UDP) Manual Computer 4

Distribution Profile of Inositol 1,4,5-Trisphosphate Receptor/Ca2+ Channels in α and β Cells of Pancreas: Dominant Localization in Secretory Granules and Common Error in Identification of Secretory Granule Membranes.

PubMed

Hur, Yong Suk; Yoo, Seung Hyun

2015-01-01

The α and β cells of pancreatic islet release important hormones in response to intracellular Ca increases that result from Ca releases through the inositol 1,4,5-trisphoshate receptor (IP3R)/Ca channels. Yet no systematic studies on distribution of IP3R/Ca channels have been done, prompting us to investigate the distribution of all 3 IP3R isoforms. Immunogold electron microscopy was performed to determine the presence and the relative concentrations of all 3 IP3R isoforms in 2 major organelles secretory granules (SGs) and the endoplasmic reticulum of α and β cells of rat pancreas. All 3 IP3R isoforms were present in SG membranes of both cells, and the IP3R concentrations in SGs were ∼2-fold higher than those in the endoplasmic reticulum. Moreover, large halos shown in the electron microscope images of insulin-containing SGs of β cells were gap spaces that resulted from separation of granule membranes from the surrounding cytoplasm. These results strongly suggest the important roles of SGs in IP3-induced, Ca-dependent regulatory secretory pathway in pancreas. Moreover, the accurate location of SG membranes of β cells was further confirmed by the location of another integral membrane protein synaptotagmin V and of membrane phospholipid PI(4,5)P2.

MLF1-interacting protein is mainly localized in nucleolus through N-terminal bipartite nuclear localization signal.

PubMed

Suzuki, Hideaki; Arakawa, Yasuhiro; Ito, Masaki; Saito, Shinobu; Takeda, Nobuakira; Yamada, Hisashi; Horiguchi-Yamada, Junko

2007-01-01

The myelodysplasia/myeloid leukemia factor 1-interacting protein (MLF1LP, also called KLIP1 and CENP-50) is reported to localize in both the nucleus and the cytoplasm. To investigate the functions of MLF1IP, its subnuclear localization was studied. MLF1IP was tagged with green fluorescent protein (EGFP). Fibrillarin was tagged with red fluorescent protein (DsRed). EGFP-tagged MLF1IP deletion vectors were also constructed. Plasmid-constructs were transfected into human cervical adenocarcinoma HeLa cells or monkey kidney fibroblast COS-7 cells, and the localization was studied by either confocal fluorescence microscopy or fluorescence microscopy. Ectopically expressed MLF1IP was localized mainly in the nucleolus. In some cells, small dot-like particles of MLF1IP fluorescence were observed in the nucleoplasm. Co-staining of fibrillarin disclosed that MLF1IP was co-localized with fibrillarin in the nucleolus. Deletion mutants of MLF1IP revealed that the N-terminal bipartite nuclear localization signal (NLS) was responsible for nucleolar targeting. MLF1IP was localized mainly in the nucleolus through the N-terminal bipartite NLS and partly in the nucleoplasm featuring small dot-like particles. These findings suggest that MLF1IP may have multi-functions and its different localizations may contribute to carcinogenesis.

Single-cell imaging techniques for the real-time detection of IP₃ in live cells.

PubMed

Nelson, Carl P

2013-01-01

Inositol 1,4,5-trisphosphate (IP(3)) is a ubiquitous second messenger, derived from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) by enzymes of the phospholipase C (PLC) family. Binding of IP(3) to its cognate receptor in the endoplasmic reticulum membrane leads to release of Ca(2+) into the cytoplasm, which is involved in the regulation of an array of cellular functions. Traditional techniques for the detection of IP(3) have required the extraction of a large number of cells, with limitations in the time resolution of changes in IP(3) and an inability to obtain detailed information on the dynamics of this second messenger in single cells. Recent progress in this field has led to the development of a number of genetically encoded fluorescent biosensors, which upon recombinant expression are able selectively to detect real-time changes in IP(3) in single live cells. In this chapter, I detail protocols for the expression, visualization (by confocol or fluorescence microscopy), and interpretation of data obtained with such biosensors expressed in mammalian cells.

Expression and high glucose-mediated regulation of K+ channel interacting protein 3 (KChIP3) and KV4 channels in retinal Müller glial cells.

PubMed

Chavira-Suárez, Erika; Sandoval, Alejandro; Felix, Ricardo; Lamas, Mónica

2011-01-14

Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K(+) channels K(V)4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the K(V)4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K(+) current and an alteration in the inactivation of the macroscopic outward K(+) currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of K(V)4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy. Copyright © 2010 Elsevier Inc. All rights reserved.

RdgB2 is required for dim-light input into intrinsically photosensitive retinal ganglion cells.

PubMed

Walker, Marquis T; Rupp, Alan; Elsaesser, Rebecca; Güler, Ali D; Sheng, Wenlong; Weng, Shijun; Berson, David M; Hattar, Samer; Montell, Craig

2015-10-15

A subset of retinal ganglion cells is intrinsically photosensitive (ipRGCs) and contributes directly to the pupillary light reflex and circadian photoentrainment under bright-light conditions. ipRGCs are also indirectly activated by light through cellular circuits initiated in rods and cones. A mammalian homologue (RdgB2) of a phosphoinositide transfer/exchange protein that functions in Drosophila phototransduction is expressed in the retinal ganglion cell layer. This raised the possibility that RdgB2 might function in the intrinsic light response in ipRGCs, which depends on a cascade reminiscent of Drosophila phototransduction. Here we found that under high light intensities, RdgB2(-/-) mutant mice showed normal pupillary light responses and circadian photoentrainment. Consistent with this behavioral phenotype, the intrinsic light responses of ipRGCs in RdgB2(-/-) were indistinguishable from wild-type. In contrast, under low-light conditions, RdgB2(-/-) mutants displayed defects in both circadian photoentrainment and the pupillary light response. The RdgB2 protein was not expressed in ipRGCs but was in GABAergic amacrine cells, which provided inhibitory feedback onto bipolar cells. We propose that RdgB2 is required in a cellular circuit that transduces light input from rods to bipolar cells that are coupled to GABAergic amacrine cells and ultimately to ipRGCs, thereby enabling ipRGCs to respond to dim light. © 2015 Walker et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

[Unresolved issues in the evaluation of research projects involving induced pluripotent stem cells (iPS)].

PubMed

Casado, María; de Lecuona, Itziar

2013-01-01

This paper identifies problems and analyzes those conflicts posed by the evaluation of research projects involving the collection and use of human induced pluripotent stem cells (iPS) in Spain. Current legislation is causing problems of interpretation, circular and unnecessary referrals, legal uncertainty and undue delays. Actually, this situation may cause a lack of control and monitoring, and even some paralysis in regenerative medicine and cell therapy research, that is a priority nowadays. The analysis of the current legislation and its bioethical implications, led us to conclude that the review of iPS research projects cannot be assimilated to the evaluation of research projects that involve human embryonic stem cell (hESC). In this context, our proposal is based on the review by the Research Ethics Committees and the checkout by the Spanish Comission of Guarantees for Donation and Use of Human Cells and Tissues (CGDUCTH) of human iPS cells research projects. Moreover, this article claims for a more transparent research system, by effectively articulating the Registry on Research Projects. Finally, a model of verification protocol (checklist) for checking out biomedical research projects involving human iPS cells is suggested.

The inositol trisphosphate receptor in the control of autophagy.

PubMed

Criollo, Alfredo; Vicencio, José Miguel; Tasdemir, Ezgi; Maiuri, M Chiara; Lavandero, Sergio; Kroemer, Guido

2007-01-01

The second messenger myo-inositol-1,4,5-trisphosphate (IP(3)) acts on the IP(3) receptor (IP(3)R), an IP(3)-activated Ca(2+) channel of the endoplasmic reticulum (ER). The IP(3)R agonist IP(3) inhibits starvation-induced autophagy. The IP(3)R antagonist xestospongin B induces autophagy in human cells through a pathway that requires the obligate contribution of Beclin-1, Atg5, Atg10, Atg12 and hVps34, yet is inhibited by ER-targeted Bcl-2 or Bcl-XL, two proteins that physically interact with IP(3)R. Autophagy can also be induced by depletion of the IP(3)R by small interfering RNAs. Autophagy induction by IP(3)R blockade cannot be explained by changes in steady state levels of Ca(2+) in the endoplasmic reticulum (ER) and the cytosol. Autophagy induction by IP(3)R blockade is effective in cells lacking the obligate mediator of ER stress IRE1. In contrast, IRE1 is required for autophagy induced by ER stress-inducing agents such a tunicamycin or thapsigargin. These findings suggest that there are several distinct pathways through which autophagy can be initiated at the level of the ER.

The new generation of beta-cells: replication, stem cell differentiation, and the role of small molecules.

PubMed

Borowiak, Malgorzata

2010-01-01

Diabetic patients suffer from the loss of insulin-secreting β-cells, or from an improper working β-cell mass. Due to the increasing prevalence of diabetes across the world, there is a compelling need for a renewable source of cells that could replace pancreatic β-cells. In recent years, several promising approaches to the generation of new β-cells have been developed. These include directed differentiation of pluripotent cells such as embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, or reprogramming of mature tissue cells. High yield methods to differentiate cell populations into β-cells, definitive endoderm, and pancreatic progenitors, have been established using growth factors and small molecules. However, the final step of directed differentiation to generate functional, mature β-cells in sufficient quantities has yet to be achieved in vitro. Beside the needs of transplantation medicine, a renewable source of β-cells would also be important in terms of a platform to study the pathogenesis of diabetes, and to seek alternative treatments. Finally, by generating new β-cells, we could learn more details about pancreatic development and β-cell specification. This review gives an overview of pancreas ontogenesis in the perspective of stem cell differentiation, and highlights the critical aspects of small molecules in the generation of a renewable β-cell source. Also, it discusses longer term challenges and opportunities in moving towards a therapeutic goal for diabetes.

Effects of antioxidants on the quality and genomic stability of induced pluripotent stem cells

PubMed Central

Luo, Lan; Kawakatsu, Miho; Guo, Chao-Wan; Urata, Yoshishige; Huang, Wen-Jing; Ali, Haytham; Doi, Hanako; Kitajima, Yuriko; Tanaka, Takayuki; Goto, Shinji; Ono, Yusuke; Xin, Hong-Bo; Hamano, Kimikazu; Li, Tao-Sheng

2014-01-01

Effects of antioxidants on the quality and genomic stability of induced pluripotent stem (iPS) cells were investigated with two human iPS cell lines (201B7 and 253G1). Cells used in this study were expanded from a single colony of each cell line with the addition of proprietary antioxidant supplement or homemade antioxidant cocktail in medium, and maintained in parallel for 2 months. The cells grew well in all culture conditions and kept “stemness”. Although antioxidants modestly decreased the levels of intracellular reactive oxygen species, there were no differences in the expression of 53BP1 and pATM, two critical molecules related with DNA damage and repair, under various culture conditions. CGH analysis showed that the events of genetic aberrations were decreased only in the 253G1 iPS cells with the addition of homemade antioxidant cocktail. Long-term culture will be necessary to confirm whether low dose antioxidants improve the quality and genomic stability of iPS cells. PMID:24445363

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs

PubMed Central

Chen, Yen-Ming; Chen, Li-Hua; Li, Meng-Pei; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Ling, Qing-Dong; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Chang, Yung; Benelli, Giovanni; Murugan, Kadarkarai; Umezawa, Akihiro

2017-01-01

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells. PMID:28332572

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs.

PubMed

Chen, Yen-Ming; Chen, Li-Hua; Li, Meng-Pei; Li, Hsing-Fen; Higuchi, Akon; Kumar, S Suresh; Ling, Qing-Dong; Alarfaj, Abdullah A; Munusamy, Murugan A; Chang, Yung; Benelli, Giovanni; Murugan, Kadarkarai; Umezawa, Akihiro

2017-03-23

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells.

Dependence of Na+ pump current on external monovalent cations and membrane potential in rabbit cardiac Purkinje cells.

PubMed Central

Bielen, F V; Glitsch, H G; Verdonck, F

1991-01-01

1. The effect of membrane potential and various extracellular monovalent cations on the Na+ pump current (Ip) was studied on isolated, single Purkinje cells of the rabbit heart by means of whole-cell recording. 2. Ip was identified as current activated by external K+ or its congeners NH4+ and Tl+. The current was blocked by dihydroouabain (1-5 x 10(-4) M) over the whole range of membrane potentials tested. 3. In Na(+)-containing solution half-maximum Ip activation (K0.5) occurred at 0.4 mM-Tl+, 1.9 mM-K+ and 5.7 mM-NH4+ (holding potential, -20 mV). 4. The pump current (Ip)-voltage (V) relationship of the cells in Na(+)-containing media with K+ or its congeners at the tested concentrations greater than K0.5 displayed a steep positive slope at negative membrane potentials between -120 and -20 mV. Little voltage dependence of Ip was observed at more positive potentials up to +40 mV. At even more positive potentials Ip measured at 2 and 5.4 mM-K+ decreased. 5. Lowering the concentration of K+ or its congeners below the K0.5 value in Na(+)-containing solution induced a region of negative slope of the Ip-V curve at membrane potentials positive to -20 mV. 6. The shape of the Ip-V relationship remained unchanged when the K+ concentration (5.4 mM) of the Na(+)-containing medium was replaced by NH4+ or Tl+ concentrations of similar potency to activate Ip (20 mM-NH4+ or 2 mM-Tl+). 7. In Na(+)-free, choline-containing solution half-maximum Ip activation occurred at 0.13 mM-K+ (holding potential, -20 mV). 8. At negative membrane potentials the positive slope of the Ip-V curve was flatter in Na(+)-free than in Na(+)-containing media. A reduced voltage dependence of Ip persisted, regardless of whether choline ions or Li+ were used as a Na+ substitute. 9. Lowering the K+ concentration of the Na(+)-free, choline-containing solution to 0.05 mM evoked an extended region of negative slope in the Ip-V relationship at membrane potentials between -40 and +60 mV. 10. It is concluded that the apparent affinity of the Na(+)-K+ pump towards K+ in cardiac Purkinje cells depends on both the membrane potential and the extracellular Na+ concentration. 11. The region of negative slope of the Ip-V curve observed in cells which were superfused with media containing low concentrations of K+ or its congeners strongly suggests the existence of at least two voltage-sensitive steps in the cardiac Na(+)-K+ pump cycle.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1665855

Planckian Information (Ip): A New Measure of Order in Atoms, Enzymes, Cells, Brains, Human Societies, and the Cosmos

NASA Astrophysics Data System (ADS)

Ji, Sungchul

A new mathematical formula referred to as the Planckian distribution equation (PDE) has been found to fit long-tailed histograms generated in various fields of studies, ranging from atomic physics to single-molecule enzymology, cell biology, brain neurobiology, glottometrics, econophysics, and to cosmology. PDE can be derived from a Gaussian-like equation (GLE) by non-linearly transforming its variable, x, while keeping the y coordinate constant. Assuming that GLE represents a random distribution (due to its symmetry), it is possible to define a binary logarithm of the ratio between the areas under the curves of PDE and GLE as a measure of the non-randomness (or order) underlying the biophysicochemical processes generating long-tailed histograms that fit PDE. This new function has been named the Planckian information, IP, which (i) may be a new measure of order that can be applied widely to both natural and human sciences and (ii) can serve as the opposite of the Boltzmann-Gibbs entropy, S, which is a measure of disorder. The possible rationales for the universality of PDE may include (i) the universality of the wave-particle duality embedded in PDE, (ii) the selection of subsets of random processes (thereby breaking the symmetry of GLE) as the basic mechanism of generating order, organization, and function, and (iii) the quantity-quality complementarity as the connection between PDE and Peircean semiotics.

Antisense Oligonucleotides Targeting Parasite Inositol 1,4,5-Trisphosphate Receptor Inhibits Mammalian Host Cell Invasion by Trypanosoma cruzi

NASA Astrophysics Data System (ADS)

Hashimoto, Muneaki; Nara, Takeshi; Hirawake, Hiroko; Morales, Jorge; Enomoto, Masahiro; Mikoshiba, Katsuhiko

2014-02-01

Chagas disease is caused by an intracellular parasitic protist, Trypanosoma cruzi. As there are no highly effective drugs against this agent that also demonstrate low toxicity, there is an urgent need for development of new drugs to treat Chagas disease. We have previously demonstrated that the parasite inositol 1,4,5-trisphosphate receptor (TcIP3R) is crucial for invasion of the mammalian host cell by T. cruzi. Here, we report that TcIP3R is a short-lived protein and that its expression is significantly suppressed in trypomastigotes. Treatment of trypomastigotes, an infective stage of T. cruzi, with antisense oligonucleotides specific to TcIP3R deceased TcIP3R protein levels and impaired trypomastigote invasion of host cells. Due to the resulting instability and very low expression level of TcIP3R in trypomastigotes indicates that TcIP3R is a promising target for antisense therapy in Chagas disease.

Retinoic acid-induced upregulation of the metalloendopeptidase nardilysin is accelerated by co-expression of the brain-specific protein p42(IP4) (centaurin α 1; ADAP1) in neuroblastoma cells.

PubMed

Borrmann, Claudia; Stricker, Rolf; Reiser, Georg

2011-11-01

The mainly neuronally expressed protein p42(IP4) (centaurin α1; ADAP1), which interacts with the metalloendopeptidase nardilysin (NRD) was found to be localized in neuritic plaques in Alzheimer disease (AD) brains. NRD was shown to enhance the cleavage of the amyloid precursor protein (APP) by α-secretases, thereby increasing the release of neuroprotective sAPPα. We here investigated in vitro the biochemical interaction of p42(IP4) and NRD and studied the physiological interaction in SH-SY5Y cells. NRD is a member of the M16 family of metalloendopeptidases. Some members of this M16 family act bi-functionally, as protease and as non-enzymatic scaffold protein. Here, we show that p42(IP4) enhances the enzymatic activity of NRD 3-4 times. However, p42(IP4) is not a substrate for NRD. Furthermore, we report that differentiation of SH-SY5Y cells by stimulation with 10μM retinoic acid (RA) results in upregulation of NRD protein levels, with a 6-fold rise after 15 days. NRD is expressed in the neurites of RA-stimulated SH-SY5Y cells, and localized in vesicular structures. Since p42(IP4) is not expressed in untreated SH-SY5Y cells, we could use this cell system as a model to find out, whether there is a functional interaction. Interestingly, SH-SY5Y cells, which we stably transfected with GFP-tagged-p42(IP4) showed an enhanced NRD protein expression already at an earlier time point after RA stimulation. Copyright © 2011 Elsevier B.V. All rights reserved.

T-box Transcription Regulator Tbr2 Is Essential for the Formation and Maintenance of Opn4/Melanopsin-Expressing Intrinsically Photosensitive Retinal Ganglion Cells

PubMed Central

Li, Hongyan; Zhang, Zhijing; Kiyama, Takae; Panda, Satchidananda; Hattar, Samer; Ribelayga, Christophe P.; Mills, Stephen L.

2014-01-01

Opsin 4 (Opn4)/melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) play a major role in non-image-forming visual system. Although advances have been made in understanding their morphological features and functions, the molecular mechanisms that regulate their formation and survival remain unknown. Previously, we found that mouse T-box brain 2 (Tbr2) (also known as Eomes), a T-box-containing transcription factor, was expressed in a subset of newborn RGCs, suggesting that it is involved in the formation of specific RGC subtypes. In this in vivo study, we used complex mouse genetics, single-cell dye tracing, and behavioral analyses to determine whether Tbr2 regulates ipRGC formation and survival. Our results show the following: (1) Opn4 is expressed exclusively in Tbr2-positive RGCs; (2) no ipRGCs are detected when Tbr2 is genetically ablated before RGC specification; and (3) most ipRGCs are eliminated when Tbr2 is deleted in established ipRGCs. The few remaining ipRGCs display abnormal dendritic morphological features and functions. In addition, some Tbr2-expressing RGCs can activate Opn4 expression on the loss of native ipRGCs, suggesting that Tbr2-expressing RGCs may serve as a reservoir of ipRGCs to regulate the number of ipRGCs and the expression levels of Opn4. PMID:25253855

The cooked meat-derived mammary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine promotes invasive behaviour of breast cancer cells.

PubMed

Lauber, Sandra N; Gooderham, Nigel J

2011-01-11

The cooked meat derived genotoxic carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces cancer of the colon, prostate and mammary gland when fed to rats. Epidemiology studies link these tumours to a Western diet and exposure to heterocyclic amines such as PhIP. We have shown that PhIP is also potently estrogenic and have proposed that this hormonal activity contributes to its target site carcinogenicity. We now postulate that the estrogenic properties of PhIP influence metastatic potential. We have used an in vitro assay for cell invasion based upon digestion and migration through a reconstituted basement membrane model. Zymography and immunoblotting were used to confirm PhIP-mediated changes associated with induction of the invasive phenotype. Treatment of the mammary cancer cell lines MCF-7 and T47D with PhIP induces cells to digest and migrate through a reconstituted basement membrane. The response was dose dependent, observed at sub-nanomolar concentrations of PhIP and was inhibited by the antiestrogen ICI 182,780. The PhIP-induced invasive phenotype was associated with expression of cathepsin D, cyclooxygenase-2 and matrix metalloproteinase activity. These findings emphasise the range and potency of the biological activities associated with this cooked meat product and mechanistically support the tissue-specific carcinogenicity of the chemical. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Derivation of novel human ground state naive pluripotent stem cells.

PubMed

Gafni, Ohad; Weinberger, Leehee; Mansour, Abed AlFatah; Manor, Yair S; Chomsky, Elad; Ben-Yosef, Dalit; Kalma, Yael; Viukov, Sergey; Maza, Itay; Zviran, Asaf; Rais, Yoach; Shipony, Zohar; Mukamel, Zohar; Krupalnik, Vladislav; Zerbib, Mirie; Geula, Shay; Caspi, Inbal; Schneir, Dan; Shwartz, Tamar; Gilad, Shlomit; Amann-Zalcenstein, Daniela; Benjamin, Sima; Amit, Ido; Tanay, Amos; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

2013-12-12

Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3β signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.

Inositol trisphosphate metabolism in carrot (Daucus carota L. ) cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Memon, A.R.; Rincon, M.; Boss, W.F.

1989-10-01

The metabolism of exogenously added D-myo-(1-{sup 3}H)inositol 1,4,5-trisphosphate (IP{sub 3}) has been examined in microsomal membrane and soluble fractions of carrot cells grown in suspension culture. When ({sup 3}H)IP{sub 3} was added to a microsomal membrane fraction, ({sup 3}H)IP{sub 2} was the primary metabolite consisting of approximately 83% of the total recovered ({sup 3}H) by electrophoresis. ({sup 3}H)IP was only 6% of the ({sup 3}H) recovered, and 10% of the ({sup 3}H)IP{sub 3} was not further metabolized. In contrast, when ({sup 3}H)IP{sub 3} was added to the soluble fraction, approximately equal amounts of ({sup 3}H)IP{sub 2} and ({sup 3}H)IP weremore » recovered. Ca{sup 2+} (100 micromolar) tended to enhance IP{sub 3} dephosphorylation but inhibited the IP{sub 2} dephosphorylation in the soluble fraction by about 20%. MoO{sub 4}{sup 2{minus}} (1 millimolar) inhibited the dephosphorylation of IP{sub 3} by the microsomal fraction and the dephosphorylation of IP{sub 2} by the soluble fraction. MoO{sub 4}{sup 2{minus}}, however, did not inhibit the dephosphorylation of IP{sub 3} by the soluble fraction. Li{sup +} (10 and 50 millimolar) had no effect on IP{sub 3} metabolism in either the soluble or membrane fraction; however, Li{sup +} (50 millimolar) inhibited IP{sub 2} dephosphorylation in the soluble fraction about 25%.« less

Stem cell transplantation therapy for multifaceted therapeutic benefits after stroke.

PubMed

Wei, Ling; Wei, Zheng Z; Jiang, Michael Qize; Mohamad, Osama; Yu, Shan Ping

2017-10-01

One of the exciting advances in modern medicine and life science is cell-based neurovascular regeneration of damaged brain tissues and repair of neuronal structures. The progress in stem cell biology and creation of adult induced pluripotent stem (iPS) cells has significantly improved basic and pre-clinical research in disease mechanisms and generated enthusiasm for potential applications in the treatment of central nervous system (CNS) diseases including stroke. Endogenous neural stem cells and cultured stem cells are capable of self-renewal and give rise to virtually all types of cells essential for the makeup of neuronal structures. Meanwhile, stem cells and neural progenitor cells are well-known for their potential for trophic support after transplantation into the ischemic brain. Thus, stem cell-based therapies provide an attractive future for protecting and repairing damaged brain tissues after injury and in various disease states. Moreover, basic research on naïve and differentiated stem cells including iPS cells has markedly improved our understanding of cellular and molecular mechanisms of neurological disorders, and provides a platform for the discovery of novel drug targets. The latest advances indicate that combinatorial approaches using cell based therapy with additional treatments such as protective reagents, preconditioning strategies and rehabilitation therapy can significantly improve therapeutic benefits. In this review, we will discuss the characteristics of cell therapy in different ischemic models and the application of stem cells and progenitor cells as regenerative medicine for the treatment of stroke. Copyright © 2017 Elsevier Ltd. All rights reserved.

Type 1 and 3 inositol trisphosphate receptors are required for extra-embryonic vascular development.

PubMed

Uchida, Keiko; Nakazawa, Maki; Yamagishi, Chihiro; Mikoshiba, Katsuhiko; Yamagishi, Hiroyuki

2016-10-01

The embryonic-maternal interface of the placental labyrinth, allantois, and yolk sac are vital during embryogenesis; however, the precise mechanism underlying the vascularization of these structures remains unknown. Herein we focus on the role of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R), which are intracellular Ca(2+) release channels, in placentation. Double knockout (DKO) of type 1 and 3 IP3Rs (IP3R1 and IP3R3, respectively) in mice resulted in embryonic lethality around embryonic day (E) 11.5. Because IP3R1 and IP3R3 were co-expressed in endothelial cells in the labyrinth, allantois, and yolk sac, we investigated extra-embryonic vascular development in IP3R1- and IP3R3-DKO mice. The formation of chorionic plates and yolk sac vessels seemed dysregulated around the timing of the chorio-allantoic attachment, immediately followed by the disorganization of allantoic vessels, the decreased expression of the spongiotrophoblast cell marker Tpbpa and the growth retardation of the embryos in DKO mice. Fluorescent immunohistochemistry demonstrated downregulation of a vascular endothelial marker, CD31, in labyrinth embryonic vessels and poor elongation of extra-embryonic mesoderm into the labyrinth layer in DKO placenta, whereas the branching of the DKO chorionic trophoblast was initiated. In addition, allantoic and yolk sac vessels in extra-embryonic tissues were less remodeled in DKO mice. In vitro endothelial cord formation and migration activities of cultured vascular endothelial cells derived from human umbilical vein were downregulated under the inhibition of IP3R. Our results suggest that IP3R1 and IP3R3 are required for extra-embryonic vascularization in the placenta, allantois, and yolk sac. This is the first demonstration of the essential role of IP3/IP3Rs signaling in the development of the vasculature at the embryonic-maternal interface. Copyright © 2016 Elsevier Inc. All rights reserved.

Temperature and nucleotide dependence of calcium release by myo-inositol 1,4,5-trisphosphate in cultured vascular smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, J.B.; Smith, L.; Higgins, B.L.

1985-11-25

Inositol 1,4,5-trisphosphate (IP3) rapidly increased UVCaS efflux from a nonmitochondrial organelle in cultured vascular smooth muscle cells that were permeabilized with saponin. A nucleotide, preferably ATP, was essential for IP3-evoked UVCaS release. Two nonhydrolyzable ATP analogues satisfied the nucleotide requirement for IP3-evoked UVCaS release. IP3 strongly stimulated UVCaS efflux at low temperatures (1 to 15 degrees C). Decreasing the temperature from 37 to 4 degrees C inhibited the rate of IP3-stimulated efflux by only about 33%. The failure of such low temperatures to strongly inhibit IP3-induced UVCaS efflux suggests that IP3 activated a CaS channel, rather than a carrier, bymore » a ligand-binding, rather than a metabolic, reaction.« less

Hematopoietic transcriptional mechanisms: from locus-specific to genome-wide vantage points.

PubMed

DeVilbiss, Andrew W; Sanalkumar, Rajendran; Johnson, Kirby D; Keles, Sunduz; Bresnick, Emery H

2014-08-01

Hematopoiesis is an exquisitely regulated process in which stem cells in the developing embryo and the adult generate progenitor cells that give rise to all blood lineages. Master regulatory transcription factors control hematopoiesis by integrating signals from the microenvironment and dynamically establishing and maintaining genetic networks. One of the most rudimentary aspects of cell type-specific transcription factor function, how they occupy a highly restricted cohort of cis-elements in chromatin, remains poorly understood. Transformative technologic advances involving the coupling of next-generation DNA sequencing technology with the chromatin immunoprecipitation assay (ChIP-seq) have enabled genome-wide mapping of factor occupancy patterns. However, formidable problems remain; notably, ChIP-seq analysis yields hundreds to thousands of chromatin sites occupied by a given transcription factor, and only a fraction of the sites appear to be endowed with critical, non-redundant function. It has become en vogue to map transcription factor occupancy patterns genome-wide, while using powerful statistical tools to establish correlations to inform biology and mechanisms. With the advent of revolutionary genome editing technologies, one can now reach beyond correlations to conduct definitive hypothesis testing. This review focuses on key discoveries that have emerged during the path from single loci to genome-wide analyses, specifically in the context of hematopoietic transcriptional mechanisms. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

Systematic evaluation of the impact of ChIP-seq read designs on genome coverage, peak identification, and allele-specific binding detection.

PubMed

Zhang, Qi; Zeng, Xin; Younkin, Sam; Kawli, Trupti; Snyder, Michael P; Keleş, Sündüz

2016-02-24

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) experiments revolutionized genome-wide profiling of transcription factors and histone modifications. Although maturing sequencing technologies allow these experiments to be carried out with short (36-50 bps), long (75-100 bps), single-end, or paired-end reads, the impact of these read parameters on the downstream data analysis are not well understood. In this paper, we evaluate the effects of different read parameters on genome sequence alignment, coverage of different classes of genomic features, peak identification, and allele-specific binding detection. We generated 101 bps paired-end ChIP-seq data for many transcription factors from human GM12878 and MCF7 cell lines. Systematic evaluations using in silico variations of these data as well as fully simulated data, revealed complex interplay between the sequencing parameters and analysis tools, and indicated clear advantages of paired-end designs in several aspects such as alignment accuracy, peak resolution, and most notably, allele-specific binding detection. Our work elucidates the effect of design on the downstream analysis and provides insights to investigators in deciding sequencing parameters in ChIP-seq experiments. We present the first systematic evaluation of the impact of ChIP-seq designs on allele-specific binding detection and highlights the power of pair-end designs in such studies.

Distribution profile of inositol 1,4,5-trisphosphate receptor isoforms in adrenal chromaffin cells.

PubMed

Huh, Yang Hoon; Yoo, Jie Ae; Bahk, Sook Jin; Yoo, Seung Hyun

2005-05-09

Given the importance of inositol 1,4,5-trisphosphate receptor (IP(3)R)/Ca(2+) channels in the control of intracellular Ca(2+) concentrations, we determined the relative concentrations of the IP(3)R isoforms in subcellular organelles, based on serially sectioned electron micrographs. The endoplasmic reticulum (ER) was estimated to contain 15-20% of each of the three IP(3)R isoforms while secretory granules contained 58-69%. The nucleus contained approximately 15% each of IP(3)R-1 and -2, but 25% of IP(3)R-3, whereas the plasma membrane contained approximately 1% or less of each. These suggested that secretory granules, the nucleus and ER are at the center of IP(3)-dependent intracellular Ca(2+) control mechanisms in chromaffin cells.

Molecular imaging of labile iron(II) pools in living cells with a turn-on fluorescent probe.

PubMed

Au-Yeung, Ho Yu; Chan, Jefferson; Chantarojsiri, Teera; Chang, Christopher J

2013-10-09

Iron is an essential metal for living organisms, but misregulation of its homeostasis at the cellular level can trigger detrimental oxidative and/or nitrosative stress and damage events. Motivated to help study the physiological and pathological consequences of biological iron regulation, we now report a reaction-based strategy for monitoring labile Fe(2+) pools in aqueous solution and living cells. Iron Probe 1 (IP1) exploits a bioinspired, iron-mediated oxidative C-O bond cleavage reaction to achieve a selective turn-on response to Fe(2+) over a range of cellular metal ions in their bioavailable forms. We show that this first-generation chemical tool for fluorescence Fe(2+) detection can visualize changes in exchangeable iron stores in living cells upon iron supplementation or depletion, including labile iron pools at endogenous, basal levels. Moreover, IP1 can be used to identify reversible expansion of labile iron pools by stimulation with vitamin C or the iron regulatory hormone hepcidin, providing a starting point for further investigations of iron signaling and stress events in living systems as well as future probe development.

Demethylation of induced pluripotent stem cells from type 1 diabetic patients enhances differentiation into functional pancreatic β cells.

PubMed

Manzar, Gohar S; Kim, Eun-Mi; Zavazava, Nicholas

2017-08-25

Type 1 diabetes (T1D) can be managed by transplanting either the whole pancreas or isolated pancreatic islets. However, cadaveric pancreas is scarcely available for clinical use, limiting this approach. As such, there is a great need to identify alternative sources of clinically usable pancreatic tissues. Here, we used induced pluripotent stem (iPS) cells derived from patients with T1D to generate glucose-responsive, insulin-producing cells (IPCs) via 3D culture. Initially, T1D iPS cells were resistant to differentiation, but transient demethylation treatment significantly enhanced IPC yield. The cells responded to high-glucose stimulation by secreting insulin in vitro The shape, size, and number of their granules, as observed by transmission electron microscopy, were identical to those found in cadaveric β cells. When the IPCs were transplanted into immunodeficient mice that had developed streptozotocin-induced diabetes, they promoted a dramatic decrease in hyperglycemia, causing the mice to become normoglycemic within 28 days. None of the mice died or developed teratomas. Because the cells are derived from "self," immunosuppression is not required, providing a much safer and reliable treatment option for T1D patients. Moreover, these cells can be used for drug screening, thereby accelerating drug discovery. In conclusion, our approach eliminates the need for cadaveric pancreatic tissue.

Distinctive functions of Syk N-terminal and C-terminal SH2 domains in the signaling cascade elicited by oxidative stress in B cells.

PubMed

Ding, J; Takano, T; Hermann, P; Gao, S; Han, W; Noda, C; Yanagi, S; Yamamura, H

2000-05-01

Syk plays a crucial role in the transduction of oxidative stress signaling. In this paper, we investigated the roles of Src homology 2 (SH2) domains of Syk in oxidative stress signaling, using Syk-negative DT40 cells expressing the N- or C-terminal SH2 domain mutant [mSH2(N) or mSH2(C)] of Syk. Tyrosine phosphorylation of Syk in cells expressing mSH2(N) Syk after H(2)O(2) treatment was higher than that in cells expressing wild-type Syk or mSH2(C) Syk. The tyrosine phosphorylation of wild-type Syk and mSH2(C) Syk, but not that of mSH2(N), was sensitive to PP2, a specific inhibitor of Src-family protein-tyrosine kinase. In oxidative stress, the C-terminal SH2 domain of Syk was demonstrated to be required for induction of tyrosine phosphorylation of cellular proteins, phospholipase C (PLC)-gamma2 phosphorylation, inositol 1,4, 5-triphosphate (IP(3)) generation, Ca(2)(+) release from intracellular stores, and c-Jun N-terminal kinase activation. In contrast, in mSH2(N) Syk-expressing cells, tyrosine phosphorylation of intracellular proteins including PLC-gamma2 was markedly induced in oxidative stress. The enhanced phosphorylation of mSH2(N) Syk and PLC-gamma2, however, did not link to Ca(2)(+) mobilization from intracellular pools and IP(3) generation. Thus, the N- and C-terminal SH2 domains of Syk possess distinctive functions in oxidative stress signaling.

A unique stylopod patterning mechanism by Shox2-controlled osteogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Wenduo; Song, Yingnan; Huang, Zhen

Here, vertebrate appendage patterning is programmed by Hox-TALE factorbound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clusteringmore » around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.« less

A unique stylopod patterning mechanism by Shox2-controlled osteogenesis

DOE PAGES

Ye, Wenduo; Song, Yingnan; Huang, Zhen; ...

2016-06-10

Here, vertebrate appendage patterning is programmed by Hox-TALE factorbound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clusteringmore » around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.« less

Identification of Epigenetic Changes in Prostate Cancer using Induced Pluripotent Stem Cells

DTIC Science & Technology

2014-04-01

somatic cells in human iPS cells. Nat Cell Bioi, 13: 541, 2011 5. Polo, J. M., Liu, S., Figueroa , M. E. et al.: Cell type of origin influences the...human iPS cells. Nat Cell Bioi 13: 5•1 \\ - 5•19. 18. Poloj:\\ə, Lu S, Figueroa ME, Kulalert W, Eminli S, ct a!. (20 10) Cell type of origin

Potassium Channel Interacting Protein 2 (KChIP2) is not a transcriptional regulator of cardiac electrical remodeling

PubMed Central

Winther, Sine V.; Tuomainen, Tomi; Borup, Rehannah; Tavi, Pasi; Antoons, Gudrun; Thomsen, Morten B.

2016-01-01

The heart-failure relevant Potassium Channel Interacting Protein 2 (KChIP2) augments CaV1.2 and KV4.3. KChIP3 represses CaV1.2 transcription in cardiomyocytes via interaction with regulatory DNA elements. Hence, we tested nuclear presence of KChIP2 and if KChIP2 translocates into the nucleus in a Ca2+ dependent manner. Cardiac biopsies from human heart-failure patients and healthy donor controls showed that nuclear KChIP2 abundance was significantly increased in heart failure; however, this was secondary to a large variation of total KChIP2 content. Administration of ouabain did not increase KChIP2 content in nuclear protein fractions in anesthetized mice. KChIP2 was expressed in cell lines, and Ca2+ ionophores were applied in a concentration- and time-dependent manner. The cell lines had KChIP2-immunoreactive protein in the nucleus in the absence of treatments to modulate intracellular Ca2+ concentration. Neither increasing nor decreasing intracellular Ca2+ concentrations caused translocation of KChIP2. Microarray analysis did not identify relief of transcriptional repression in murine KChIP2−/− heart samples. We conclude that although there is a baseline presence of KChIP2 in the nucleus both in vivo and in vitro, KChIP2 does not directly regulate transcriptional activity. Moreover, the nuclear transport of KChIP2 is not dependent on Ca2+. Thus, KChIP2 does not function as a conventional transcription factor in the heart. PMID:27349185

Generation of polyclonal antibodies against a chemically synthesized N-terminal fragment of the bacteriocin pediocin PA-1.

PubMed

Martínez, M I; Rodríguez, J M; Suárez, A; Martínez, J M; Azcona, J I; Hernández, P E

1997-06-01

Six mice were immunized intraperitoneally (i.p.) with a chemically synthesized 9-mer fragment (PH1) designed from the N-terminal part of the bacteriocin pediocin PA-1 and conjugated to keyhole limpet haemocyanin (KLH). After three doses of the immunogen had been administered, serum-specific antibodies were detected by a competitive direct ELISA. Myeloma cells were injected i.p. into mice in order to obtain ascites polyclonal antibodies. Although four mice developed ascites, only mouse 2 had detectable specific antibodies in the ascites fluid. The serum and ascites antibodies were specific for PH1 but they did not recognize the whole pediocin PA-1 molecule. This is the first attempt to generate antibodies against bacteriocins with a chemically synthesized oligopeptide as immunogen. This approach still remains attractive for detection, quantification, mode of action studies and purification of bacteriocins, especially those for which the purification process is difficult or inefficient at present.

Genome Therapy of Myotonic Dystrophy Type 1 iPS Cells for Development of Autologous Stem Cell Therapy.

PubMed

Gao, Yuanzheng; Guo, Xiuming; Santostefano, Katherine; Wang, Yanlin; Reid, Tammy; Zeng, Desmond; Terada, Naohiro; Ashizawa, Tetsuo; Xia, Guangbin

2016-08-01

Myotonic dystrophy type 1 (DM1) is caused by expanded Cytosine-Thymine-Guanine (CTG) repeats in the 3'-untranslated region (3' UTR) of the Dystrophia myotonica protein kinase (DMPK) gene, for which there is no effective therapy. The objective of this study is to develop genome therapy in human DM1 induced pluripotent stem (iPS) cells to eliminate mutant transcripts and reverse the phenotypes for developing autologous stem cell therapy. The general approach involves targeted insertion of polyA signals (PASs) upstream of DMPK CTG repeats, which will lead to premature termination of transcription and elimination of toxic mutant transcripts. Insertion of PASs was mediated by homologous recombination triggered by site-specific transcription activator-like effector nuclease (TALEN)-induced double-strand break. We found genome-treated DM1 iPS cells continue to maintain pluripotency. The insertion of PASs led to elimination of mutant transcripts and complete disappearance of nuclear RNA foci and reversal of aberrant splicing in linear-differentiated neural stem cells, cardiomyocytes, and teratoma tissues. In conclusion, genome therapy by insertion of PASs upstream of the expanded DMPK CTG repeats prevented the production of toxic mutant transcripts and reversal of phenotypes in DM1 iPS cells and their progeny. These genetically-treated iPS cells will have broad clinical application in developing autologous stem cell therapy for DM1.

Development of fully non-inductive plasmas heated by medium and high-harmonic fast waves in the national spherical torus experiment upgrade

NASA Astrophysics Data System (ADS)

Taylor, G.; Poli, F.; Bertelli, N.; Harvey, R. W.; Hosea, J. C.; Mueller, D.; Perkins, R. J.; Phillips, C. K.; Raman, R.

2015-12-01

A major challenge for spherical tokamak development is to start-up and ramp-up the plasma current (Ip) without using a central solenoid. Experiments in the National Spherical Torus eXperiment (NSTX) demonstrated that 1.4 MW of 30 MHz high-harmonic fast wave (HHFW) power could generate an Ip = 300 kA H-mode discharge with a non-inductive Ip fraction, fNI ˜ 0.7. The discharge had an axial toroidal magnetic field (BT(0)) of 0.55 T, the maximum BT(0) available on NSTX. NSTX has undergone a major upgrade (NSTX-U), that will eventually allow the generation of BT(0) ≤ 1 T and Ip ≤ 2 MA plasmas. Full wave simulations of 30 MHz HHFW and medium harmonic fast wave (MHFW) heating in NSTX-U predict significantly reduced FW power loss in the plasma edge at the higher BT(0) achievable in NSTX-U. HHFW experiments will aim to generate stable, fNI ˜ 1, Ip = 300 kA H-mode plasmas and to ramp Ip from 250 to 400 kA with FW power. Time-dependent TRANSP simulations are used to develop non-inductive Ip ramp-up and sustainment using 30 MHz FW power. This paper presents results from these RF simulations and plans for developing non-inductive plasmas heated by FW power.

Hindered cytoplasmic diffusion of inositol trisphosphate restricts its cellular range of action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dickinson, G. D.; Ellefsen, K. L.; Dawson, S. P.

The range of action of intracellular messengers is determined by their rates of diffusion and degradation. Previous measurements in oocyte cytoplasmic extracts indicated that the Ca 2+-liberating second messenger inositol trisphosphate (IP 3) diffuses with a coefficient (~280 μm 2 s -1) similar to that in water, corresponding to a range of action of ~25 μm. Consequently, IP 3 is generally considered a “global” cellular messenger. We also reexamined this issue by measuring local IP 3-evoked Ca 2+ puffs to monitor IP 3 diffusing from spot photorelease in neuroblastoma cells. Fitting these data by numerical simulations yielded a diffusion coefficientmore » (≤10 μm 2 s -1) about 30-fold slower than that previously reported. Here, we propose that diffusion of IP 3 in mammalian cells is hindered by binding to immobile, functionally inactive receptors that were diluted in oocyte extracts. The predicted range of action of IP 3 (<5 μm) is thus smaller than the size of typical mammalian cells, indicating that IP 3 should better be considered as a local rather than a global cellular messenger.« less

Hindered cytoplasmic diffusion of inositol trisphosphate restricts its cellular range of action

DOE PAGES

Dickinson, G. D.; Ellefsen, K. L.; Dawson, S. P.; ...

2016-11-08

The range of action of intracellular messengers is determined by their rates of diffusion and degradation. Previous measurements in oocyte cytoplasmic extracts indicated that the Ca 2+-liberating second messenger inositol trisphosphate (IP 3) diffuses with a coefficient (~280 μm 2 s -1) similar to that in water, corresponding to a range of action of ~25 μm. Consequently, IP 3 is generally considered a “global” cellular messenger. We also reexamined this issue by measuring local IP 3-evoked Ca 2+ puffs to monitor IP 3 diffusing from spot photorelease in neuroblastoma cells. Fitting these data by numerical simulations yielded a diffusion coefficientmore » (≤10 μm 2 s -1) about 30-fold slower than that previously reported. Here, we propose that diffusion of IP 3 in mammalian cells is hindered by binding to immobile, functionally inactive receptors that were diluted in oocyte extracts. The predicted range of action of IP 3 (<5 μm) is thus smaller than the size of typical mammalian cells, indicating that IP 3 should better be considered as a local rather than a global cellular messenger.« less

Inhibition of pulmonary fibrosis by the chemokine IP-10/CXCL10.

PubMed

Tager, Andrew M; Kradin, Richard L; LaCamera, Peter; Bercury, Scott D; Campanella, Gabriele S V; Leary, Carol P; Polosukhin, Vasiliy; Zhao, Long-Hai; Sakamoto, Hideo; Blackwell, Timothy S; Luster, Andrew D

2004-10-01

Pulmonary fibrosis is an enigmatic and devastating disease with few treatment options, now thought to result from abnormal wound healing in the lung in response to injury. We have previously noted a role for the chemokine interferon gamma-inducible protein of 10 kD (IP-10)/CXC chemokine ligand 10 in the regulation of cutaneous wound healing, and consequently investigated whether IP-10 regulates pulmonary fibrosis. We found that IP-10 is highly expressed in a mouse model of pulmonary fibrosis induced by bleomycin. IP-10-deficient mice exhibited increased pulmonary fibrosis after administration of bleomycin, suggesting that IP-10 limits the development of fibrosis in this model. Substantial fibroblast chemoattractant and proliferative activities were generated in the lung after bleomycin exposure. IP-10 significantly inhibited fibroblast responses to the chemotactic, but not the proliferative activity generated, suggesting that IP-10 may attenuate fibroblast accumulation in bleomycin-induced pulmonary fibrosis by limiting fibroblast migration. Consistent with this inhibitory activity of IP-10 on fibroblast migration, fibroblast accumulation in the lung after bleomycin exposure was dramatically increased in IP-10-deficient mice compared with wild-type mice. Conversely, transgenic mice overexpressing IP-10 were protected from mortality after bleomycin exposure, and demonstrated decreased fibroblast accumulation in the lung after challenge compared with wild-type mice. Our findings suggest that interruption of fibroblast recruitment may represent a novel therapeutic strategy for pulmonary fibrosis, which could have applicability to a wide range of fibrotic illnesses.

Light as a central modulator of circadian rhythms, sleep and affect

PubMed Central

LeGates, T.A.; Fernandez, D.C.; Hattar, S

2014-01-01

Light has profoundly influenced the evolution of life on earth. As widely appreciated, light allows us to generate images of our environment. However, light, through the atypical intrinsically photosensitive retinal ganglion cells (ipRGCs; Box 1), also influences behaviors that are essential for our health and quality of life, yet are independent of image formation. These include the synchronization of the circadian clock to the solar day, tracking of seasonal changes, and regulation of sleep. Irregular light environments lead to problems in circadian rhythms and sleep, which eventually cause mood and learning deficits. Recently, it was found that irregular light can also directly impact mood and learning without producing major disruptions in circadian rhythms and sleep. Here, we will discuss the indirect and direct influence of light on mood and learning and provide a model for how light, the circadian clock, and sleep interact to influence mood and cognitive functions. Box 1Intrinsically photosensitive retinal ganglion cells (ipRGCs)Retinal photoreceptors transduce light energy into electrical signals that initiate vision. The classical photoreceptors, rods and cones, possess modified cilia that consist of stacks of membranes in which photopigments (rhodopsin and cone opsins) are concentrated. Rods are exquisitely sensitive and are able to detect even a few photons. Rods are therefore used for night vision. Cones are less sensitive than rods and are used for day and color vision. Color vision is mediated by cone photoreceptors that express cone-opsins with sensitivity peaks at different wavelengths (colors) of light. Humans have three cone types: short, mid and long wavelength sensitive cones (for simplicity, we will refer to these as blue, green and red cones, respectively). Rods and cones relay photic information through multisynaptic pathways to retinal ganglion cells (RGCs), which innervate different areas in the brain for complex visual processing13.A surprising discovery showed that a subpopulation of RGCs is intrinsically photosensitive and express the photopigment melanopsin. These cells were thus termed ipRGCs17–19. The melanopsin gene (Opn4) was originally cloned from Xenopus laevis dermal melanophores, and was shown to have orthologs in many mammalian species, including humans141. Sequence analysis shows that melanopsin shares more homology with invertebrate opsins than with vertebrate opsins, suggesting that melanopsin may use a different mechanism for light signaling than that used by the photopigments present in the rods and cones of vertebrates142. ipRGCs do not have modified membranes in which the photopigment can be concentrated: thus, melanopsin protein is expressed uniformly throughout the soma, dendrites, and the initial segment of the axon143. The lack of membrane specialization makes ipRGCs less sensitive to light than rods and cones. However, ipRGCs are able to incorporate light signals over extended period of time, resulting in an increase in their sensitivity during prolonged light stimulation. ipRGCs are most sensitive to wavelengths of light that are in the blue region of the light spectrum144, 145. As ganglion cells, ipRGCs also convey light information from rods and cones in addition to their intrinsic melanopsin-dependent pathway and can control a variety of light-mediated behaviors30.Originally, ipRGCs were thought to comprise a uniform population, however, recent discoveries revealed that ipRGCs are highly diverse, comprising at least five distinct subtypes (M1-M5) in rodents based on morphological and electrophysiological analyses22–29. The originally identified population is now known as M1 ipRGCs and project predominantly to brain regions involved in non-image forming visual functions, whereas the non-M1 ipRGCs show widespread projections to areas in the brain important for image formation. ipRGC subtypes express varying levels of the melanopsin protein and have different patterns of dendrite stratification in the inner plexiform layer (IPL)27, 28, 146, 147, indicating that each subtype could play a particular role in detecting light intrinsically and in signaling rod and cone information to the brain. PMID:24917305

Mapping of transcription factor binding regions in mammalian cells by ChIP: Comparison of array- and sequencing-based technologies

PubMed Central

Euskirchen, Ghia M.; Rozowsky, Joel S.; Wei, Chia-Lin; Lee, Wah Heng; Zhang, Zhengdong D.; Hartman, Stephen; Emanuelsson, Olof; Stolc, Viktor; Weissman, Sherman; Gerstein, Mark B.; Ruan, Yijun; Snyder, Michael

2007-01-01

Recent progress in mapping transcription factor (TF) binding regions can largely be credited to chromatin immunoprecipitation (ChIP) technologies. We compared strategies for mapping TF binding regions in mammalian cells using two different ChIP schemes: ChIP with DNA microarray analysis (ChIP-chip) and ChIP with DNA sequencing (ChIP-PET). We first investigated parameters central to obtaining robust ChIP-chip data sets by analyzing STAT1 targets in the ENCODE regions of the human genome, and then compared ChIP-chip to ChIP-PET. We devised methods for scoring and comparing results among various tiling arrays and examined parameters such as DNA microarray format, oligonucleotide length, hybridization conditions, and the use of competitor Cot-1 DNA. The best performance was achieved with high-density oligonucleotide arrays, oligonucleotides ≥50 bases (b), the presence of competitor Cot-1 DNA and hybridizations conducted in microfluidics stations. When target identification was evaluated as a function of array number, 80%–86% of targets were identified with three or more arrays. Comparison of ChIP-chip with ChIP-PET revealed strong agreement for the highest ranked targets with less overlap for the low ranked targets. With advantages and disadvantages unique to each approach, we found that ChIP-chip and ChIP-PET are frequently complementary in their relative abilities to detect STAT1 targets for the lower ranked targets; each method detected validated targets that were missed by the other method. The most comprehensive list of STAT1 binding regions is obtained by merging results from ChIP-chip and ChIP-sequencing. Overall, this study provides information for robust identification, scoring, and validation of TF targets using ChIP-based technologies. PMID:17568005

Central melanopsin projections in the diurnal rodent, Arvicanthis niloticus

PubMed Central

Langel, Jennifer L.; Smale, Laura; Esquiva, Gema; Hannibal, Jens

2015-01-01

The direct effects of photic stimuli on behavior are very different in diurnal and nocturnal species, as light stimulates an increase in activity in the former and a decrease in the latter. Studies of nocturnal mice have implicated a select population of retinal ganglion cells that are intrinsically photosensitive (ipRGCs) in mediation of these acute responses to light. ipRGCs are photosensitive due to the expression of the photopigment melanopsin; these cells use glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitters. PACAP is useful for the study of central ipRGC projections because, in the retina, it is found exclusively within melanopsin cells. Little is known about the central projections of ipRGCs in diurnal species. Here, we first characterized these cells in the retina of the diurnal Nile grass rat using immunohistochemistry (IHC). The same basic subtypes of melanopsin cells that have been described in other mammals were present, but nearly 25% of them were displaced, primarily in its superior region. PACAP was present in 87.7% of all melanopsin cells, while 97.4% of PACAP cells contained melanopsin. We then investigated central projections of ipRGCs by examining the distribution of immunoreactive PACAP fibers in intact and enucleated animals. This revealed evidence that these cells project to the suprachiasmatic nucleus, lateral geniculate nucleus (LGN), pretectum, and superior colliculus. This distribution was confirmed with injections of cholera toxin subunit β coupled with Alexa Fluor 488 in one eye and Alexa Fluor 594 in the other, combined with IHC staining of PACAP. These studies also revealed that the ventral and dorsal LGN and the caudal olivary pretectal nucleus receive less innervation from ipRGCs than that reported in nocturnal rodents. Overall, these data suggest that although ipRGCs and their projections are very similar in diurnal and nocturnal rodents, they may not be identical. PMID:26236201

Central melanopsin projections in the diurnal rodent, Arvicanthis niloticus.

PubMed

Langel, Jennifer L; Smale, Laura; Esquiva, Gema; Hannibal, Jens

2015-01-01

The direct effects of photic stimuli on behavior are very different in diurnal and nocturnal species, as light stimulates an increase in activity in the former and a decrease in the latter. Studies of nocturnal mice have implicated a select population of retinal ganglion cells that are intrinsically photosensitive (ipRGCs) in mediation of these acute responses to light. ipRGCs are photosensitive due to the expression of the photopigment melanopsin; these cells use glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP) as neurotransmitters. PACAP is useful for the study of central ipRGC projections because, in the retina, it is found exclusively within melanopsin cells. Little is known about the central projections of ipRGCs in diurnal species. Here, we first characterized these cells in the retina of the diurnal Nile grass rat using immunohistochemistry (IHC). The same basic subtypes of melanopsin cells that have been described in other mammals were present, but nearly 25% of them were displaced, primarily in its superior region. PACAP was present in 87.7% of all melanopsin cells, while 97.4% of PACAP cells contained melanopsin. We then investigated central projections of ipRGCs by examining the distribution of immunoreactive PACAP fibers in intact and enucleated animals. This revealed evidence that these cells project to the suprachiasmatic nucleus, lateral geniculate nucleus (LGN), pretectum, and superior colliculus. This distribution was confirmed with injections of cholera toxin subunit β coupled with Alexa Fluor 488 in one eye and Alexa Fluor 594 in the other, combined with IHC staining of PACAP. These studies also revealed that the ventral and dorsal LGN and the caudal olivary pretectal nucleus receive less innervation from ipRGCs than that reported in nocturnal rodents. Overall, these data suggest that although ipRGCs and their projections are very similar in diurnal and nocturnal rodents, they may not be identical.

Induced Pluripotent Stem Cell-Derived Red Blood Cells and Platelet Concentrates: From Bench to Bedside.

PubMed

Focosi, Daniele; Amabile, Giovanni

2017-12-27

Red blood cells and platelets are anucleate blood components indispensable for oxygen delivery and hemostasis, respectively. Derivation of these blood elements from induced pluripotent stem (iPS) cells has the potential to develop blood donor-independent and genetic manipulation-prone products to complement or replace current transfusion banking, also minimizing the risk of alloimmunization. While the production of erythrocytes from iPS cells has challenges to overcome, such as differentiation into adult-type phenotype that functions properly after transfusion, platelet products are qualitatively and quantitatively approaching a clinically-applicable level owing to advances in expandable megakaryocyte (MK) lines, platelet-producing bioreactors, and novel reagents. Guidelines that assure the quality of iPS cells-derived blood products for clinical application represent a novel challenge for regulatory agencies. Considering the minimal risk of tumorigenicity and the expected significant demand of such products, ex vivo production of iPS-derived blood components can pave the way for iPS translation into the clinic.

Detecting Single-Nucleotide Substitutions Induced by Genome Editing.

PubMed

Miyaoka, Yuichiro; Chan, Amanda H; Conklin, Bruce R

2016-08-01

The detection of genome editing is critical in evaluating genome-editing tools or conditions, but it is not an easy task to detect genome-editing events-especially single-nucleotide substitutions-without a surrogate marker. Here we introduce a procedure that significantly contributes to the advancement of genome-editing technologies. It uses droplet digital polymerase chain reaction (ddPCR) and allele-specific hydrolysis probes to detect single-nucleotide substitutions generated by genome editing (via homology-directed repair, or HDR). HDR events that introduce substitutions using donor DNA are generally infrequent, even with genome-editing tools, and the outcome is only one base pair difference in 3 billion base pairs of the human genome. This task is particularly difficult in induced pluripotent stem (iPS) cells, in which editing events can be very rare. Therefore, the technological advances described here have implications for therapeutic genome editing and experimental approaches to disease modeling with iPS cells. © 2016 Cold Spring Harbor Laboratory Press.

Blocking IP3 signal transduction pathways inhibits melatonin-induced Ca2+ signals and impairs P. falciparum development and proliferation in erythrocytes.

PubMed

Pecenin, Mateus Fila; Borges-Pereira, Lucas; Levano-Garcia, Julio; Budu, Alexandre; Alves, Eduardo; Mikoshiba, Katsuhiko; Thomas, Andrew; Garcia, Celia R S

2018-03-14

Inositol 1,4,5 trisphosphate (IP 3 ) signaling plays a crucial role in a wide range of eukaryotic processes. In Plasmodium falciparum, IP 3 elicits Ca 2+ release from intracellular Ca 2+ stores, even though no IP 3 receptor homolog has been identified to date. The human host hormone melatonin plays a key role in entraining the P. falciparum life cycle in the intraerythrocytic stages, apparently through an IP 3 -dependent Ca 2+ signal. The melatonin-induced cytosolic Ca 2+ ([Ca 2+ ] cyt ) increase and malaria cell cycle can be blocked by the IP 3 receptor blocker 2-aminoethyl diphenylborinate (2-APB). However, 2-APB also inhibits store-operated Ca 2+ entry (SOCE). Therefore, we have used two novel 2-APB derivatives, DPB162-AE and DPB163-AE, which are 100-fold more potent than 2-APB in blocking SOCE in mammalian cells, and appear to act by interfering with clustering of STIM proteins. In the present work we report that DPB162-AE and DPB163-AE block the [Ca 2+ ] cyt rise in response to melatonin in P. falciparum, but only at high concentrations. These compounds also block SOCE in the parasite at similarly high concentrations suggesting that P. falciparum SOCE is not activated in the same way as in mammalian cells. We further find that DPB162-AE and DPB163-AE affect the development of the intraerythrocytic parasites and invasion of new red blood cells. Our efforts to episomally express proteins that compete with native IP 3 receptor like IP 3 -sponge and an IP 3 sensor such as IRIS proved to be lethal to P. falciparum during intraerythrocytic cycle. The present findings point to an important role of IP 3 -induced Ca 2+ release in intraerythrocytic stage of P. falciparum. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Mobility management techniques for the next-generation wireless networks

NASA Astrophysics Data System (ADS)

Sun, Junzhao; Howie, Douglas P.; Sauvola, Jaakko J.

2001-10-01

The tremendous demands from social market are pushing the booming development of mobile communications faster than ever before, leading to plenty of new advanced techniques emerging. With the converging of mobile and wireless communications with Internet services, the boundary between mobile personal telecommunications and wireless computer networks is disappearing. Wireless networks of the next generation need the support of all the advances on new architectures, standards, and protocols. Mobility management is an important issue in the area of mobile communications, which can be best solved at the network layer. One of the key features of the next generation wireless networks is all-IP infrastructure. This paper discusses the mobility management schemes for the next generation mobile networks through extending IP's functions with mobility support. A global hierarchical framework model for the mobility management of wireless networks is presented, in which the mobility management is divided into two complementary tasks: macro mobility and micro mobility. As the macro mobility solution, a basic principle of Mobile IP is introduced, together with the optimal schemes and the advances in IPv6. The disadvantages of the Mobile IP on solving the micro mobility problem are analyzed, on the basis of which three main proposals are discussed as the micro mobility solutions for mobile communications, including Hierarchical Mobile IP (HMIP), Cellular IP, and Handoff-Aware Wireless Access Internet Infrastructure (HAWAII). A unified model is also described in which the different micro mobility solutions can coexist simultaneously in mobile networks.

myo-Inositol hexakisphosphate is a major component of an extracellular structure in the parasitic cestode Echinococcus granulosus.

PubMed Central

Irigoín, Florencia; Ferreira, Fernando; Fernández, Cecilia; Sim, Robert B; Díaz, Alvaro

2002-01-01

myo-Inositol hexakisphosphate (IP(6)) is an abundant intracellular component of animal cells. In this study we describe the presence of extracellular IP(6) in the hydatid cyst wall (HCW) of the larval stage of the cestode parasite Echinococcus granulosus. The HCW comprises an inner cellular layer and an outer, acellular (laminated) layer up to 2 mm in thickness that protects the parasite from host immune cells. A compound, subsequently identified as IP(6), was detected in and purified from an HCW extract on the basis of its capacity to inhibit complement activation. The identification of the isolated compound was carried out by a combination of NMR, MS and TLC. The majority of IP(6) in the HCW was found in the acellular layer, with only a small fraction of the compound being extracted from cells. In the laminated layer, IP(6) was present in association with calcium, and accounted for up to 15% of the total dry mass of the HCW. IP(6) was not detected in any other structures or stages of the parasite. Our results imply that IP(6) is secreted by the larval stage of the parasite in a polarized fashion towards the interface with the host. This is the first report of the secretion of IP(6), and the possible implications beyond the biology of E. granulosus are discussed. PMID:11853537

[DAB2IP expression in bladder transitional cell carcinoma and its correlation with clinical outcome].

PubMed

Zhu, Jian-Ning; Wu, Kai-Jie; Guan, Zhen-Feng; Liu, Li-Xia; Ning, Zhong-Yun; Zhou, Jian-Cheng; Wang, Xin-Yang; Fan, Jin-Hai

2014-07-01

To investigate the expression of DAB2IP in bladder transitional cell carcinoma (BTCC) and its correlation with clinical characteristics and prognosis of BTCC patients. Immunohistochemical staining was applied to detect DAB2IP protein level in 79 cases of TCCB tissues and 11 cases of normal bladder tissues, and the relationships of the staining results with pathological grade, stage, lymph node metastasis, gender, age and the 3-year survival rate of the patients were analyzed. The expression of DAB2IP in BTCC tissues was significantly lower than that in normal bladder epithelium, and the expression score and rate of DAB2IP in the high-grade, invasive and metastatic BTCC were significantly lower than those in low-grade, superficial and non-metastatic BTCC (P < 0.05). The 3-year survival rate of the patients with high DAB2IP expression was significantly higher than that of the patients with low DAB2IP expression. DAB2IP may be one of the important inhibitory factors during the occurrence and progression of BTCC.

Prevalence of human papillomavirus, Epstein-Barr virus, p21, and p53 expression in sinonasal inverted papilloma, nasal polyp, and hypertrophied turbinate in Hong Kong patients.

PubMed

Sham, C L; To, K F; Chan, Paul K S; Lee, Dennis L Y; Tong, Michael C F; van Hasselt, C Andrew

2012-04-01

The purpose of this study of human papillomavirus (HPV), Epstein-Barr virus (EBV), p21, and p53 in sinonasal inverted papilloma (IP) was to help elucidate its pathogenesis. Seventy-three IPs, 48 nasal polyps, and 85 hypertrophied turbinates were subjected to HPV polymerase chain reaction (PCR) study. Seventy-three IPs, 30 nasal polyps, and 32 hypertrophied turbinates were subjected to EBV in situ hybridization (ISH), p21, and p53 immunohistochemical (IHC) studies. HPV was positive in 3 of 73 IPs (4.1%). All specimens were EBV negative. In all, 99% of IPs showed strong and diffuse p21 nuclear reactivity. Most nasal polyps and hypertrophied turbinates showed weak to moderate immunoreactivity of the basal and parabasal cells. Only focal p53 immunoreactivity of the basal and parabasal cells was found in 19% of IPs and 40% of nasal polyps. HPV prevalence of our IP is low. EBV is not present in IP. High p21 and low p53 expression in IP suggests a non-p53-dependent regulation pathway. Copyright © 2011 Wiley Periodicals, Inc.

Inhibitory effects of albuterol and fenoterol on RANTES and IP-10 expression in bronchial epithelial cells.

PubMed

Lam, Ka-Pan; Chu, Yu-Te; Lee, Min-Sheng; Chen, Huan-Nan; Wang, Wei-Li; Tok, Teck-Siang; Chin, Yow-Yue; Chen, Solomon Chih-Cheng; Kuo, Chang-Hung; Hung, Chih-Hsing

2011-06-01

Short-acting β2-adrenoreceptor agonist (SABA) is the major asthma reliever as indicated in the GINA guidelines. Regulated on activation, normal T expressed and secreted (RANTES) is a chemokine that attracts eosinophils, mast cells, and basophils toward site of allergic inflammation. Interferon γ-inducible protein (IP)-10 is a Th1-related chemokine that is also important in asthmatic inflammation and also involved in our immune defense against pathogens. Bronchial epithelial cells are first-line barrier against invasive pathogen and also have immunomodulatory function. However, whether albuterol and fenoterol (two SABAs) have modulatory effects on RANTES and IP-10 expression in bronchial epithelial cells is unknown. The human bronchial epithelial cell lines, BEAS-2B cells, were pre-treated with different concentrations of albuterol, fenoterol or dibutyryl-cAMP (a cyclic AMP analog) before polyinosinic-polycytidylic acid (poly I:C) stimulation. In some condition, BEAS-2B cells were pre-treated with ICI-118551, a selective β2-adrenoreceptor antagonist, 30 min before albuterol or fenoterol treatment. The levels of RANTES and IP-10 were measured by ELISA. Intracellular signaling was investigated using cAMP assay, mitogen-activated protein kinase (MAPK) inhibitor, nuclear factor (NF)-κB inhibitor, and western blot. Albuterol and fenoterol suppressed poly I:C-induced RANTES and IP-10 expression of BEAS-2B cells. ICI-118551 could partly reverse the suppressive effects of albuterol and fenoterol on RANTES and IP-10 expression. Albuterol and fenoterol increased intracellular cAMP levels. Dibutyryl-cAMP conferred the similar effects of albuterol and fenoterol. Western blot revealed that albuterol suppressed p-ERK, p-JNK and pp38, and also their associated kinase expression. Albuterol had no effect on pp65 expression. Albuterol and fenoterol could suppress poly I:C-induced RANTES and IP-10 expression in human bronchial epithelial cells via at least partly the β2-adrenoreceptor-cAMP and the MAPK pathways, implicating that albuterol and fenoterol could exert anti-inflammatory effect and benefit asthmatic patients by suppressing RANTES and IP-10 expression. However, these suppressive effects of albuterol and fenoterol may inhibit the defense against viral infection. © 2010 John Wiley & Sons A/S.

Akt/PKB activation and insulin signaling: a novel insulin signaling pathway in the treatment of type 2 diabetes.

PubMed

Mackenzie, Richard Wa; Elliott, Bradley T

2014-01-01

Type 2 diabetes is a metabolic disease categorized primarily by reduced insulin sensitivity, β-cell dysfunction, and elevated hepatic glucose production. Treatments reducing hyperglycemia and the secondary complications that result from these dysfunctions are being sought after. Two distinct pathways encourage glucose transport activity in skeletal muscle, ie, the contraction-stimulated pathway reliant on Ca(2+)/5'-monophosphate-activated protein kinase (AMPK)-dependent mechanisms and an insulin-dependent pathway activated via upregulation of serine/threonine protein kinase Akt/PKB. Metformin is an established treatment for type 2 diabetes due to its ability to increase peripheral glucose uptake while reducing hepatic glucose production in an AMPK-dependent manner. Peripheral insulin action is reduced in type 2 diabetics whereas AMPK signaling remains largely intact. This paper firstly reviews AMPK and its role in glucose uptake and then focuses on a novel mechanism known to operate via an insulin-dependent pathway. Inositol hexakisphosphate (IP6) kinase 1 (IP6K1) produces a pyrophosphate group at the position of IP6 to generate a further inositol pyrophosphate, ie, diphosphoinositol pentakisphosphate (IP7). IP7 binds with Akt/PKB at its pleckstrin homology domain, preventing interaction with phosphatidylinositol 3,4,5-trisphosphate, and therefore reducing Akt/PKB membrane translocation and insulin-stimulated glucose uptake. Novel evidence suggesting a reduction in IP7 production via IP6K1 inhibition represents an exciting therapeutic avenue in the treatment of insulin resistance. Metformin-induced activation of AMPK is a key current intervention in the management of type 2 diabetes. However, this treatment does not seem to improve peripheral insulin resistance. In light of this evidence, we suggest that inhibition of IP6K1 may increase insulin sensitivity and provide a novel research direction in the treatment of insulin resistance.

The antiproliferative cytostatic effects of a self-activating viridin prodrug

PubMed Central

Smith, Adam; Blois, Joseph; Yuan, Hushan; Aikawa, Elena; Ellson, Christian; Figueiredo, Jose-Luiz; Weissleder, Ralph; Kohler, Rainer; Yaffe, Michael B.; Cantley, Lewis C.; Josephson, Lee

2009-01-01

Although viridins like wortmannin (Wm) have long been examined as anticancer agents, their ability to self-activate has only recently been recognized. Here, we describe the cytostatic effects of a self-activating viridin (SAV), which is an inactive, polymeric prodrug. SAV self-activates to generate a bioactive, fluorescent viridin NBD-Wm with a half-time of 9.2 hours. With cultured A549 cells, 10 µmol/L SAV caused growth arrest without inducing apoptosis or cell death, a cytostatic action markedly different from other chemotherapeutic agents (vinblastine, camptothecin, and paclitaxel). In vivo, a SAV dosing of 1 mg/kg once in 48 hours (i.p.) resulted in growth arrest of an A549 tumor xenograft, with growth resuming when dosing ceased. With a peak serum concentration of SAV of 2.36 µmol/L (at 2 hours post i.p. injection), the concentration of bioactive NBD-Wm was 41 nmol/L based on the partial inhibition of neutrophil respiratory burst. Therefore, SAV was present as an inactive prodrug in serum (peak = 2.36 µmol/L), which generated low concentrations of active viridin (41 nmol/L). SAV is a prodrug, the slowrelease and cytostatic activities of which suggest that it might be useful as a component of metronomic-based chemotherapeutic strategies. PMID:19509266

Genome-wide profiling of DNA-binding proteins using barcode-based multiplex Solexa sequencing.

PubMed

Raghav, Sunil Kumar; Deplancke, Bart

2012-01-01

Chromatin immunoprecipitation (ChIP) is a commonly used technique to detect the in vivo binding of proteins to DNA. ChIP is now routinely paired to microarray analysis (ChIP-chip) or next-generation sequencing (ChIP-Seq) to profile the DNA occupancy of proteins of interest on a genome-wide level. Because ChIP-chip introduces several biases, most notably due to the use of a fixed number of probes, ChIP-Seq has quickly become the method of choice as, depending on the sequencing depth, it is more sensitive, quantitative, and provides a greater binding site location resolution. With the ever increasing number of reads that can be generated per sequencing run, it has now become possible to analyze several samples simultaneously while maintaining sufficient sequence coverage, thus significantly reducing the cost per ChIP-Seq experiment. In this chapter, we provide a step-by-step guide on how to perform multiplexed ChIP-Seq analyses. As a proof-of-concept, we focus on the genome-wide profiling of RNA Polymerase II as measuring its DNA occupancy at different stages of any biological process can provide insights into the gene regulatory mechanisms involved. However, the protocol can also be used to perform multiplexed ChIP-Seq analyses of other DNA-binding proteins such as chromatin modifiers and transcription factors.

miR-14 regulates autophagy during developmental cell death by targeting ip3-kinase 2.

PubMed

Nelson, Charles; Ambros, Victor; Baehrecke, Eric H

2014-11-06

Macroautophagy (autophagy) is a lysosome-dependent degradation process that has been implicated in age-associated diseases. Autophagy is involved in both cell survival and cell death, but little is known about the mechanisms that distinguish its use during these distinct cell fates. Here, we identify the microRNA miR-14 as being both necessary and sufficient for autophagy during developmentally regulated cell death in Drosophila. Loss of miR-14 prevented induction of autophagy during salivary gland cell death, but had no effect on starvation-induced autophagy in the fat body. Moreover, misexpression of miR-14 was sufficient to prematurely induce autophagy in salivary glands, but not in the fat body. Importantly, miR-14 regulates this context-specific autophagy through its target, inositol 1,4,5-trisphosphate kinase 2 (ip3k2), thereby affecting inositol 1,4,5-trisphosphate (IP3) signaling and calcium levels during salivary gland cell death. This study provides in vivo evidence of microRNA regulation of autophagy through modulation of IP3 signaling. Copyright © 2014 Elsevier Inc. All rights reserved.

NANOG priming before full reprogramming may generate germ cell tumours.

PubMed

Grad, I; Hibaoui, Y; Jaconi, M; Chicha, L; Bergström-Tengzelius, R; Sailani, M R; Pelte, M F; Dahoun, S; Mitsiadis, T A; Töhönen, V; Bouillaguet, S; Antonarakis, S E; Kere, J; Zucchelli, M; Hovatta, O; Feki, A

2011-11-09

Reprogramming somatic cells into a pluripotent state brings patient-tailored, ethical controversy-free cellular therapy closer to reality. However, stem cells and cancer cells share many common characteristics; therefore, it is crucial to be able to discriminate between them. We generated two induced pluripotent stem cell (iPSC) lines, with NANOG pre-transduction followed by OCT3/4, SOX2, and LIN28 overexpression. One of the cell lines, CHiPS W, showed normal pluripotent stem cell characteristics, while the other, CHiPS A, though expressing pluripotency markers, failed to differentiate and gave rise to germ cell-like tumours in vivo. Comparative genomic hybridisation analysis of the generated iPS lines revealed that they were genetically more stable than human embryonic stem cell counterparts. This analysis proved to be predictive for the differentiation potential of analysed cells. Moreover, the CHiPS A line expressed a lower ratio of p53/p21 when compared to CHiPS W. NANOG pre-induction followed by OCT3/4, SOX2, MYC, and KLF4 induction resulted in the same tumour-inducing phenotype. These results underline the importance of a re-examination of the role of NANOG during reprogramming. Moreover, this reprogramming method may provide insights into primordial cell tumour formation and cancer stem cell transformation.

DNA damage response curtails detrimental replication stress and chromosomal instability induced by the dietary carcinogen PhIP

PubMed Central

Mimmler, Maximilian; Peter, Simon; Kraus, Alexander; Stroh, Svenja; Nikolova, Teodora; Seiwert, Nina; Hasselwander, Solveig; Neitzel, Carina; Haub, Jessica; Monien, Bernhard H.; Nicken, Petra; Steinberg, Pablo; Shay, Jerry W.; Kaina, Bernd; Fahrer, Jörg

2016-01-01

PhIP is an abundant heterocyclic aromatic amine (HCA) and important dietary carcinogen. Following metabolic activation, PhIP causes bulky DNA lesions at the C8-position of guanine. Although C8-PhIP-dG adducts are mutagenic, their interference with the DNA replication machinery and the elicited DNA damage response (DDR) have not yet been studied. Here, we analyzed PhIP-triggered replicative stress and elucidated the role of the apical DDR kinases ATR, ATM and DNA-PKcs in the cellular defense response. First, we demonstrate that PhIP induced C8-PhIP-dG adducts and DNA strand breaks. This stimulated ATR-CHK1 signaling, phosphorylation of histone 2AX and the formation of RPA foci. In proliferating cells, PhIP treatment increased the frequency of stalled replication forks and reduced fork speed. Inhibition of ATR in the presence of PhIP-induced DNA damage strongly promoted the formation of DNA double-strand breaks, activation of the ATM-CHK2 pathway and hyperphosphorylation of RPA. The abrogation of ATR signaling potentiated the cell death response and enhanced chromosomal aberrations after PhIP treatment, while ATM and DNA-PK inhibition had only marginal effects. These results strongly support the notion that ATR plays a key role in the defense against cancer formation induced by PhIP and related HCAs. PMID:27599846

ChIP-seq.

PubMed

Kim, Tae Hoon; Dekker, Job

2018-05-01

Owing to its digital nature, ChIP-seq has become the standard method for genome-wide ChIP analysis. Using next-generation sequencing platforms (notably the Illumina Genome Analyzer), millions of short sequence reads can be obtained. The densities of recovered ChIP sequence reads along the genome are used to determine the binding sites of the protein. Although a relatively small amount of ChIP DNA is required for ChIP-seq, the current sequencing platforms still require amplification of the ChIP DNA by ligation-mediated PCR (LM-PCR). This protocol, which involves linker ligation followed by size selection, is the standard ChIP-seq protocol using an Illumina Genome Analyzer. The size-selected ChIP DNA is amplified by LM-PCR and size-selected for the second time. The purified ChIP DNA is then loaded into the Genome Analyzer. The ChIP DNA can also be processed in parallel for ChIP-chip results. © 2018 Cold Spring Harbor Laboratory Press.

A CI-Independent Form of Replicative Inhibition: Turn Off of Early Replication of Bacteriophage Lambda

PubMed Central

Hayes, Sidney; Horbay, Monique A.; Hayes, Connie

2012-01-01

Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, pO is required for IP, as are iterons ITN3–4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop + oriλ+ sequence. PMID:22590552

Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins.

PubMed

Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

2016-04-01

The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis. © 2016 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Chromatin immunoprecipitation assays: application of ChIP-on-chip for defining dynamic transcriptional mechanisms in bone cells.

PubMed

van der Deen, Margaretha; Hassan, Mohammad Q; Pratap, Jitesh; Teplyuk, Nadiya M; Young, Daniel W; Javed, Amjad; Zaidi, Sayyed K; Lian, Jane B; Montecino, Martin; Stein, Janet L; Stein, Gary S; van Wijnen, Andre J

2008-01-01

Normal cell growth and differentiation of bone cells requires the sequential expression of cell type specific genes to permit lineage specification and development of cellular phenotypes. Transcriptional activation and repression of distinct sets of genes support the anabolic functions of osteoblasts and the catabolic properties of osteoclasts. Furthermore, metastasis of tumors to the bone environment is controlled by transcriptional mechanisms. Insights into the transcriptional regulation of genes in bone cells may provide a conceptual basis for improved therapeutic approaches to treat bone fractures, genetic osteopathologies, and/or cancer metastases to bone. Chromatin immunoprecipitation (ChIP) is a powerful technique to establish in vivo binding of transcription factors to the promoters of genes that are either activated or repressed in bone cells. Combining ChIP with genomic microarray analysis, colloquially referred to as "ChIP-on-chip," has become a valuable method for analysis of endogenous protein/DNA interactions. This technique permits assessment of chromosomal binding sites for transcription factors or the location of histone modifications at a genomic scale. This chapter discusses protocols for performing chromatin immunoprecipitation experiments, with a focus on ChIP-on-chip analysis. The information presented is based on the authors' experience with defining interactions of Runt-related (RUNX) transcription factors with bone-related genes within the context of the native nucleosomal organization of intact osteoblastic cells.

Differential methylation of tissue- and cancer-specific CpG island shores distinguishes human induced pluripotent stem cells, embryonic stem cells and fibroblasts

PubMed Central

Doi, Akiko; Park, In-Hyun; Wen, Bo; Murakami, Peter; Aryee, Martin J; Irizarry, Rafael; Herb, Brian; Ladd-Acosta, Christine; Rho, Junsung; Loewer, Sabine; Miller, Justine; Schlaeger, Thorsten; Daley, George Q; Feinberg, Andrew P

2010-01-01

Induced pluripotent stem (iPS) cells are derived by epigenetic reprogramming, but their DNA methylation patterns have not yet been analyzed on a genome-wide scale. Here, we find substantial hypermethylation and hypomethylation of cytosine-phosphate-guanine (CpG) island shores in nine human iPS cell lines as compared to their parental fibroblasts. The differentially methylated regions (DMRs) in the reprogrammed cells (denoted R-DMRs) were significantly enriched in tissue-specific (T-DMRs; 2.6-fold, P < 10−4) and cancer-specific DMRs (C-DMRs; 3.6-fold, P < 10−4). Notably, even though the iPS cells are derived from fibroblasts, their R-DMRs can distinguish between normal brain, liver and spleen cells and between colon cancer and normal colon cells. Thus, many DMRs are broadly involved in tissue differentiation, epigenetic reprogramming and cancer. We observed colocalization of hypomethylated R-DMRs with hypermethylated C-DMRs and bivalent chromatin marks, and colocalization of hypermethylated R-DMRs with hypomethylated C-DMRs and the absence of bivalent marks, suggesting two mechanisms for epigenetic reprogramming in iPS cells and cancer. PMID:19881528

Melanopsin-expressing retinal ganglion-cell photoreceptors: cellular diversity and role in pattern vision

PubMed Central

Ecker, Jennifer L.; Dumitrescu, Olivia N.; Wong, Kwoon Y.; Alam, Nazia M.; Chen, Shih-Kuo; LeGates, Tara; Renna, Jordan M.; Prusky, Glen T.; Berson, David M.; Hattar, Samer

2010-01-01

Using the photopigment melanopsin, intrinsically photosensitive retinal ganglion cells (ipRGCs) respond directly to light to drive circadian clock resetting and pupillary constriction. We now report that ipRGCs are more abundant and diverse than previously appreciated, project more widely within the brain, and can support spatial visual perception. A Cre-based melanopsin reporter mouse line revealed at least five subtypes of ipRGCs with distinct morphological and physiological characteristics. Collectively, these cells project beyond the known brain targets of ipRGCs to heavily innervate the superior colliculus and dorsal lateral geniculate nucleus, retinotopically-organized nuclei mediating object localization and discrimination. Mice lacking classical rod-cone photoreception, and thus entirely dependent on melanopsin for light detection, were able to discriminate grating stimuli from equiluminant gray, and had measurable visual acuity. Thus, non-classical retinal photoreception occurs within diverse cell types, and influences circuits and functions encompassing luminance as well as spatial information. PMID:20624591

Rapid increase of inositol 1,4,5-trisphosphate in the HeLa cells after hypergravity exposure

NASA Technical Reports Server (NTRS)

Kumei, Yasuhiro; Whitson, Peggy A.; Cintron, Nitza M.; Sato, Atsushige

1990-01-01

The IP3 level in HeLa cells has been elevated through the application in hypergravity in a time-dependent manner. The data obtained for the hydrolytic products of PIP2, IP3, and DG are noted to modulate c-myc gene expression. It is also established that the cAMP accumulation by the IBMX in hypergravity-exposed cells was suppressed relative to the control. In light of IP3 increase and cAMP decrease results, a single GTP-binding protein may play a role in the hypergravity signal transduction of HeLa cells by stimulating PLC while inhibiting adenylate cyclase.

Angiotensin II stimulates calcineurin activity in proximal tubule epithelia through AT-1 receptor-mediated tyrosine phosphorylation of the PLC-gamma1 isoform.

PubMed

Lea, Janice P; Jin, Shao G; Roberts, Brian R; Shuler, Michael S; Marrero, Mario B; Tumlin, James A

2002-07-01

Angiotensin II (AngII) contributes to the maintenance of extracellular fluid volume by regulating sodium transport in the nephron. In nonepithelial cells, activation of phospholipase C (PLC) by AT-1 receptors stimulates the generation of 1,4,5-trisphosphate (IP(3)) and the release of intracellular calcium. Calcineurin, a serine-threonine phosphatase, is activated by calcium and calmodulin, and both PLC and calcineurin have been linked to sodium transport in the proximal tubule. An examination of whether AngII activates calcineurin in a model of proximal tubule epithelia (LLC-PK1 cells) was performed; AngII increased calcineurin activity within 30 s. An examination of whether AngII activates PLC in proximal tubule epithelia was also performed after first showing that all three families of PLC isoforms are present in LLC-PK1 cells. Application of AngII increased IP(3) generation by 60% within 15 s, which coincided with AngII-induced tyrosine phosphorylation of the PLC-gamma1 isoform also observed at 15 s. AngII-induced tyrosine phosphorylation was blocked by the AT-1 receptor antagonist, Losartan. Subsequently, an inhibitor of tyrosine phosphorylation blocked the AngII-induced activation of calcineurin, as did coincubation with an inhibitor of PLC activity and with an antagonist of the AT-1 receptor. It is therefore concluded that AngII stimulates calcineurin phosphatase activity in proximal tubule epithelial cells through a mechanism involving AT-1 receptor-mediated tyrosine phosphorylation of the PLC isoform.

The synergistic effect of micro/nano-structured and Cu2+-doped hydroxyapatite particles to promote osteoblast viability and antibacterial activity.

PubMed

Shi, Feng; Liu, Yumei; Zhi, Wei; Xiao, Dongqin; Li, Hongyu; Duan, Ke; Qu, Shuxin; Weng, Jie

2017-06-06

Microstructure and chemical constitution are important factors affecting the biological activity of biomaterials. This study aimed to fabricate hydroxyapatite (HAp) particles with both micro/nanohybrid structure and Cu 2+ doping to promote osteogenic differentiation and antibacterial property. In the presence of inositol hexakisphosphate (IP6), micro/nano-structured and Cu 2+ -doped HAp (HAp-IP6-Cu) microspheres were successfully fabricated via hydrothermal method. Morphological observation showed that HAp-IP6-Cu microspheres with a diameter of 3.1-4.1 μm were chrysanthemum-like and composed of nano-flakes approximately 50 nm in thickness. Compared with the HAp micro-rods or IP6 modified HAp (HAp-IP6) microspheres, HAp-IP6-Cu microspheres had a larger specific surface area, better hydrophilicity and stronger ability to adsorb bovine serum albumin. To evaluate the synergistic effects of micro/nanohybrid structure and Cu 2+ on cell behavior, rat calvarial osteoblasts (RCOs) were cultured on HAp-IP6-Cu, HAp-IP6 and HAp layers as well as their extracts, respectively. Results demonstrated that HAp-IP6-Cu layer promoted the adhesion, proliferation and osteogenic differentiation of RCOs. The cells grew on HAp-IP6-Cu and HAp-IP6 layers exhibited greater spreading than those on HAp layer. In addition, quantitative test by the agar disk diffusion technique found that HAp-IP6-Cu microspheres were effectively against S taphylococcus aureus and E scherichia coli. These results demonstrated that HAp-IP6-Cu microspheres may be a potential candidate as a bioactive and anti-infective biomaterial for bone regeneration.

Security for IP Multimedia Services in the 3GPP Third Generation Mobile System.

ERIC Educational Resources Information Center

Horn, G.; Kroselberg, D.; Muller, K.

2003-01-01

Presents an overview of the security architecture of the IP multimedia core network subsystem (IMS) of the third generation mobile system, known in Europe as UMTS. Discusses IMS security requirements; IMS security architecture; authentication between IMS user and home network; integrity and confidentiality for IMS signalling; and future aspects of…

Depletion of mRNA export regulator DBP5/DDX19, GLE1 or IPPK that is a key enzyme for the production of IP6, resulting in differentially altered cytoplasmic mRNA expression and specific cell defect

PubMed Central

Okamura, Masumi; Yamanaka, Yasutaka; Shigemoto, Maki; Kitadani, Yuya; Kobayashi, Yuhko; Kambe, Taiho; Nagao, Masaya; Kobayashi, Issei; Okumura, Katsuzumi

2018-01-01

DBP5, also known as DDX19, GLE1 and inositol hexakisphosphate (IP6) function in messenger RNA (mRNA) export at the cytoplasmic surface of the nuclear pore complex in eukaryotic cells. DBP5 is a DEAD-box RNA helicase, and its activity is stimulated by interactions with GLE1 and IP6. In addition, these three factors also have unique role(s). To investigate how these factors influenced the cytoplasmic mRNA expression and cell phenotype change, we performed RNA microarray analysis to detect the effect and function of DBP5, GLE1 and IP6 on the cytoplasmic mRNA expression. The expression of some cytoplasmic mRNA subsets (e.g. cell cycle, DNA replication) was commonly suppressed by the knock-down of DBP5, GLE1 and IPPK (IP6 synthetic enzyme). The GLE1 knock-down selectively reduced the cytoplasmic mRNA expression required for mitotic progression, results in an abnormal spindle phenotype and caused the delay of mitotic process. Meanwhile, G1/S cell cycle arrest was observed in DBP5 and IPPK knock-down cells. Several factors that function in immune response were also down-regulated in DBP5 or IPPK knock-down cells. Thereby, IFNβ-1 mRNA transcription evoked by poly(I:C) treatment was suppressed. These results imply that DBP5, GLE1 and IP6 have a conserved and individual function in the cytoplasmic mRNA expression. Variations in phenotype are due to the difference in each function of DBP5, GLE1 and IPPK in intracellular mRNA metabolism. PMID:29746542

Immunopotentiator from Pantoea agglomerans 1 (IP-PA1) Promotes Murine Hair Growth and Human Dermal Papilla Cell Gene Expression.

PubMed

Wakame, Koji; Okawa, Hiroshi; Komatsu, Ken-Ich; Nakata, Akifumi; Sato, Keisuke; Ingawa, Hiroyuki; Kohchi, Chie; Nishizawa, Takashi; Soma, Gen-Ichiro

2016-07-01

The lipopolysaccharide (LPS)-like compound derived from Pantoea agglomerans (immunopotentiator from Pantoea agglomerans 1 (IP-PA1)) has been used not only as dietary supplement or cosmetic for humans, but also by Japanese veterinarians as an anti-tumor, anti-allergy, "keep a fine coat of fur" and hair growth-promoting functional food for dogs and cats. In the present study, we focused on the hair growth-promoting effects of IP-PA1 on a hair-shaved animal model and its mechanism of action. We also investigated its potential on gene expression after stimulating human dermal papilla cells with IP-PA1. The hair on the back of a C3H/HeN mouse was shaved and IP-PA1 was orally administered or applied to the skin. The status of hair growth was observed and recorded for 14 days. Skin was collected and histological tissue examination was performed with respect to hair growth status using hematoxylin and eosin staining. After IP-PA1 administration (2 and 10 μg/ml) to human dermal papilla cell culture system for 24 h, fibroblast growth factor-7 (FGF-7) and vascular endothelial growth factor (VEGF) mRNA expression were measured using real-time polymerase chain reaction (PCR) analysis. IP-PA1, when given orally, showed a tendency to promote hair growth in mice. In addition, skin application also significantly promoted hair growth, while histopathological examinations further demonstrated hair elongation from dermal papilla cells. In the human dermal papilla cell culture system, significant FGF-7 and VEGF mRNA expressions were observed (p<0.05). An underlying mechanism of gene expression by which IP-PA1 promotes hair growth was suggested to be different from that of medicine and traditional hair tonics, such as minoxidil and adenosine. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

The expression of asparaginyl endopeptidase promotes growth potential in epithelial ovarian cancer.

PubMed

Zhu, Qinyi; Tang, Meiling; Wang, Xipeng

2017-04-03

Epithelial ovarian cancer (EOC) is the most common and lethal cancer-related death among females in the world. Asparaginyl endopeptidase (AEP) is a member of C13 family peptidases and expressed in the extracellular matrix and tumor cells. The aim of this article is to explore the function of asparaginyl endopeptidase in epithelial ovarian cancer. The expression of AEP was examined in 20 EOC samples, 3 EOC metastasis samples, 6 fallopian tube metastasis samples, 4 peritoneum metastasis samples and 20 benign ovarian tumor samples by immunohistochemistry. The expression of AEP was also evaluated in serum and ascites of EOC patients by elisa. And we used a lentiviral vector to overexpress AEP in human epithelial ovarian cancer cell lines SKOV3ip and detected the function of AEP-SKOV3ip cells both in vitro and in vivo. The growth of AEP-SKOV3ip cells was observed by MTT, migration and tube formation assays in vitro. Additionally, the subcutaneous mice model was used to identify the tumor growth and metastasis in vivo. Mice tumors were stained for CD31 to determine the microvessel density (MVD). We demonstrated that AEP was highly expressed in the EOC patient tissues and ascites. The AEP transfected SKOV3ip cells could both promote tumor growth in vitro and in vivo. The MVD in AEP-SKOV3ip group was higher than that in NC-SKOV3ip group. Therefore, our results demonstrated that AEP could induce EOC growth and progressionboth in vitro and in vivo.

Mission Applicability Assessment of Integrated Power Components and Systems

NASA Technical Reports Server (NTRS)

Raffaelle, R. P.; Hepp, A. F.; Landis, G. A.; Hoffman, D. J.

2002-01-01

The need for smaller lightweight autonomous power systems has recently increased with the increasing focus on micro- and nanosatellites. Small area high-efficiency thin film batteries and solar cells are an attractive choice for such applications. The NASA Glenn Research Center, Johns Hopkins Applied Physics Laboratory, Lithium Power Technologies, MicroSat Systems, and others, have been working on the development of autonomous monolithic packages combining these elements or what are called integrated power supplies (IPS). These supplies can be combined with individual satellite components and are capable of providing continuous power even under intermittent illumination associated with a spinning or Earth orbiting satellite. This paper discusses the space mission applicability, benefits, and current development efforts associated with integrated power supply components and systems. The characteristics and several mission concepts for an IPS that combines thin-film photovoltaic power generation with thin-film lithium ion energy storage are described. Based on this preliminary assessment, it is concluded that the most likely and beneficial application of an IPS will be for small "nanosatellites" or in specialized applications serving as a decentralized or as a distributed power source or uninterruptible power supply.

Isoflurane-Induced Caspase-3 Activation Is Dependent on Cytosolic Calcium and Can Be Attenuated by Memantine

PubMed Central

Zhang, Guohua; Dong, Yuanlin; Zhang, Bin; Ichinose, Fumito; Wu, Xu; Culley, Deborah J.; Crosby, Gregory

2008-01-01

Increasing evidence indicates that caspase activation and apoptosis are associated with a variety of neurodegenerative disorders, including Alzheimer's disease. We reported that anesthetic isoflurane can induce apoptosis, alter processing of the amyloid precursor protein (APP), and increase amyloid-β protein (Aβ) generation. However, the mechanism by which isoflurane induces apoptosis is primarily unknown. We therefore set out to assess effects of extracellular calcium concentration on isoflurane-induced caspase-3 activation in H4 human neuroglioma cells stably transfected to express human full-length APP (H4-APP cells). In addition, we tested effects of RNA interference (RNAi) silencing of IP3 receptor, NMDA receptor, and endoplasmic reticulum (ER) calcium pump, sacro-/ER calcium ATPase (SERCA1). Finally, we examined the effects of the NMDA receptor partial antagonist, memantine, in H4-APP cells and brain tissue of naive mice. EDTA (10 mm), BAPTA (10 μm), and RNAi silencing of IP3 receptor, NMDA receptor, or SERCA1 attenuated capase-3 activation. Memantine (4 μm) inhibited isoflurane-induced elevations in cytosolic calcium levels and attenuated isoflurane-induced caspase-3 activation, apoptosis, and cell viability. Memantine (20 mg/kg, i.p.) reduced isoflurane-induced caspase-3 activation in brain tissue of naive mice. These results suggest that disruption of calcium homeostasis underlies isoflurane-induced caspase activation and apoptosis. We also show for the first time that the NMDA receptor partial antagonist, memantine, can prevent isoflurane-induced caspase-3 activation and apoptosis in vivo and in vitro. These findings, indicating that isoflurane-induced caspase activation and apoptosis are dependent on cytosolic calcium levels, should facilitate the provision of safer anesthesia care, especially for Alzheimer's disease and elderly patients. PMID:18434534

Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells

PubMed Central

Linehan, Erin K.; Schrader, Carol E.; Stavnezer, Janet

2015-01-01

Activation-induced cytidine deaminase (AID) is required for initiation of Ig class switch recombination (CSR) and somatic hypermutation (SHM) of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs) involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP) for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq). We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID. PMID:26263206

A demonstration of the H3 trimethylation ChIP-seq analysis of galline follicular mesenchymal cells and male germ cells.

PubMed

Chokeshaiusaha, Kaj; Puthier, Denis; Nguyen, Catherine; Sananmuang, Thanida

2018-06-01

Trimethylation of histone 3 (H3) at 4th lysine N-termini (H3K4me3) in gene promoter region was the universal marker of active genes specific to cell lineage. On the contrary, coexistence of trimethylation at 27th lysine (H3K27me3) in the same loci-the bivalent H3K4m3/H3K27me3 was known to suspend the gene transcription in germ cells, and could also be inherited to the developed stem cell. In galline species, throughout example of H3K4m3 and H3K27me3 ChIP-seq analysis was still not provided. We therefore designed and demonstrated such procedures using ChIP-seq and mRNA-seq data of chicken follicular mesenchymal cells and male germ cells. Analytical workflow was designed and provided in this study. ChIP-seq and RNA-seq datasets of follicular mesenchymal cells and male germ cells were acquired and properly preprocessed. Peak calling by Model-based analysis of ChIP-seq 2 was performed to identify H3K4m3 or H3K27me3 enriched regions (Fold-change≥2, FDR≤0.01) in gene promoter regions. Integrative genomics viewer was utilized for cellular retinoic acid binding protein 1 ( CRABP1 ), growth differentiation factor 10 ( GDF10 ), and gremlin 1 ( GREM1 ) gene explorations. The acquired results indicated that follicular mesenchymal cells and germ cells shared several unique gene promoter regions enriched with H3K4me3 (5,704 peaks) and also unique regions of bivalent H3K4m3/H3K27me3 shared between all cell types and germ cells (1,909 peaks). Subsequent observation of follicular mesenchyme-specific genes- CRABP1 , GDF10 , and GREM1 correctly revealed vigorous transcriptions of these genes in follicular mesenchymal cells. As expected, bivalent H3K4m3/H3K27me3 pattern was manifested in gene promoter regions of germ cells, and thus suspended their transcriptions. According the results, an example of chicken H3K4m3/H3K27me3 ChIP-seq data analysis was successfully demonstrated in this study. Hopefully, the provided methodology should hereby be useful for galline ChIP-seq data analysis in the future.

Space exploration by dendritic cells requires maintenance of myosin II activity by IP3 receptor 1

PubMed Central

Solanes, Paola; Heuzé, Mélina L; Maurin, Mathieu; Bretou, Marine; Lautenschlaeger, Franziska; Maiuri, Paolo; Terriac, Emmanuel; Thoulouze, Maria-Isabel; Launay, Pierre; Piel, Matthieu; Vargas, Pablo; Lennon-Duménil, Ana-Maria

2015-01-01

Dendritic cells (DCs) patrol the interstitial space of peripheral tissues. The mechanisms that regulate their migration in such constrained environment remain unknown. We here investigated the role of calcium in immature DCs migrating in confinement. We found that they displayed calcium oscillations that were independent of extracellular calcium and more frequently observed in DCs undergoing strong speed fluctuations. In these cells, calcium spikes were associated with fast motility phases. IP3 receptors (IP3Rs) channels, which allow calcium release from the endoplasmic reticulum, were identified as required for immature DCs to migrate at fast speed. The IP3R1 isoform was further shown to specifically regulate the locomotion persistence of immature DCs, that is, their capacity to maintain directional migration. This function of IP3R1 results from its ability to control the phosphorylation levels of myosin II regulatory light chain (MLC) and the back/front polarization of the motor protein. We propose that by upholding myosin II activity, constitutive calcium release from the ER through IP3R1 maintains DC polarity during migration in confinement, facilitating the exploration of their environment. PMID:25637353

ASN reputation system model

NASA Astrophysics Data System (ADS)

Hutchinson, Steve; Erbacher, Robert F.

2015-05-01

Network security monitoring is currently challenged by its reliance on human analysts and the inability for tools to generate indications and warnings for previously unknown attacks. We propose a reputation system based on IP address set membership within the Autonomous System Number (ASN) system. Essentially, a metric generated based on the historic behavior, or misbehavior, of nodes within a given ASN can be used to predict future behavior and provide a mechanism to locate network activity requiring inspection. This will provide reinforcement of notifications and warnings and lead to inspection for ASNs known to be problematic even if initial inspection leads to interpretation of the event as innocuous. We developed proof of concept capabilities to generate the IP address to ASN set membership and analyze the impact of the results. These results clearly show that while some ASNs are one-offs with individual or small numbers of misbehaving IP addresses, there are definitive ASNs with a history of long term and wide spread misbehaving IP addresses. These ASNs with long histories are what we are especially interested in and will provide an additional correlation metric for the human analyst and lead to new tools to aid remediation of these IP address blocks.

Mini-dystrophin Expression Down-regulates IP3-mediated Calcium Release Events in Resting Dystrophin-deficient Muscle Cells

PubMed Central

Balghi, Haouaria; Sebille, Stéphane; Mondin, Ludivine; Cantereau, Anne; Constantin, Bruno; Raymond, Guy; Cognard, Christian

2006-01-01

We present here evidence for the enhancement, at rest, of an inositol 1,4,5-trisphosphate (IP3)–mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, the number of sites discharging calcium (release site density [RSD]) was quantified and found more elevated in SolC1(−) than in SolD(+) myotubes. Variations of membrane potential had no significant effect on this difference, and higher resting [Ca2+]i in SolC1(−) (Marchand, E., B. Constantin, H. Balghi, M.C. Claudepierre, A. Cantereau, C. Magaud, A. Mouzou, G. Raymond, S. Braun, and C. Cognard. 2004. Exp. Cell Res. 297:363–379) cannot explain alone higher RSD. The exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) and phospholipase C inhibitor (U73122) significantly reduced RSD in both cell types but with a stronger effect in dystrophin-deficient SolC1(−) myotubes. Immunocytochemistry allowed us to localize ryanodine receptors (RyRs) as well as IP3 receptors (IP3Rs), IP3R-1 and IP3R-2 isoforms, indicating the presence of both RyRs-dependent and IP3-dependent release systems in both cells. We previously reported evidence for the enhancement, through a Gi protein, of the IP3-mediated calcium signaling pathway in SolC1(−) as compared to SolD(+) myotubes during a high K+ stimulation (Balghi, H., S. Sebille, B. Constantin, S. Patri, V. Thoreau, L. Mondin, E. Mok, A. Kitzis, G. Raymond, and C. Cognard. 2006. J. Gen. Physiol. 127:171–182). Here we show that, at rest, these regulation mechanisms are also involved in the modulation of calcium release activities. The enhancement of resting release activity may participate in the calcium overload observed in dystrophin-deficient myotubes, and our findings support the hypothesis of the regulatory role of mini-dystrophin on intracellular signaling. PMID:16847098

Induced pluripotent stem cells in hematology: current and future applications

PubMed Central

Focosi, D; Amabile, G; Di Ruscio, A; Quaranta, P; Tenen, D G; Pistello, M

2014-01-01

Reprogramming somatic cells into induced pluripotent stem (iPS) cells is nowadays approaching effectiveness and clinical grade. Potential uses of this technology include predictive toxicology, drug screening, pathogenetic studies and transplantation. Here, we review the basis of current iPS cell technology and potential applications in hematology, ranging from disease modeling of congenital and acquired hemopathies to hematopoietic stem and other blood cell transplantation. PMID:24813079

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations*

PubMed Central

Bartlett, Paula J.; Metzger, Walson; Gaspers, Lawrence D.; Thomas, Andrew P.

2015-01-01

How Ca2+ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca2+ oscillations report signal strength via frequency, whereas Ca2+ spike amplitude and wave velocity remain constant. IP3 uncaging also triggers oscillatory Ca2+ release, but, in contrast to hormones, Ca2+ spike amplitude, width, and wave velocity were dependent on [IP3] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP3 uncaging are driven by the biphasic regulation of the IP3 receptor by Ca2+, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca2+ did not perturb Ca2+ oscillations elicited by IP3 uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca2+ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca2+ oscillations but had no effect on Ca2+ increases induced by uncaging IP3. Importantly, PKC activation and inhibition differentially affected Ca2+ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP3 metabolism to attenuate IP3 levels and suppress the generation of Ca2+ oscillations. Inhibition of PKC relieves negative feedback regulation of IP3 accumulation and, thereby, shifts Ca2+ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca2+ wave velocity, whereas responses to IP3 uncaging are enhanced. The ability to assess Ca2+ responses in the absence of PLC activity indicates that IP3 receptor modulation by PKC regulates Ca2+ release and wave velocity. PMID:26078455

Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells

PubMed Central

Mach, François; Sauty, Alain; Iarossi, Albert S.; Sukhova, Galina K.; Neote, Kuldeep; Libby, Peter; Luster, Andrew D.

1999-01-01

Activated T lymphocytes accumulate early in atheroma formation and persist at sites of lesion growth and rupture, suggesting that they may play an important role in the pathogenesis of atherosclerosis. Moreover, atherosclerotic lesions contain the Th1-type cytokine IFN-γ, a potentiator of atherosclerosis. The present study demonstrates the differential expression of the 3 IFN-γ–inducible CXC chemokines — IFN-inducible protein 10 (IP-10), monokine induced by IFN-γ (Mig), and IFN-inducible T-cell α chemoattractant (I-TAC) — by atheroma-associated cells, as well as the expression of their receptor, CXCR3, by all T lymphocytes within human atherosclerotic lesions in situ. Atheroma-associated endothelial cells (ECs), smooth muscle cells (SMCs), and macrophages (MØ) all expressed IP-10, whereas Mig and I-TAC were mainly expressed in ECs and MØ, as detected by double immunofluorescence staining. ECs of microvessels within lesions also expressed abundant I-TAC. In vitro experiments supported these results and showed that IL-1β, TNF-α, and CD40 ligand potentiated IP-10 expression from IFN-γ–stimulated ECs. In addition, nitric oxide (NO) treatment decreased IFN-γ induction of IP-10. Our findings suggest that the differential expression of IP-10, Mig, and I-TAC by atheroma-associated cells plays a role in the recruitment and retention of activated T lymphocytes observed within vascular wall lesions during atherogenesis. PMID:10525042

[Stem cell research and science and technology policy in Japan].

PubMed

Yashiro, Yoshimi

2011-12-01

In this paper I review the present condition of the regeneration medicine research using pluripotency and a somatic stem cell, and I describe the subject of the science and technology policy in Japan towards realization of regeneration medicine. The Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) supported research promotion by the prompt action in 2007 when establishment of the iPS cell was reported by Shinya Yamanaka. Although the hospitable support of the Japanese government to an iPS cell is continued still now, there are some problems in respect of the support to other stem cell researches, and industrialization of regeneration medicine. In order to win a place in highly competitive area of investigation, MEXT needs to change policy so that funds may be widely supplied also to stem cell researches other than iPS cell research.

Sulforaphane inhibits the interferon-γ-induced expression of MIG, IP-10 and I-TAC in INS‑1 pancreatic β-cells through the downregulation of IRF-1, STAT-1 and PKB.

PubMed

Park, Yu-Kyoung; Ramalingam, Mahesh; Kim, Shin; Jang, Byeong-Churl; Park, Jong Wook

2017-09-01

Sulforaphane (SFN) is a dietary isothiocyanate abundantly available in cruciferous vegetables and has been shown to possess anti-inflammatory and immunomodulatory activities. Chemokines are important mediators of inflammation and immune responses due to their ability to recruit and activate macrophages and leukocytes. To date, little is known about the SFN-mediated regulation of chemokine expression in pancreatic β-cells. In this study, we investigated the inhibitory effects and mechanisms of SFN on the interferon-γ (IFN-γ)-induced expression of a subset of chemokines, including monokine induced by IFN-γ (MIG), IFN-inducible protein of 10 kDa (IP-10) and IFN-inducible T‑cell alpha chemoattractant (I-TAC), in INS‑1 cells, a rat pancreatic β-cell line. Notably, IFN-γ treatment led to an increase in the mRNA expression levels of MIG, IP-10 and I-TAC in the INS‑1 cells. However, SFN strongly blocked the mRNA expressions of MIG, IP-10 and I-TAC induced by IFN-γ in INS‑1 cells. On the mechanistic level, SFN significanlty decreased not only the mRNA expression levels of interferon regulatory factor-1 (IRF-1), but also the phosphorylation levels of signal transducer and activator of transcription-1 (STAT-1) and protein kinase B (PKB) which were induced by IFN-γ in the INS‑1 cells. Pharmacological inhibition experiments further revealed that treatment with JAK inhibitor I weakly inhibited the IFN-γ-induced expression of IP-10, whereas it strongly suppressed the IFN-γ-induced expression of MIG and I-TAC in the INS‑1 cells. Moreover, treatment with LY294002, a PI3K/PKB inhibitor, was able to slightly repress IFN‑γ‑induced expressions of MIG and I-TAC, but not IP-10, in INS‑1 cells. Importantly, the IFN-γ-induced increase in the expression levels of MIG, IP-10 and I-TAC in the INS-1 cells was strongly inhibited by SFN, but not by other natural substances, such as curcumin, sanguinarine, resveratrol, triptolide and epigallocatechin gallate (EGCG), suggesting the specificity of SFN in downregulating the levels of these chemokines. To the best of our knowledge, these results collectively demonstrate for the first time that SFN strongly inhibits the IFN-γ-induced expression of MIG, IP-10 and I-TAC in INS‑1 cells and this inhibition is, at least in part, mediated through the reduced expression and phosphorylation levels of IRF-1, STAT-1 and PKB.

Endopolysaccharides from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor affect differently the proliferation rate of HepG2 cells.

PubMed

Silva, Amélia M; Miranda, Andreia; Fernandes, Elisabete; Santos, Susana; Fraga, Irene; Santos, Dario L; Dias, Albino A; Bezerra, Rui M

2013-03-01

Fungi have been used for medicinal purposes for long time by Asian countries, being a putative source of powerful new phytopharmaceuticals such as polysaccharides. The aim of this study was to extract endopolysaccharides (IPS) from Ganoderma resinaceum, Phlebia rufa, and Trametes versicolor, grown under submerged culture, to compare crude IPS production, total carbohydrate, and protein yield, and to study the effect of these IPS on HepG2 cells proliferation rate. Total biomass produced by G. resinaceum, P. rufa, and T. versicolor was (in gram per liter) 3.32 ± 0.80, 5.42 ± 0.58, and 4.2 ± 1.29 and the IPS yield (as the biomass percent) was 9.9 ± 0.05, 29.0 ± 6.3, and 9.1 ± 3.1 %, respectively. Characterization of IPS has shown different proportion between total sugar and protein being, on average 6.04, 10.74, and 22.62, for G. resinaceum, T. versicolor, and P. rufa, respectively. The IPS effect, at 50, 100, and 200 μg mL(-1) on HepG2 cell growth and viability was negligible for G. resinaceum and P. rufa but, in the case of T. versicolor, 200 μg mL(-1) of IPS evoked 40 % reduction on cell growth. The results suggest that the intracellular polysaccharides from T. versicolor are a potential source for bioactive molecules with anti-proliferative properties.

Bombesin and muscarinic receptor activation in rat pancreas generate cyclic inositol monophosphate: possible involvement of different phospholipase C isoenzymes.

PubMed

Sekar, M C; Sambandam, V; McDonald, J M

1993-05-14

Phospholipase C isoenzymes can generate different proportions of cyclic and non-cyclic inositol phosphates. Stimulation of [3H]-inositol labeled pancreatic minilobules by buffer, bombesin, neuromedin B or carbachol in presence of 10 mM lithium, followed by separation of inositol phosphates, yielded the following results for cyclic inositol monophosphate (cIP) [DPM/mg protein; Mean +/- SEM (n)]: control [21 +/- 6, (9)]; bombesin [145 +/- 24, (12)]; neuromedin B (99 +/- 22 (9)] and carbachol [512 +/- 60, (12)]. The generation of cIP and IP were significantly correlated [r2 = 0.72 (p < 0.05)] following carbachol activation, while no significant correlation was obtained following bombesin receptor activation by either bombesin or neuromedin B. Presence of zinc (100 microM) in the final incubation medium failed to amplify the bombesin-stimulated cIP accumulation. Based on our studies we postulate that different phospholipase C isoenzymes may be activated following muscarinic and bombesin receptor stimulation in pancrea.

Preparation of Low-Input and Ligation-Free ChIP-seq Libraries Using Template-Switching Technology.

PubMed

Bolduc, Nathalie; Lehman, Alisa P; Farmer, Andrew

2016-10-10

Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low-abundance transcription factors, the small amount of DNA recovered makes ligation of adapters very challenging. In this unit, we describe a ChIP-seq workflow that can be applied to small cell numbers, including a robust single-tube and ligation-free method for preparation of sequencing libraries from sub-nanogram amounts of ChIP DNA. An example ChIP protocol is first presented, resulting in selective enrichment of DNA-binding proteins and cross-linked DNA fragments immobilized on beads via an antibody bridge. This is followed by a protocol for fast and easy cross-linking reversal and DNA recovery. Finally, we describe a fast, ligation-free library preparation protocol, featuring DNA SMART technology, resulting in samples ready for Illumina sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Impact of artifact removal on ChIP quality metrics in ChIP-seq and ChIP-exo data

PubMed Central

Carroll, Thomas S.; Liang, Ziwei; Salama, Rafik; Stark, Rory; de Santiago, Ines

2014-01-01

With the advent of ChIP-seq multiplexing technologies and the subsequent increase in ChIP-seq throughput, the development of working standards for the quality assessment of ChIP-seq studies has received significant attention. The ENCODE consortium's large scale analysis of transcription factor binding and epigenetic marks as well as concordant work on ChIP-seq by other laboratories has established a new generation of ChIP-seq quality control measures. The use of these metrics alongside common processing steps has however not been evaluated. In this study, we investigate the effects of blacklisting and removal of duplicated reads on established metrics of ChIP-seq quality and show that the interpretation of these metrics is highly dependent on the ChIP-seq preprocessing steps applied. Further to this we perform the first investigation of the use of these metrics for ChIP-exo data and make recommendations for the adaptation of the NSC statistic to allow for the assessment of ChIP-exo efficiency. PMID:24782889

cDNA cloning and characterization of a novel gene encoding the MLF1-interacting protein MLF1IP.

PubMed

Hanissian, Silva H; Akbar, Umar; Teng, Bin; Janjetovic, Zorica; Hoffmann, Anne; Hitzler, Johann K; Iscove, Norman; Hamre, Kristin; Du, Xiaoping; Tong, Yiai; Mukatira, Suraj; Robertson, Jon H; Morris, Stephan W

2004-04-29

Myelodysplasia/acute myeloid leukemia (MDS/AML) is characterized by a t(3;5)(q25.1;q34) chromosomal translocation that forms a fusion gene between nucleophosmin (NPM) and MDS/myeloid leukemia factor 1 (MLF1). We identified a novel protein, MLF1-interacting protein (MLF1IP), that specifically associates with MLF1 by yeast two-hybrid analysis and in pulldown assays, and colocalizes with it in both the nuclei and cytoplasm of cells. The MLF1IP gene locus is at chromosome 4q35.1 and is composed of 14 exons spanning 75.8 kb of genomic DNA. The MLF1IP cDNA encodes a 46-kDa protein that contains two bipartite and two classical nuclear localization signals, two nuclear receptor-binding motifs (LXXLL), two leucine zippers, two PEST residues and several potential phosphorylation sites. MLF1IP transcripts are expressed in a variety of tissues (e.g. fetal liver, bone marrow, thymus and testis). MLF1IP appears to be a lineage-specific gene whose expression is confined exclusively to the CFU-E erythroid precursor cells, but not in mature erythrocytes. These observations, together with previous data demonstrating a role for MLF1 in suppressing red cell maturation, suggest a possible role for MLF1IP and MLF1 deregulation in the genesis of erythroleukemias.

The crystal structure of mammalian inositol 1,3,4,5,6-pentakisphosphate 2-kinase reveals a new zinc-binding site and key features for protein function

PubMed Central

Franco-Echevarría, Elsa; Sanz-Aparicio, Julia; Brearley, Charles A.; González-Rubio, Juana M.; González, Beatriz

2017-01-01

Inositol 1,3,4,5,6-pentakisphosphate 2-kinases (IP5 2-Ks) are part of a family of enzymes in charge of synthesizing inositol hexakisphosphate (IP6) in eukaryotic cells. This protein and its product IP6 present many roles in cells, participating in mRNA export, embryonic development, and apoptosis. We reported previously that the full-length IP5 2-K from Arabidopsis thaliana is a zinc metallo-enzyme, including two separated lobes (the N- and C-lobes). We have also shown conformational changes in IP5 2-K and have identified the residues involved in substrate recognition and catalysis. However, the specific features of mammalian IP5 2-Ks remain unknown. To this end, we report here the first structure for a murine IP5 2-K in complex with ATP/IP5 or IP6. Our structural findings indicated that the general folding in N- and C-lobes is conserved with A. thaliana IP5 2-K. A helical scaffold in the C-lobe constitutes the inositol phosphate-binding site, which, along with the participation of the N-lobe, endows high specificity to this protein. However, we also noted large structural differences between the orthologues from these two eukaryotic kingdoms. These differences include a novel zinc-binding site and regions unique to the mammalian IP5 2-K, as an unexpected basic patch on the protein surface. In conclusion, our findings have uncovered distinct features of a mammalian IP5 2-K and set the stage for investigations into protein-protein or protein-RNA interactions important for IP5 2-K function and activity. PMID:28450399

Immunosuppression induced in vivo by 15 hydroxyeicosatetraenoic acid (15 HETE).

PubMed

Aldigier, J C; Gualde, N; Mexmain, S; Chable-Rabinovitch, H; Ratinaud, M H; Rigaud, M

1984-01-01

We have investigated the in vivo effects of 15 HETE on C57Bl/6 (H-2b) mice injected IP daily with this product. After that the 15 HETE treated animals and the controls were challenged in vivo by DBA/2 (H-2d) cells. Splenocytes from 15 HETE injected animals were either stimulated in vitro by lectins or cocultivated with DBA/2 irradiated splenocytes. It was observed that the response of splenocytes from in vivo treated animals is weaker than the control's response. The data suggest that 15 HETE induce the generation of suppressor cells.

Mercury-arc photolysis: a method for examining second messenger regulation of endothelial cell monolayer integrity.

PubMed

Patton, W F; Alexander, J S; Dodge, A B; Patton, R J; Hechtman, H B; Shepro, D

1991-07-01

Cell-cell apposition in bovine pulmonary endothelial cell monolayers was modulated by inducing transient increases in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) and 1,4,5-inositol triphosphate (IP3). This was accomplished by mercury-arc flash photolysis of o-nitrobenzyl derivatives of the second messengers (caged compounds). Second messenger release by the mercury-arc lamp was determined by radioimmunoassay of cAMP to have a t1/2 of approximately 8 min. Each second messenger induced the phosphorylation of a distinct subset of cytoskeletal proteins; however, both IP3 and cAMP increased vimentin phosphorylation. Actin isoform patterns were not altered by the second messengers. Intracellular pulses of IP3 in pulmonary endothelial cells caused disruption of endothelial monolayer integrity as determined by phase-contrast microscopy and by visualization of actin stress fibers with rhodamine-phalloidin. Intracellular pulses of cAMP increased cell-cell contact, cell surface area, and apposition. IP3 appeared to have its greatest effect on the actin peripheral band. In silicone rubber contractility assays this agent caused contraction of pulmonary microvascular endothelial cells as visualized by an increase in wrinkles beneath the cells. On the other hand, cAMP appeared to effect both the peripheral band and centralized actin domains. Caged cAMP caused relaxation of endothelial cells as visualized by a disappearance of wrinkles beneath the cells.

Extracellular Matrix from Periodontal Ligament Cells Could Induce the Differentiation of Induced Pluripotent Stem Cells to Periodontal Ligament Stem Cell-Like Cells.

PubMed

Hamano, Sayuri; Tomokiyo, Atsushi; Hasegawa, Daigaku; Yoshida, Shinichiro; Sugii, Hideki; Mitarai, Hiromi; Fujino, Shoko; Wada, Naohisa; Maeda, Hidefumi

2018-01-15

The periodontal ligament (PDL) plays an important role in anchoring teeth in the bone socket. Damage to the PDL, such as after severe inflammation, can be treated with a therapeutic strategy that uses stem cells derived from PDL tissue (PDLSCs), a strategy that has received intense scrutiny over the past decade. However, there is an insufficient number of PDLSCs within the PDL for treating such damage. Therefore, we sought to induce the differentiation of induced pluripotent stem (iPS) cells into PDLSCs as an initial step toward PDL therapy. To this end, we first induced iPS cells into neural crest (NC)-like cells. We then captured the p75 neurotrophic receptor-positive cells (iPS-NC cells) and cultured them on an extracellular matrix (ECM) produced by human PDL cells (iPS-NC-PDL cells). These iPS-NC-PDL cells showed reduced expression of embryonic stem cell and NC cell markers as compared with iPS and iPS-NC cells, and enrichment of mesenchymal stem cell markers. The cells also had a higher proliferative capacity, multipotency, and elevated expression of PDL-related markers than iPS-NC cells cultured on fibronectin and laminin (iPS-NC-FL cells) or ECM produced by human skin fibroblast cells (iPS-NC-SF cells). Overall, we present a culture method to produce high number of PDLSC-like cells from iPS cells as a first step toward a strategy for PDL regeneration.

Telebation: next-generation telemedicine in remote airway management using current wireless technologies.

PubMed

Mosier, Jarrod; Joseph, Bellal; Sakles, John C

2013-02-01

Since the first remote intubation with telemedicine guidance, wireless technology has advanced to enable more portable methods of telemedicine involvement in remote airway management. Three voice over Internet protocol (VoIP) services were evaluated for quality of image transmitted, data lag, and audio quality with remotely observed and assisted intubations in an academic emergency department. The VoIP clients evaluated were Apple (Cupertino, CA) FaceTime(®), Skype™ (a division of Microsoft, Luxembourg City, Luxembourg), and Tango(®) (TangoMe, Palo Alto, CA). Each client was tested over a Wi-Fi network as well as cellular third generation (3G) (Skype and Tango). All three VoIP clients provided acceptable image and audio quality. There is a significant data lag in image transmission and quality when VoIP clients are used over cellular broadband (3G) compared with Wi-Fi. Portable remote telemedicine guidance is possible with newer technology devices such as a smartphone or tablet, as well as VoIP clients used over Wi-Fi or cellular broadband.

A dual role for the anti-apoptotic Bcl-2 protein in cancer: mitochondria versus endoplasmic reticulum.

PubMed

Akl, Haidar; Vervloessem, Tamara; Kiviluoto, Santeri; Bittremieux, Mart; Parys, Jan B; De Smedt, Humbert; Bultynck, Geert

2014-10-01

Anti-apoptotic Bcl-2 contributes to cancer formation and progression by promoting the survival of altered cells. Hence, it is a prime target for novel specific anti-cancer therapeutics. In addition to its canonical anti-apoptotic role, Bcl-2 has an inhibitory effect on cell-cycle progression. Bcl-2 acts at two different intracellular compartments, the mitochondria and the endoplasmic reticulum (ER). At the mitochondria, Bcl-2 via its hydrophobic cleft scaffolds the Bcl-2-homology (BH) domain 3 (BH3) of pro-apoptotic Bcl-2-family members. Small molecules (like BH3 mimetics) can disrupt this interaction, resulting in apoptotic cell death in cancer cells. At the ER, Bcl-2 modulates Ca(2+) signaling, thereby promoting proliferation while increasing resistance to apoptosis. Bcl-2 at the ER acts via its N-terminal BH4 domain, which directly binds and inhibits the inositol 1,4,5-trisphosphate receptor (IP3R), the main intracellular Ca(2+)-release channel. Tools targeting the BH4 domain of Bcl-2 reverse Bcl-2's inhibitory action on IP3Rs and trigger pro-apoptotic Ca(2+) signaling in cancer B-cells, including chronic lymphocytic leukemia (CLL) cells and diffuse large B-cell lymphoma (DLBCL) cells. The sensitivity of DLBCL cells to BH4-domain targeting tools strongly correlated with the expression levels of the IP3R2 channel, the IP3R isoform with the highest affinity for IP3. Interestingly, bio-informatic analysis of a database of primary CLL patient cells also revealed a transcriptional upregulation of IP3R2. Finally, this review proposes a model, in which cancer cell survival depends on Bcl-2 at the mitochondria and/or the ER. This dependence likely will have an impact on their responses to BH3-mimetic drugs and BH4-domain targeting tools. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau. Copyright © 2014 Elsevier B.V. All rights reserved.

Electrical remodelling maintains firing properties in cortical pyramidal neurons lacking KCND2-encoded A-type K+ currents.

PubMed

Nerbonne, Jeanne M; Gerber, Benjamin R; Norris, Aaron; Burkhalter, Andreas

2008-03-15

Considerable experimental evidence has accumulated demonstrating a role for voltage-gated K(+) (Kv) channel pore-forming (alpha) subunits of the Kv4 subfamily in the generation of fast transient outward K(+), I(A), channels. Immunohistochemical data suggest that I(A) channels in hippocampal and cortical pyramidal neurons reflect the expression of homomeric Kv4.2 channels. The experiments here were designed to define directly the role of Kv4.2 in the generation of I(A) in cortical pyramidal neurons and to determine the functional consequences of the targeted deletion of Kv4.2 on the resting and active membrane properties of these cells. Whole-cell voltage-clamp recordings, obtained from visual cortical pyramidal neurons isolated from mice in which the KCND2 (Kv4.2) locus was disrupted (Kv4.2-/- mice), revealed that I(A) is indeed eliminated. In addition, the densities of other Kv current components, specifically I(K) and I(ss), are increased significantly (P < 0.001) in most ( approximately 80%) Kv4.2-/- cells. The deletion of KCND2 (Kv4.2) and the elimination of I(A) is also accompanied by the loss of the Kv4 channel accessory protein KChIP3, suggesting that in the absence of Kv4.2, the KChIP3 protein is targeted for degradation. The expression levels of several Kv alpha subunits (Kv4.3, Kv1.4, Kv2.1, Kv2.2), however, are not measurably altered in Kv4.2-/- cortices. Although I(A) is eliminated in Kv4.2-/- pyramidal neurons, the mean +/- s.e.m. current threshold for action potential generation and the waveforms of action potentials are indistinguishable from those recorded from wild-type cells. Repetitive firing is also maintained in Kv4.2-/- cortical pyramidal neurons, suggesting that the increased densities of I(K) and I(ss) compensate for the in vivo loss of I(A).

Combined DC resistivity and induced polarization (DC-IP) for mapping the internal composition of a mine waste rock pile in Nova Scotia, Canada

NASA Astrophysics Data System (ADS)

Power, Christopher; Tsourlos, Panagiotis; Ramasamy, Murugan; Nivorlis, Aristeidis; Mkandawire, Martin

2018-03-01

Mine waste rock piles (WRPs) can contain sulfidic minerals whose interaction with oxygen and water can generate acid mine drainage (AMD). Thus, WRPs can be a long-term source of environmental pollution. Since the generation of AMD and its release into the environment is dependent on the net volume and bulk composition of waste rock, effective characterization of WRPs is necessary for successful remedial design and monitoring. In this study, a combined DC resistivity and induced polarization (DC-IP) approach was employed to characterize an AMD-generating WRP in the Sydney Coalfield, Nova Scotia, Canada. Two-dimensional (2D) DC-IP imaging with 6 survey lines was performed to capture the full WRP landform. 2D DC results indicated a highly heterogeneous and moderately conductive waste rock underlain by a resistive bedrock containing numerous fractures. 2D IP (chargeability) results identified several highly-chargeable regions within the waste, with normalized chargeability delineating regions specific to waste mineralogy only. Three-dimensional (3D) DC-IP imaging, using 17 parallel lines on the plateau of the pile, was then used to focus on the composition of the waste rock. The full 3D inverted DC-IP distributions were used to identify coincident and continuous zones (isosurfaces) of low resistivity (<30 Ω-m) and high normalized chargeability (>0.4 mS/m) that were inferred as generated AMD (leachate) and stored AMD (sulfides), respectively. Integrated geological, hydrogeological and geochemical data increased confidence in the geoelectrical interpretations. Knowledge on the location of potentially more reactive waste material is extremely valuable for improved long-term AMD monitoring at the WRP.

Formation of Polymer Networks for Fast In-Plane Switching of Liquid Crystals at Low Temperatures

NASA Astrophysics Data System (ADS)

Yu, Byeong-Hun; Song, Dong Han; Kim, Ki-Han; Wok Park, Byung; Choi, Sun-Wook; Park, Sung Il; Kang, Sung Gu; Yoon, Jeong Hwan; Kim, Byeong Koo; Yoon, Tae-Hoon

2013-09-01

We formed a polymer structure to enable fast in-plane switching of liquid crystals at low temperatures. The problem of the inevitable slow response at low temperatures was reduced by the formation of in-cell polymer networks in in-plane switching (IPS) cells. The electro-optic characteristics of polymer-networked IPS cells were measured at temperatures ranging from -10 to 20 °C. The turn-on and turn-off times of an IPS cell were reduced by 44.5 and 47.2% at -10 °C by the formation of polymer networks. We believe that the proposed technology can be applied to emerging display devices such as mobile phones and automotive displays that may be used at low temperatures.

Cell Penetrating Peptides and Cationic Antibacterial Peptides

PubMed Central

Rodriguez Plaza, Jonathan G.; Morales-Nava, Rosmarbel; Diener, Christian; Schreiber, Gabriele; Gonzalez, Zyanya D.; Lara Ortiz, Maria Teresa; Ortega Blake, Ivan; Pantoja, Omar; Volkmer, Rudolf; Klipp, Edda; Herrmann, Andreas; Del Rio, Gabriel

2014-01-01

Cell penetrating peptides (CPP) and cationic antibacterial peptides (CAP) have similar physicochemical properties and yet it is not understood how such similar peptides display different activities. To address this question, we used Iztli peptide 1 (IP-1) because it has both CPP and CAP activities. Combining experimental and computational modeling of the internalization of IP-1, we show it is not internalized by receptor-mediated endocytosis, yet it permeates into many different cell types, including fungi and human cells. We also show that IP-1 makes pores in the presence of high electrical potential at the membrane, such as those found in bacteria and mitochondria. These results provide the basis to understand the functional redundancy of CPPs and CAPs. PMID:24706763

Buffer kinetics shape the spatiotemporal patterns of IP3-evoked Ca2+ signals

PubMed Central

Dargan, Sheila L; Parker, Ian

2003-01-01

Ca2+ liberation through inositol 1,4,5-trisphosphate receptors (IP3Rs) plays a universal role in cell regulation, and specificity of cell signalling is achieved through the spatiotemporal patterning of Ca2+ signals. IP3Rs display Ca2+-induced Ca2+ release (CICR), but are grouped in clusters so that regenerative Ca2+ signals may remain localized to individual clusters, or propagate globally between clusters by successive cycles of Ca2+ diffusion and CICR. We used confocal microscopy and photoreleased IP3 in Xenopus oocytes to study how these properties are modulated by mobile cytosolic Ca2+ buffers. EGTA (a buffer with slow ‘on-rate’) speeded Ca2+ signals and ‘balkanized’ Ca2+ waves by dissociating them into local signals. In contrast, BAPTA (a fast buffer with similar affinity) slowed Ca2+ responses and promoted ‘globalization’ of spatially uniform Ca2+ signals. These actions are likely to arise through differential effects on Ca2+ feedback within and between IP3R clusters, because Ca2+ signals evoked by influx through voltage-gated channels were little affected. We propose that cell-specific expression of Ca2+-binding proteins with distinct kinetics may shape the time course and spatial distribution of IP3-evoked Ca2+ signals for specific physiological roles. PMID:14555715

ChIP-Chip Identifies SEC23A, CFDP1, and NSD1 as TFII-I Target Genes in Human Neural Crest Progenitor Cells.

PubMed

Makeyev, Aleksandr V; Bayarsaihan, Dashzeveg

2013-05-01

Objectives :  GTF2I and GTF2IRD1 genes located in Williams-Beuren syndrome (WBS) critical region encode TFII-I family transcription factors. The aim of this study was to map genomic sites bound by these proteins across promoter regions of developmental regulators associated with craniofacial development. Design :  Chromatin was isolated from human neural crest progenitor cells and the DNA-binding profile was generated using the human RefSeq tiling promoter ChIP-chip arrays. Results :  TFII-I transcription factors are recruited to the promoters of SEC23A, CFDP1, and NSD1 previously defined as TFII-I target genes. Moreover, our analysis revealed additional binding elements that contain E-boxes and initiator-like motifs. Conclusions :  Genome-wide promoter binding studies revealed SEC23A, CFDP1, and NSD1 linked to craniofacial or dental development as direct TFII-I targets. Developmental regulation of these genes by TFII-I factors could contribute to the WBS-specific facial dysmorphism.

Real-time capture and reconstruction system with multiple GPUs for a 3D live scene by a generation from 4K IP images to 8K holograms.

PubMed

Ichihashi, Yasuyuki; Oi, Ryutaro; Senoh, Takanori; Yamamoto, Kenji; Kurita, Taiichiro

2012-09-10

We developed a real-time capture and reconstruction system for three-dimensional (3D) live scenes. In previous research, we used integral photography (IP) to capture 3D images and then generated holograms from the IP images to implement a real-time reconstruction system. In this paper, we use a 4K (3,840 × 2,160) camera to capture IP images and 8K (7,680 × 4,320) liquid crystal display (LCD) panels for the reconstruction of holograms. We investigate two methods for enlarging the 4K images that were captured by integral photography to 8K images. One of the methods increases the number of pixels of each elemental image. The other increases the number of elemental images. In addition, we developed a personal computer (PC) cluster system with graphics processing units (GPUs) for the enlargement of IP images and the generation of holograms from the IP images using fast Fourier transform (FFT). We used the Compute Unified Device Architecture (CUDA) as the development environment for the GPUs. The Fast Fourier transform is performed using the CUFFT (CUDA FFT) library. As a result, we developed an integrated system for performing all processing from the capture to the reconstruction of 3D images by using these components and successfully used this system to reconstruct a 3D live scene at 12 frames per second.

Modulation of Endoplasmic Reticulum Stress Controls CD4+ T-cell Activation and Antitumor Function.

PubMed

Thaxton, Jessica E; Wallace, Caroline; Riesenberg, Brian; Zhang, Yongliang; Paulos, Chrystal M; Beeson, Craig C; Liu, Bei; Li, Zihai

2017-08-01

The endoplasmic reticulum (ER) is an energy-sensing organelle with intimate ties to programming cell activation and metabolic fate. T-cell receptor (TCR) activation represents a form of acute cell stress and induces mobilization of ER Ca 2+ stores. The role of the ER in programming T-cell activation and metabolic fate remains largely undefined. Gp96 is an ER protein with functions as a molecular chaperone and Ca 2+ buffering protein. We hypothesized that the ER stress response may be important for CD4 + T-cell activation and that gp96 may be integral to this process. To test our hypothesis, we utilized genetic deletion of the gp96 gene Hsp90b1 in a CD4 + T cell-specific manner. We show that gp96-deficient CD4 + T cells cannot undergo activation-induced glycolysis due to defective Ca 2+ mobilization upon TCR engagement. We found that activating naïve CD4 + T cells while inhibiting ER Ca 2+ exchange, through pharmacological blockade of the ER Ca 2+ channel inositol trisphosphate receptor (IP 3 R), led to a reduction in cytosolic Ca 2+ content and generated a pool of CD62L high /CD44 low CD4 + T cells compared with wild-type (WT) matched controls. In vivo IP 3 R-inhibited CD4 + T cells exhibited elevated tumor control above WT T cells. Together, these data show that ER-modulated cytosolic Ca 2+ plays a role in defining CD4 + T-cell phenotype and function. Factors associated with the ER stress response are suitable targets for T cell-based immunotherapies. Cancer Immunol Res; 5(8); 666-75. ©2017 AACR . ©2017 American Association for Cancer Research.

Determining the Roles of Inositol Trisphosphate Receptors in Neurodegeneration: Interdisciplinary Perspectives on a Complex Topic.

PubMed

Takada, Silvia Honda; Ikebara, Juliane Midori; de Sousa, Erica; Cardoso, Débora Sterzeck; Resende, Rodrigo Ribeiro; Ulrich, Henning; Rückl, Martin; Rüdiger, Sten; Kihara, Alexandre Hiroaki

2017-11-01

It is well known that calcium (Ca 2+ ) is involved in the triggering of neuronal death. Ca 2+ cytosolic levels are regulated by Ca 2+ release from internal stores located in organelles, such as the endoplasmic reticulum. Indeed, Ca 2+ transit from distinct cell compartments follows complex dynamics that are mediated by specific receptors, notably inositol trisphosphate receptors (IP3Rs). Ca 2+ release by IP3Rs plays essential roles in several neurological disorders; however, details of these processes are poorly understood. Moreover, recent studies have shown that subcellular location, molecular identity, and density of IP3Rs profoundly affect Ca 2+ transit in neurons. Therefore, regulation of IP3R gene products in specific cellular vicinities seems to be crucial in a wide range of cellular processes from neuroprotection to neurodegeneration. In this regard, microRNAs seem to govern not only IP3Rs translation levels but also subcellular accumulation. Combining new data from molecular cell biology with mathematical modelling, we were able to summarize the state of the art on this topic. In addition to presenting how Ca 2+ dynamics mediated by IP3R activation follow a stochastic regimen, we integrated a theoretical approach in an easy-to-apply, cell biology-coherent fashion. Following the presented premises and in contrast to previously tested hypotheses, Ca 2+ released by IP3Rs may play different roles in specific neurological diseases, including Alzheimer's disease and Parkinson's disease.

IP3R1 deficiency in the cerebellum/brainstem causes basal ganglia-independent dystonia by triggering tonic Purkinje cell firings in mice

PubMed Central

Hisatsune, Chihiro; Miyamoto, Hiroyuki; Hirono, Moritoshi; Yamaguchi, Naohide; Sugawara, Takeyuki; Ogawa, Naoko; Ebisui, Etsuko; Ohshima, Toshio; Yamada, Masahisa; Hensch, Takao K.; Hattori, Mitsuharu; Mikoshiba, Katsuhiko

2013-01-01

The type 1 inositol 1,4,5- trisphosphate receptor (IP3R1) is a Ca2+ channel on the endoplasmic reticulum and is a predominant isoform in the brain among the three types of IP3Rs. Mice lacking IP3R1 show seizure-like behavior; however the cellular and neural circuit mechanism by which IP3R1 deletion causes the abnormal movements is unknown. Here, we found that the conditional knockout mice lacking IP3R1 specifically in the cerebellum and brainstem experience dystonia and show that cerebellar Purkinje cell (PC) firing patterns were coupled to specific dystonic movements. Recordings in freely behaving mice revealed epochs of low and high frequency PC complex spikes linked to body extension and rigidity, respectively. Remarkably, dystonic symptoms were independent of the basal ganglia, and could be rescued by inactivation of the cerebellum, inferior olive or in the absence of PCs. These findings implicate IP3R1-dependent PC firing patterns in cerebellum in motor coordination and the expression of dystonia through the olivo-cerebellar pathway. PMID:24109434

Marmoset induced pluripotent stem cells: Robust neural differentiation following pretreatment with dimethyl sulfoxide.

PubMed

Qiu, Zhifang; Mishra, Anuja; Li, Miao; Farnsworth, Steven L; Guerra, Bernadette; Lanford, Robert E; Hornsby, Peter J

2015-07-01

The marmoset is an important nonhuman primate model for regenerative medicine. For experimental autologous cell therapy based on induced pluripotent (iPS) cells in the marmoset, cells must be able to undergo robust and reliable directed differentiation that will not require customization for each specific iPS cell clone. When marmoset iPS cells were aggregated in a hanging drop format for 3 days, followed by exposure to dual SMAD inhibitors and retinoic acid in monolayer culture for 3 days, we found substantial variability in the response of different iPS cell clones. However, when clones were pretreated with 0.05-2% dimethyl sulfoxide (DMSO) for 24 hours, all clones showed a very similar maximal response to the directed differentiation scheme. Peak responses were observed at 0.5% DMSO in two clones and at 1% DMSO in a third clone. When patterns of gene expression were examined by microarray analysis, hierarchical clustering showed very similar responses in all 3 clones when they were pretreated with optimal DMSO concentrations. The change in phenotype following exposure to DMSO and the 6 day hanging drop/monolayer treatment was confirmed by immunocytochemistry. Analysis of DNA content in DMSO-exposed cells indicated that it is unlikely that DMSO acts by causing cells to exit from the cell cycle. This approach should be generally valuable in the directed neural differentiation of pluripotent cells for experimental cell therapy. Copyright © 2015. Published by Elsevier B.V.

Using Human Induced Pluripotent Stem Cells to Model Skeletal Diseases.

PubMed

Barruet, Emilie; Hsiao, Edward C

2016-01-01

Musculoskeletal disorders affecting the bones and joints are major health problems among children and adults. Major challenges such as the genetic origins or poor diagnostics of severe skeletal disease hinder our understanding of human skeletal diseases. The recent advent of human induced pluripotent stem cells (human iPS cells) provides an unparalleled opportunity to create human-specific models of human skeletal diseases. iPS cells have the ability to self-renew, allowing us to obtain large amounts of starting material, and have the potential to differentiate into any cell types in the body. In addition, they can carry one or more mutations responsible for the disease of interest or be genetically corrected to create isogenic controls. Our work has focused on modeling rare musculoskeletal disorders including fibrodysplasia ossificans progressive (FOP), a congenital disease of increased heterotopic ossification. In this review, we will discuss our experiences and protocols differentiating human iPS cells toward the osteogenic lineage and their application to model skeletal diseases. A number of critical challenges and exciting new approaches are also discussed, which will allow the skeletal biology field to harness the potential of human iPS cells as a critical model system for understanding diseases of abnormal skeletal formation and bone regeneration.

Human induced pluripotent stem cell differentiation and direct transdifferentiation into corneal epithelial-like cells

PubMed Central

Cieślar-Pobuda, Artur; Rafat, Mehrdad; Knoflach, Viktoria; Skonieczna, Magdalena; Hudecki, Andrzej; Małecki, Andrzej; Urasińska, Elżbieta; Ghavami, Seaid; Łos, Marek J.

2016-01-01

The corneal epithelium is maintained by a small pool of tissue stem cells located at the limbus. Through certain injuries or diseases this pool of stem cells may get depleted. This leads to visual impairment. Standard treatment options include autologous or allogeneic limbal stem cell (LSC) transplantation, however graft rejection and chronic inflammation lowers the success rate over long time. Induced pluripotent stem (iPS) cells have opened new possibilities for treating various diseases with patient specific cells, eliminating the risk of immune rejection. In recent years, several protocols have been developed, aimed at the differentiation of iPS cells into the corneal epithelial lineage by mimicking the environmental niche of limbal stem cells. However, the risk of teratoma formation associated with the use of iPS cells hinders most applications from lab into clinics. Here we show that the differentiation of iPS cells into corneal epithelial cells results in the expression of corneal epithelial markers showing a successful differentiation, but the process is long and the level of gene expression for the pluripotency markers does not vanish completely. Therefore we set out to determine a direct transdifferentiation approach to circumvent the intermediate state of pluripotency (iPS-stage). The resulting cells, obtained by direct transdifferentiation of fibroblasts into limbal cells, exhibited corneal epithelial cell morphology and expressed corneal epithelial markers. Hence we shows for the first time a direct transdifferentiation of human dermal fibroblasts into the corneal epithelial lineage that may serve as source for corneal epithelial cells for transplantation approaches. PMID:27275539

Measuring Sister Chromatid Cohesion Protein Genome Occupancy in Drosophila melanogaster by ChIP-seq.

PubMed

Dorsett, Dale; Misulovin, Ziva

2017-01-01

This chapter presents methods to conduct and analyze genome-wide chromatin immunoprecipitation of the cohesin complex and the Nipped-B cohesin loading factor in Drosophila cells using high-throughput DNA sequencing (ChIP-seq). Procedures for isolation of chromatin, immunoprecipitation, and construction of sequencing libraries for the Ion Torrent Proton high throughput sequencer are detailed, and computational methods to calculate occupancy as input-normalized fold-enrichment are described. The results obtained by ChIP-seq are compared to those obtained by ChIP-chip (genomic ChIP using tiling microarrays), and the effects of sequencing depth on the accuracy are analyzed. ChIP-seq provides similar sensitivity and reproducibility as ChIP-chip, and identifies the same broad regions of occupancy. The locations of enrichment peaks, however, can differ between ChIP-chip and ChIP-seq, and low sequencing depth can splinter broad regions of occupancy into distinct peaks.

Science spin: iPS cell research in the news.

PubMed

Caulfield, T; Rachul, C

2011-05-01

Big scientific developments have always been spun to meet particular social agendas. We have seen it in the context of global warming, nuclear power, and genetically modified organisms. But few stories illustrate the phenomenon of spin as well as the reaction, and concomitant media coverage, that surrounded the November 2007 announcement regarding the reprogramming of skin cells to produce cells with qualities comparable to those of human embryonic stem cells (hESCs) known as induced pluripotent stem (iPS) cells.

The ChIP-exo Method: Identifying Protein-DNA Interactions with Near Base Pair Precision.

PubMed

Perreault, Andrea A; Venters, Bryan J

2016-12-23

Chromatin immunoprecipitation (ChIP) is an indispensable tool in the fields of epigenetics and gene regulation that isolates specific protein-DNA interactions. ChIP coupled to high throughput sequencing (ChIP-seq) is commonly used to determine the genomic location of proteins that interact with chromatin. However, ChIP-seq is hampered by relatively low mapping resolution of several hundred base pairs and high background signal. The ChIP-exo method is a refined version of ChIP-seq that substantially improves upon both resolution and noise. The key distinction of the ChIP-exo methodology is the incorporation of lambda exonuclease digestion in the library preparation workflow to effectively footprint the left and right 5' DNA borders of the protein-DNA crosslink site. The ChIP-exo libraries are then subjected to high throughput sequencing. The resulting data can be leveraged to provide unique and ultra-high resolution insights into the functional organization of the genome. Here, we describe the ChIP-exo method that we have optimized and streamlined for mammalian systems and next-generation sequencing-by-synthesis platform.

NAADP Activates Two-Pore Channels on T Cell Cytolytic Granules to Stimulate Exocytosis and Killing

PubMed Central

Davis, Lianne C.; Morgan, Anthony J.; Chen, Ji-Li; Snead, Charlotte M.; Bloor-Young, Duncan; Shenderov, Eugene; Stanton-Humphreys, Megan N.; Conway, Stuart J.; Churchill, Grant C.; Parrington, John; Cerundolo, Vincenzo; Galione, Antony

2012-01-01

Summary A cytotoxic T lymphocyte (CTL) kills an infected or tumorigenic cell by Ca2+-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. Although inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum activates the store-operated Ca2+-influx pathway that is necessary for exocytosis, it is not a sufficient stimulus [1–4]. Here we identify the Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and its recently identified molecular target, two-pore channels (TPCs) [5–7], as being important for T cell receptor signaling in CTLs. We demonstrate that cytolytic granules are not only reservoirs of cytolytic proteins but are also the acidic Ca2+ stores mobilized by NAADP via TPC channels on the granules themselves, so that TPCs migrate to the immunological synapse upon CTL activation. Moreover, NAADP activates TPCs to drive exocytosis in a way that is not mimicked by global Ca2+ signals induced by IP3 or ionomycin, suggesting that critical, local Ca2+ nanodomains around TPCs stimulate granule exocytosis. Hence, by virtue of the NAADP/TPC pathway, cytolytic granules generate Ca2+ signals that lead to their own exocytosis and to cell killing. This study highlights a selective role for NAADP in stimulating exocytosis crucial for immune cell function and may impact on stimulus-secretion coupling in wider cellular contexts. PMID:23177477

Vaccination with vascular progenitor cells derived from induced pluripotent stem cells elicits antitumor immunity targeting vascular and tumor cells.

PubMed

Koido, Shigeo; Ito, Masaki; Sagawa, Yukiko; Okamoto, Masato; Hayashi, Kazumi; Nagasaki, Eijiro; Kan, Shin; Komita, Hideo; Kamata, Yuko; Homma, Sadamu

2014-05-01

Vaccination of BALB/c mice with dendritic cells (DCs) loaded with the lysate of induced vascular progenitor (iVP) cells derived from murine-induced pluripotent stem (iPS) cells significantly suppressed the tumor of CMS-4 fibrosarcomas and prolonged the survival of CMS-4-inoculated mice. This prophylactic antitumor activity was more potent than that of immunization with DCs loaded with iPS cells or CMS-4 tumor cells. Tumors developed slowly in mice vaccinated with DCs loaded with iVP cells (DC/iVP) and exhibited a limited vascular bed. Immunohistochemistry and a tomato-lectin perfusion study demonstrated that the tumors that developed in the iVP-immunized mice showed a marked decrease in tumor vasculature. Immunization with DC/iVP induced a potent suppressive effect on vascular-rich CMS-4 tumors, a weaker effect on BNL tumors with moderate vasculature, and nearly no effect on C26 tumors with poor vasculature. Treatment of DC/iVP-immunized mice with a monoclonal antibody against CD4 or CD8, but not anti-asialo GM1, inhibited the antitumor activity. CD8(+) T cells from DC/iVP-vaccinated mice showed significant cytotoxic activity against murine endothelial cells and CMS-4 cells, whereas CD8(+) T cells from DC/iPS-vaccinated mice did not. DNA microarray analysis showed that the products of 29 vasculature-associated genes shared between genes upregulated by differentiation from iPS cells into iVP cells and genes shared by iVP cells and isolated Flk-1(+) vascular cells in CMS-4 tumor tissue might be possible targets in the immune response. These results suggest that iVP cells from iPS cells could be used as a cancer vaccine targeting tumor vascular cells and tumor cells.

A Paracrine Mechanism Accelerating Expansion of Human Induced Pluripotent Stem Cell-Derived Hepatic Progenitor-Like Cells

PubMed Central

Tsuruya, Kota; Chikada, Hiromi; Ida, Kinuyo; Anzai, Kazuya; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tetsuya

2015-01-01

Hepatic stem/progenitor cells in liver development have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In this study, we focused on the cell surface molecules of human induced pluripotent stem (iPS) cell-derived hepatic progenitor-like cells (HPCs) and analyzed how these molecules modulate expansion of these cells. Human iPS cells were differentiated into immature hepatic lineage cells by cytokines. In addition to hepatic progenitor markers (CD13 and CD133), the cells were coimmunostained for various cell surface markers (116 types). The cells were analyzed by flow cytometry and in vitro colony formation culture with feeder cells. Twenty types of cell surface molecules were highly expressed in CD13+CD133+ cells derived from human iPS cells. Of these molecules, CD221 (insulin-like growth factor receptor), which was expressed in CD13+CD133+ cells, was quickly downregulated after in vitro expansion. The proliferative ability was suppressed by a neutralizing antibody and specific inhibitor of CD221. Overexpression of CD221 increased colony-forming ability. We also found that inhibition of CD340 (erbB2) and CD266 (fibroblast growth factor-inducible 14) signals suppressed proliferation. In addition, both insulin-like growth factor (a ligand of CD221) and tumor necrosis factor-like weak inducer of apoptosis (a ligand of CD266) were provided by feeder cells in our culture system. This study revealed the expression profiles of cell surface molecules in human iPS cell-derived HPCs and that the paracrine interactions between HPCs and other cells through specific receptors are important for proliferation. PMID:25808356

A Paracrine Mechanism Accelerating Expansion of Human Induced Pluripotent Stem Cell-Derived Hepatic Progenitor-Like Cells.

PubMed

Tsuruya, Kota; Chikada, Hiromi; Ida, Kinuyo; Anzai, Kazuya; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tetsuya; Kamiya, Akihide

2015-07-15

Hepatic stem/progenitor cells in liver development have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In this study, we focused on the cell surface molecules of human induced pluripotent stem (iPS) cell-derived hepatic progenitor-like cells (HPCs) and analyzed how these molecules modulate expansion of these cells. Human iPS cells were differentiated into immature hepatic lineage cells by cytokines. In addition to hepatic progenitor markers (CD13 and CD133), the cells were coimmunostained for various cell surface markers (116 types). The cells were analyzed by flow cytometry and in vitro colony formation culture with feeder cells. Twenty types of cell surface molecules were highly expressed in CD13(+)CD133(+) cells derived from human iPS cells. Of these molecules, CD221 (insulin-like growth factor receptor), which was expressed in CD13(+)CD133(+) cells, was quickly downregulated after in vitro expansion. The proliferative ability was suppressed by a neutralizing antibody and specific inhibitor of CD221. Overexpression of CD221 increased colony-forming ability. We also found that inhibition of CD340 (erbB2) and CD266 (fibroblast growth factor-inducible 14) signals suppressed proliferation. In addition, both insulin-like growth factor (a ligand of CD221) and tumor necrosis factor-like weak inducer of apoptosis (a ligand of CD266) were provided by feeder cells in our culture system. This study revealed the expression profiles of cell surface molecules in human iPS cell-derived HPCs and that the paracrine interactions between HPCs and other cells through specific receptors are important for proliferation.

Interferon-gamma inducible protein 10 (IP10) induced cisplatin resistance of HCC after liver transplantation through ER stress signaling pathway

PubMed Central

Geng, Wei; Lo, Chung-Mau; Ng, Kevin T.P.; Ling, Chang-Chun; Qi, Xiang; Li, Chang-Xian; Zhai, Yuan; Liu, Xiao-Bing; Ma, Yuen-Yuen; Man, Kwan

2015-01-01

Tumor recurrence remains an obstacle after liver surgery, especially in living donor liver transplantation (LDLT) for patients with hepatocellular carcinoma (HCC). The acute-phase liver graft injury might potentially induce poor response to chemotherapy in recurrent HCC after liver transplantation. We here intended to explore the mechanism and to identify a therapeutic target to overcome such chemoresistance. The associations among graft injury, overexpression of IP10 and multidrug resistant genes were investigated in a rat liver transplantation model, and further validated in clinical cohort. The role of IP10 on HCC cell proliferation and tumor growth under chemotherapy was studied both in vitro and in vivo. The underlying mechanism was revealed by detecting the activation of endoplasmic reticulum (ER) stress signaling pathways. Moreover, the effect of IP10 neutralizing antibody sensitizing cisplatin treatment was further explored. In rat liver transplantation model, significant up-regulation of IP10 associated with multidrug resistant genes was found in small-for-size liver graft. Clinically, high expression of circulating IP10 was significant correlated with tumor recurrence in HCC patients underwent LDLT. Overexpression of IP10 promoted HCC cell proliferation and tumor growth under cisplatin treatment by activation of ATF6/Grp78 signaling. IP10 neutralizing antibody sensitized cisplatin treatment in nude mice. The overexpression of IP10, which induced by liver graft injury, may lead to cisplatin resistance via ATF6/Grp78 ER stress signaling pathway. IP10 neutralizing antibody could be a potential adjuvant therapy to sensitize cisplatin treatment. PMID:26336986

Engraftment of donor mesenchymal stem cells in chimeric BXSB includes vascular endothelial cells and hepatocytes.

PubMed

Jones, Olcay Y; Gok, Faysal; Rushing, Elisabeth J; Horkayne-Szakaly, Iren; Ahmed, Atif A

2011-01-01

Somatic tissue engraftment was studied in BXSB mice treated with mesenchymal stem cell transplantation. Hosts were conditioned with nonlethal radiation prior to introducing donor cells from major histocompatibility complex-matched green fluorescent protein transgenic mice. Transplant protocols differed for route of injection, ie, intravenous (i.v.) versus intraperitoneal (i.p.), and source of mesenchymal stem cells, ie, unfractionated bone marrow cells, ex vivo expanded mesenchymal stem cells, or bone chips. Tissue chimerism was determined after short (10-12 weeks) or long (62 weeks) posttransplant follow-up by immunohistochemistry for green fluorescent protein. Engraftment of endothelial cells was seen in several organs including liver sinusoidal cells in i.v. treated mice with ex vivo expanded mesenchymal stem cells or with unfractionated bone marrow cells. Periportal engraftment of liver hepatocytes, but not engraftment of endothelial cells, was found in mice injected i.p. with bone chips. Engraftment of adipocytes was a common denominator in both i.v. and i.p. routes and occurred during early phases post-transplant. Disease control was more robust in mice that received both i.v. bone marrow and i.p. bone chips compared to mice that received i.v. bone marrow alone. Thus, the data support potential use of mesenchymal stem cell transplant for treatment of severe lupus. Future studies are needed to optimize transplant conditions and tailor protocols that may in part be guided by fat and endothelial biomarkers. Furthermore, the role of liver chimerism in disease control and the nature of cellular communication among donor hematopoietic and mesenchymal stem cells in a chimeric host merit further investigation.

Localization and socialization: Experimental insights into the functional architecture of IP3 receptors

NASA Astrophysics Data System (ADS)

Diambra, Luis; Marchant, Jonathan S.

2009-09-01

Inositol 1,4,5-trisphosphate (IP3)-evoked Ca2+ signals display great spatiotemporal malleability. This malleability depends on diversity in both the cellular organization and in situ functionality of IP3 receptors (IP3Rs) that regulate Ca2+ release from the endoplasmic reticulum (ER). Recent experimental data imply that these considerations are not independent, such that—as with other ion channels—the local organization of IP3Rs impacts their functionality, and reciprocally IP3R activity impacts their organization within native ER membranes. Here, we (i) review experimental data that lead to our understanding of the "functional architecture" of IP3Rs within the ER, (ii) propose an updated terminology to span the organizational hierarchy of IP3Rs observed in intact cells, and (iii) speculate on the physiological significance of IP3R socialization in Ca2+ dynamics, and consequently the emerging need for modeling studies to move beyond gridded, planar, and static simulations of IP3R clustering even over short experimental timescales.

DROMPA: easy-to-handle peak calling and visualization software for the computational analysis and validation of ChIP-seq data.

PubMed

Nakato, Ryuichiro; Itoh, Tahehiko; Shirahige, Katsuhiko

2013-07-01

Chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) can identify genomic regions that bind proteins involved in various chromosomal functions. Although the development of next-generation sequencers offers the technology needed to identify these protein-binding sites, the analysis can be computationally challenging because sequencing data sometimes consist of >100 million reads/sample. Herein, we describe a cost-effective and time-efficient protocol that is generally applicable to ChIP-seq analysis; this protocol uses a novel peak-calling program termed DROMPA to identify peaks and an additional program, parse2wig, to preprocess read-map files. This two-step procedure drastically reduces computational time and memory requirements compared with other programs. DROMPA enables the identification of protein localization sites in repetitive sequences and efficiently identifies both broad and sharp protein localization peaks. Specifically, DROMPA outputs a protein-binding profile map in pdf or png format, which can be easily manipulated by users who have a limited background in bioinformatics. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Reduced incidence of interstitial pneumonitis after allogeneic hematopoietic stem cell transplantation using a modified technique of total body irradiation.

PubMed

Chiang, Yun; Tsai, Cheng-Hong; Kuo, Sung-Hsin; Liu, Chieh-Yu; Yao, Ming; Li, Chi-Cheng; Huang, Shang-Yi; Ko, Bor-Sheng; Lin, Chien-Ting; Hou, Hsin-An; Chou, Wen-Chien; Liu, Jia-Hau; Lin, Chien-Chin; Wu, Shang-Ju; Hsu, Szu-Chun; Chen, Yao-Chang; Lin, Kai-Hsin; Lin, Dong-Tsamn; Chou, Hsien-Tang; Lu, Meng-Yu; Yang, Yung-Li; Chang, Hsiu-Hao; Liu, Ming-Chih; Liao, Xiu-Wen; Wu, Jian-Kuen; Chou, Sheng-Chieh; Cheng, Chieh-Lung; Chen, Chien-Yuan; Tsay, Woei; Tien, Hwei-Fang; Tang, Jih-Luh; Chen, Yu-Hsuan

2016-11-10

Allogeneic hematopoietic stem cell transplantation is a curative-intent treatment for patients with high-risk hematologic diseases. However, interstitial pneumonitis (IP) and other toxicities remain major concerns after total body irradiation (TBI). We have proposed using linear accelerators with rice-bag compensators for intensity modulation (IM-TBI), as an alternative to the traditional cobalt-60 teletherapy with lung-shielding technique (Co-TBI). Patients who received a TBI-based myeloablative conditioning regimen between 1995 and 2014 were recruited consecutively. Before March 2007, TBI was delivered using Co-TBI (n = 181); afterward, TBI was administered using IM-TBI (n = 126). Forty-four patients developed IP; of these cases, 19 were idiopathic. The IP-related mortality rate was 50% in the total IP cohort and 63% in the idiopathic subgroup. The 1-year cumulative incidences of IP and idiopathic IP were 16.5% and 7.4%, respectively; both rates were significantly higher in the Co-TBI group than in the IM-TBI group. Multivariate analysis revealed that Co-TBI was an independent prognostic factor for both total and idiopathic IP. In the acute myeloid leukemia subgroup, patients with different TBI techniques had similar outcomes for both overall and relapse-free survival. In conclusion, IM-TBI is an easy and effective TBI technique that could substantially reduce the complication rate of IP without compromising treatment efficacy.

The prevalence of human papillomavirus infection in Iranian patients with sinonasal inverted papilloma.

PubMed

Jalilvand, Somayeh; Saidi, Masoumeh; Shoja, Zabihollah; Ghavami, Nastaran; Hamkar, Rasool

2016-03-01

Inverted papilloma (IP) is an uncommon disease which arises in the mucosal membrane of the nasal cavity and paranasal sinus. It has been proposed that human papillomavirus (HPV) is the causal agent in the pathogenesis of IP and plays a key role in the progression from benign IP to malignancy. As there are no prior studies that focus on an Iranian population, this study intended to characterize the prevalence of HPV types in benign and malignant forms of IP. In this retrospective study, we included a total of 40 IP patients [37 benign IP and 3 IP/squamous cell carcinoma (SCC)] who were referred to Amiralam Hospital in Tehran from 2004-2006. HPV was detected in 18.9% and 100% of IP and IP/SCC cases, respectively. In all HPV positive cases of IP and IP/SCC cases, HPV6/11 and HPV16/18 were detected, respectively. Therefore, HPV types were different between the IP and IP/SCC patients, and this difference was statistically significant (p = 0.002). This study suggests that HPV6 and 11 may be involved in the development of IP, but HPV16 and 18 likely play an important role in the progression from benign to malignant form of IP. Copyright © 2015. Published by Elsevier Taiwan LLC.

Impact of Environmental Exposures on the Mutagenicity/Carcinogenicity of Heterocyclic Amines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Felton, J S; Knize, M G; Bennett, L M

2003-12-19

Carcinogenic heterocyclic amines are produced from overcooked foods and are highly mutagenic in most short-term test systems. One of the most abundant of these amines, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), induces breast, colon and prostate tumors in rats. Human dietary epidemiology studies suggest a strong correlation between either meat consumption or well-done muscle meat consumption and cancers of the colon, breast, stomach, lung and esophagus. For over 20 years our laboratory has helped define the human exposure to these dietary carcinogens. In this report we describe how various environmental exposures may modulate the risk from exposure to heterocyclic amines, especially PhIP. To assessmore » the impact of foods on PhIP metabolism in humans, we developed an LC/MS/MS method to analyze the four major PhIP urinary metabolites following the consumption of a single portion of grilled chicken. Adding broccoli to the volunteers' diet altered the kinetics of PhIP metabolism. At the cellular level we have found that PhIP itself stimulates a significant estrogenic response in MCF-7 cells, but even more interestingly, co-incubation of the cells with herbal teas appear to enhance the response. Numerous environmental chemicals found in food or the atmosphere can impact the exposure, metabolism, and cell proliferation response of heterocyclic amines.« less

Chloride secretagogues stimulate inositol phosphate formation in shark rectal gland tubules cultured in suspension

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ecay, T.W.; Valentich, J.D.

1991-03-01

Neuroendocrine activation of transepithelial chloride secretion by shark rectal gland cells is associated with increases in cellular cAMP, cGMP, and free calcium concentrations. We report here on the effects of several chloride secretagogues on inositol phosphate formation in cultured rectal gland tubules. Vasoactive intestinal peptide (VIP), atriopeptin (AP), and ionomycin increase the total inositol phosphate levels of cultured tubules, as measured by ion exchange chromatography. Forskolin, a potent chloride secretagogue, has no effect on inositol phosphate formation. The uptake of {sup 3}H-myo-inositol into phospholipids is very slow, preventing the detection of increased levels of inositol trisphosphate. However, significant increases inmore » inositol monophosphate (IP1) and inositol biphosphate (IP2) were measured. The time course of VIP- and AP-stimulated IP1 and IP2 formation is similar to the effects of these agents on the short-circuit current responses of rectal gland monolayer cultures. In addition, aluminum fluoride, an artificial activator of guanine nucleotide-binding proteins, stimulates IP1 and IP2 formation. We conclude that rectal gland cells contain VIP and AP receptors coupled to the activation of phospholipase C. Coupling may be mediated by G-proteins. Receptor-stimulated increases in inositol phospholipid metabolism is one mechanism leading to increased intracellular free calcium concentrations, an important regulatory event in the activation of transepithelial chloride secretion by shark rectal gland epithelial cells.« less

The influence of implantoplasty on the diameter, chemical surface composition, and biocompatibility of titanium implants.

PubMed

Schwarz, Frank; John, Gordon; Becker, Jürgen

2017-09-01

The objective of the study was to assess the influence of implantoplasty (IP) on the diameter, chemical surface composition, and biocompatibility of titanium implants in vitro. Twenty soft tissue-level (TL; machined transmucosal-M and rough endosseous part-SLA) and 20 bone-level (BL; SLA) implants were allocated to IP covering 3 or 6 mm of the structured surface (SLA) area. The samples were subjected to diameter, energy-dispersive X-ray spectroscopy (EDX), and cell viability (ginigval fibroblasts, 6 days) assessments. Median diameter reductions varied between 0.1 (TL 3 mm) and 0.2 mm (TL 6 mm). EDX analysis revealed that IP and M surfaces were characterized by a comparable quantity (Wt%) of elements C, O, Na, Cl, K, and Si, but a significantly different quantity of elements Ti and Al. When compared to SLA surfaces, significant differences were noted for elements C, O, Na, Ti, and Al. At BL implants, the extension of IP (i.e., 3 to 6 mm) was associated with a significant increase in cell viability. IP applied to SLA implants was associated with (i) a minimal diameter reduction, (ii) an undisturbed cell viability, and (iii) a chemical elemental composition comparable to M surfaces. This specific IP procedure appears to be suitable for the management of exposed SLA implant surfaces.

IP3R deficit underlies loss of salivary fluid secretion in Sjögren’s Syndrome

PubMed Central

Teos, Leyla Y.; Zhang, Yu; Cotrim, Ana P.; Swaim, William; Won, Jon H.; Ambrus, Julian; Shen, Long; Bebris, Lolita; Grisius, Margaret; Jang, Shyh-Ing; Yule, David I.; Ambudkar, Indu S.; Alevizos, Ilias

2015-01-01

The autoimmune exocrinopathy, Sjögren’s syndrome (SS), is associated with secretory defects in patients, including individuals with mild lymphocytic infiltration and minimal glandular damage. The mechanism(s) underlying the secretory dysfunction is not known. We have used minor salivary gland biopsies from SS patients and healthy individuals to assess acinar cell function in morphologically intact glandular areas. We report that agonist-regulated intracellular Ca2+ release, critically required for Ca2+ entry and fluid secretion, is defective in acini from SS patients. Importantly, these acini displayed reduction in IP3R2 and IP3R3, but not AQP5 or STIM1. Similar decreases in IP3R and carbachol (CCh)-stimulated [Ca2+]i elevation were detected in acinar cells from lymphotoxin-alpha (LTα) transgenic (TG) mice, a model for (SS). Treatment of salivary glands from healthy individuals with LT α, a cytokine linked to disease progression in SS and IL14α mice, reduced Ca2+ signaling. Together, our findings reveal novel IP3R deficits in acinar cells that underlie secretory dysfunction in SS patients. PMID:26365984

ePIANNO: ePIgenomics ANNOtation tool.

PubMed

Liu, Chia-Hsin; Ho, Bing-Ching; Chen, Chun-Ling; Chang, Ya-Hsuan; Hsu, Yi-Chiung; Li, Yu-Cheng; Yuan, Shin-Sheng; Huang, Yi-Huan; Chang, Chi-Sheng; Li, Ker-Chau; Chen, Hsuan-Yu

2016-01-01

Recently, with the development of next generation sequencing (NGS), the combination of chromatin immunoprecipitation (ChIP) and NGS, namely ChIP-seq, has become a powerful technique to capture potential genomic binding sites of regulatory factors, histone modifications and chromatin accessible regions. For most researchers, additional information including genomic variations on the TF binding site, allele frequency of variation between different populations, variation associated disease, and other neighbour TF binding sites are essential to generate a proper hypothesis or a meaningful conclusion. Many ChIP-seq datasets had been deposited on the public domain to help researchers make new discoveries. However, researches are often intimidated by the complexity of data structure and largeness of data volume. Such information would be more useful if they could be combined or downloaded with ChIP-seq data. To meet such demands, we built a webtool: ePIgenomic ANNOtation tool (ePIANNO, http://epianno.stat.sinica.edu.tw/index.html). ePIANNO is a web server that combines SNP information of populations (1000 Genomes Project) and gene-disease association information of GWAS (NHGRI) with ChIP-seq (hmChIP, ENCODE, and ROADMAP epigenomics) data. ePIANNO has a user-friendly website interface allowing researchers to explore, navigate, and extract data quickly. We use two examples to demonstrate how users could use functions of ePIANNO webserver to explore useful information about TF related genomic variants. Users could use our query functions to search target regions, transcription factors, or annotations. ePIANNO may help users to generate hypothesis or explore potential biological functions for their studies.

External light activates hair follicle stem cells through eyes via an ipRGC-SCN-sympathetic neural pathway.

PubMed

Fan, Sabrina Mai-Yi; Chang, Yi-Ting; Chen, Chih-Lung; Wang, Wei-Hung; Pan, Ming-Kai; Chen, Wen-Pin; Huang, Wen-Yen; Xu, Zijian; Huang, Hai-En; Chen, Ting; Plikus, Maksim V; Chen, Shih-Kuo; Lin, Sung-Jan

2018-06-29

Changes in external light patterns can alter cell activities in peripheral tissues through slow entrainment of the central clock in suprachiasmatic nucleus (SCN). It remains unclear whether cells in otherwise photo-insensitive tissues can achieve rapid responses to changes in external light. Here we show that light stimulation of animals' eyes results in rapid activation of hair follicle stem cells with prominent hair regeneration. Mechanistically, light signals are interpreted by M1-type intrinsically photosensitive retinal ganglion cells (ipRGCs), which signal to the SCN via melanopsin. Subsequently, efferent sympathetic nerves are immediately activated. Increased norepinephrine release in skin promotes hedgehog signaling to activate hair follicle stem cells. Thus, external light can directly regulate tissue stem cells via an ipRGC-SCN autonomic nervous system circuit. Since activation of sympathetic nerves is not limited to skin, this circuit can also facilitate rapid adaptive responses to external light in other homeostatic tissues.

Cigarette Smoke–Induced CXCR3 Receptor Up-Regulation Mediates Endothelial Apoptosis

PubMed Central

Green, Linden A.; Petrusca, Daniela; Rajashekhar, Gangaraju; Gianaris, Tom; Schweitzer, Kelly S.; Wang, Liang; Justice, Matthew J.; Petrache, Irina

2012-01-01

Endothelial monocyte–activating polypeptide II (EMAP II) and interferon-inducible protein (IP)–10 are proinflammatory mediators, which in addition to their chemokine activities, selectively induce apoptosis in endothelial cells and are up-regulated in the lungs of cigarette smoke–exposed humans. Previously, we showed that EMAP II is an essential mediator of cigarette smoke–induced lung emphysema in mice linking endothelial cell apoptosis with inflammation. Here we addressed the role of the CXCR3 receptor in EMAP II–induced and IP-10–induced apoptosis in endothelial cells and its regulation by cigarette smoke. We found that both neutralizing antibodies and small inhibitory RNA to CXCR3 abrogated EMAP II–induced and IP-10–induced endothelial caspase-3 activation and DNA fragmentation. CXCR3 receptor surface expression in human lung microvascular endothelial cells and in lung tissue endothelium was up-regulated by exposure to cigarette smoke. In tissue culture conditions, EMAP II–induced and IP-10–induced apoptosis was enhanced by preincubation with cigarette smoke extract. Interestingly, serum starvation also induced CXCR3 up-regulation and enhanced EMAP II–induced endothelial apoptosis. Signal transduction via p38 mitogen-activated protein kinase activation was essential for CXCR3-induced cell death, but not for CXCR3 receptor up-regulation by cigarette smoke. In turn, protein nitration was required for CXCR3 receptor up-regulation by cigarette smoke and consequently for subsequent CXCR3-induced cell death. In conclusion, the concerted up-regulation of proinflammatory EMAP II, IP-10, and CXCR3 by cigarette smoke could sustain a cascade of cell death that may promote the alveolar tissue loss noted in human emphysema. PMID:22936405

Discrimination Between Human Leukocyte Antigen Class I-Bound and Co-Purified HIV-Derived Peptides in Immunopeptidomics Workflows

PubMed Central

Partridge, Thomas; Nicastri, Annalisa; Kliszczak, Anna E.; Yindom, Louis-Marie; Kessler, Benedikt M.; Ternette, Nicola; Borrow, Persephone

2018-01-01

Elucidation of novel peptides presented by human leukocyte antigen (HLA) class I alleles by immunopeptidomics constitutes a powerful approach that can inform the rational design of CD8+ T cell inducing vaccines to control infection with pathogens such as human immunodeficiency virus type 1 (HIV-1) or to combat tumors. Recent advances in the sensitivity of liquid chromatography tandem mass spectrometry instrumentation have facilitated the discovery of thousands of natural HLA-restricted peptides in a single measurement. However, the extent of contamination of class I-bound peptides identified using HLA immunoprecipitation (IP)-based immunopeptidomics approaches with peptides from other sources has not previously been evaluated in depth. Here, we investigated the specificity of the IP-based immunopeptidomics methodology using HLA class I- or II-deficient cell lines and membrane protein-specific antibody IPs. We demonstrate that the 721.221 B lymphoblastoid cell line, widely regarded to be HLA class Ia-deficient, actually expresses and presents peptides on HLA-C*01:02. Using this cell line and the C8166 (HLA class I- and II-expressing) cell line, we show that some HLA class II-bound peptides were co-purified non-specifically during HLA class I and membrane protein IPs. Furthermore, IPs of “irrelevant” membrane proteins from HIV-1-infected HLA class I- and/or II-expressing cells revealed that unusually long HIV-1-derived peptides previously reported by us and other immunopeptidomics studies as potentially novel CD8+ T cell epitopes were non-specifically co-isolated, and so constitute a source of contamination in HLA class I IPs. For example, a 16-mer (FLGKIWPSYKGRPGNF), which was detected in all samples studied represents the full p1 segment of the abundant intracellular or virion-associated proteolytically-processed HIV-1 Gag protein. This result is of importance, as these long co-purified HIV-1 Gag peptides may not elicit CD8+ T cell responses when incorporated into candidate vaccines. These results have wider implications for HLA epitope discovery from abundant or membrane-associated antigens by immunopeptidomics in the context of infectious diseases, cancer, and autoimmunity. PMID:29780384

Induced Pluripotent Stem Cells from Nonhuman Primates.

PubMed

Mishra, Anuja; Qiu, Zhifang; Farnsworth, Steven L; Hemmi, Jacob J; Li, Miao; Pickering, Alexander V; Hornsby, Peter J

2016-01-01

Induced pluripotent stem cells from nonhuman primates (NHPs) have unique roles in cell biology and regenerative medicine. Because of the relatedness of NHPs to humans, NHP iPS cells can serve as a source of differentiated derivatives that can be used to address important questions in the comparative biology of primates. Additionally, when used as a source of cells for regenerative medicine, NHP iPS cells serve an invaluable role in translational experiments in cell therapy. Reprogramming of NHP somatic cells requires the same conditions as previously established for human cells. However, throughout the process, a variety of modifications to the human cell protocols must be made to accommodate significant species differences.

Residues within the myristoylation motif determine intracellular targeting of the neuronal Ca2+ sensor protein KChIP1 to post-ER transport vesicles and traffic of Kv4 K+ channels.

PubMed

O'Callaghan, Dermott W; Hasdemir, Burcu; Leighton, Mark; Burgoyne, Robert D

2003-12-01

KChIPs (K+ channel interacting proteins) regulate the function of A-type Kv4 potassium channels by modifying channel properties and by increasing their cell surface expression. We have explored factors affecting the localisation of Kv4.2 and the targeting of KChIP1 and other NCS proteins by using GFP-variant fusion proteins expressed in HeLa cells. ECFP-Kv4.2 expressed alone was not retained in the ER but reached the Golgi complex. In cells co-expressing ECFP-Kv4.2 and KChIP1-EYFP, the two proteins were co-localised and were mainly present on the plasma membrane. When KChIP1-EYFP was expressed alone it was instead targeted to punctate structures. This was distinct from the localisation of the NCS proteins NCS-1 and hippocalcin, which were targeted to the trans-Golgi network (TGN) and plasma membrane. The membrane localisation of each NCS protein required myristoylation and minimal myristoylation motifs of hippocalcin or KChIP1 were sufficient to target fusion proteins to either TGN/plasma membrane or to punctate structures. The existence of targeting information within the N-terminal motifs was confirmed by mutagenesis of residues corresponding to three conserved basic amino acids in hippocalcin and NCS-1 at positions 3, 7 and 9. Residues at these positions determined intracellular targeting to the different organelles. Myristoylation and correct targeting of KChIP1 was required for the efficient traffic of ECFP-Kv4.2 to the plasma membrane. Expression of KChIP1(1-11)-EYFP resulted in the formation of enlarged structures that were positive for ERGIC-53 and beta-COP. ECFP-Kv4.2 was also accumulated in these structures suggesting that KChIP1(1-11)-EYFP inhibited traffic out of the ERGIC. We suggest that KChIP1 is targeted by its myristoylation motif to post-ER transport vesicles where it could interact with and regulate the traffic of Kv4 channels to the plasma membrane under the influence of localised Ca2+ signals.

IP/sub 3/ stimulates CA/sup + +/ efflux from fusogenic carrot protoplasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rincon, M.; Boss, W.F.

1986-04-01

Polyphosphoinositide breakdown plays an important role in signal transduction in animal cells (Berridge and Irvine, 1984, Nature, 312:315). Upon stimulation, phospholipase C hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP/sub 3/) and diacylglycerol both of which act as cellular second messengers. IP/sub 3/ mobilizes Ca/sup + +/ from internal stores, hence the cytosolic free Ca/sup + +/ concentration increases and those physiological activities regulated by Ca/sup + +/ are stimulated. To test if plant cells also responded to IP/sub 3/, Ca/sup + +/ efflux studies were done with fusogenic carrot protoplasts released in EGTA. The protoplasts were preloaded with /sup 45/Ca/supmore » + +/ placed in a Ca/sup + +/-free medium, and efflux determined as /sup 45/Ca/sup + +/ loss from the protoplasts. IP/sub 3/ (10-20..mu..M) caused enhanced /sup 45/Ca/sup + +/ efflux and the response was sustained for at least 15 min. In plants, as in animals, the observed IP/sub 3/-enhanced /sup 45/Ca/sup + +/ efflux suggested that IP/sub 3/ released Ca/sup + +/ from internal stores, and the increased free cytosolic Ca/sup + +/ activated Ca/sup + +/ pumping mechanisms which restored the Ca/sup + +/ concentration in the cytosol to the normal level.« less

Molecule-Specific Imaging Analysis of Carcinogens in Breast Cancer Cells Using Time-of-Flight Secondary Ion Mass Spectrometry

DOE Office of Scientific and Technical Information (OSTI.GOV)

Quong, J N; Knize, M G; Kulp, K S

2003-08-19

Imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) is used to study the localization of heterocyclic amines in MCF7 line of human breast cancer cells. The detection sensitivities of a model rodent mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were determined. Following an established criteria for the determination of status of freeze-fracture cells, the distribution of PhIP in the MCF7 cells are reported.

Enabling IP Header Compression in COTS Routers via Frame Relay on a Simplex Link

NASA Technical Reports Server (NTRS)

Nguyen, Sam P.; Pang, Jackson; Clare, Loren P.; Cheng, Michael K.

2010-01-01

NASA is moving toward a networkcentric communications architecture and, in particular, is building toward use of Internet Protocol (IP) in space. The use of IP is motivated by its ubiquitous application in many communications networks and in available commercial off-the-shelf (COTS) technology. The Constellation Program intends to fit two or more voice (over IP) channels on both the forward link to, and the return link from, the Orion Crew Exploration Vehicle (CEV) during all mission phases. Efficient bandwidth utilization of the links is key for voice applications. In Voice over IP (VoIP), the IP packets are limited to small sizes to keep voice latency at a minimum. The common voice codec used in VoIP is G.729. This new algorithm produces voice audio at 8 kbps and in packets of 10-milliseconds duration. Constellation has designed the VoIP communications stack to use the combination of IP/UDP/RTP protocols where IP carries a 20-byte header, UDP (User Datagram Protocol) carries an 8-byte header, and RTP (Real Time Transport Protocol) carries a 12-byte header. The protocol headers total 40 bytes and are equal in length to a 40-byte G.729 payload, doubling the VoIP latency. Since much of the IP/UDP/RTP header information does not change from IP packet to IP packet, IP/UDP/RTP header compression can avoid transmission of much redundant data as well as reduce VoIP latency. The benefits of IP header compression are more pronounced at low data rate links such as the forward and return links during CEV launch. IP/UDP/RTP header compression codecs are well supported by many COTS routers. A common interface to the COTS routers is through frame relay. However, enabling IP header compression over frame relay, according to industry standard (Frame Relay IP Header Compression Agreement FRF.20), requires a duplex link and negotiations between the compressor router and the decompressor router. In Constellation, each forward to and return link from the CEV in space is treated independently as a simplex link. Without negotiation, the COTS routers are prevented from entering into the IP header compression mode, and no IP header compression would be performed. An algorithm is proposed to enable IP header compression in COTS routers on a simplex link with no negotiation or with a one-way messaging. In doing so, COTS routers can enter IP header compression mode without the need to handshake through a bidirectional link as required by FRF.20. This technique would spoof the routers locally and thereby allow the routers to enter into IP header compression mode without having the negotiations between routers actually occur. The spoofing function is conducted by a frame relay adapter (also COTS) with the capability to generate control messages according to the FRF.20 descriptions. Therefore, negotiation is actually performed between the FRF.20 adapter and the connecting COTS router locally and never occurs over the space link. Through understanding of the handshaking protocol described by FRF.20, the necessary FRF.20 negotiations messages can be generated to control the connecting router, not only to turn on IP header compression but also to adjust the compression parameters. The FRF.20 negotiation (or control) message is composed in the FRF.20 adapter by interpreting the incoming router request message. Many of the fields are simply transcribed from request to response while the control field indicating response and type are modified.

The Science and Ethics of Induced Pluripotency: What Will Become of Embryonic Stem Cells?

PubMed Central

Zacharias, David G.; Nelson, Timothy J.; Mueller, Paul S.; Hook, C. Christopher

2011-01-01

For over a decade, the field of stem cell research has advanced tremendously and gained new attention in light of novel insights and emerging developments for regenerative medicine. Invariably, multiple considerations come into play, and clinicians and researchers must weigh the benefits of certain stem cell platforms against the costs they incur. Notably, human embryonic stem (hES) cell research has been a source of continued debate, leading to differing policies and regulations worldwide. This article briefly reviews current stem cell platforms, looking specifically at the two existing pluripotent lines available for potential therapeutic applications: hES cells and induced pluripotent stem (iPS) cells. We submit iPS technology as a viable and possibly superior alternative for future medical and research endeavors as it obviates many ethical and resource-related concerns posed by hES cells while prospectively matching their potential for scientific use. However, while the clinical realities of iPS cells appear promising, we must recognize the current limitations of this technology, avoid hype, and articulate ethically acceptable medical and scientific goals. PMID:21719620

In vitro modelling of familial amyloidotic polyneuropathy allows quantitative detection of transthyretin amyloid fibril-like structures in hepatic derivatives of patient-specific induced pluripotent stem cells.

PubMed

Hoepfner, Jeannine; Kleinsorge, Mandy; Papp, Oliver; Alfken, Susanne; Heiringhoff, Robin; Pich, Andreas; Sauer, Vanessa; Zibert, Andree; Göhring, Gudrun; Schmidt, Hartmut; Sgodda, Malte; Cantz, Tobias

2017-07-26

The transthyretin protein is thermodynamically destabilised by mutations in the transthyretin gene, promoting the formation of amyloid fibrils in various tissues. Consequently, impaired autonomic organ function is observed in patients suffering from transthyretin-related familial amyloidotic polyneuropathy (FAP). The influence of individual genetic backgrounds on fibril formation as a potential cause of genotype-phenotype variations needs to be investigated in order to ensure efficient patient-specific therapies. We reprogrammed FAP patient fibroblasts to induced pluripotent stem (iPS) cells and differentiated these cells into transthyretin-expressing hepatocyte-like cells (HLCs). HLCs differentiated from FAP iPS cells and healthy control iPS cells secreted the transthyretin protein in similar concentrations. Mass spectrometry revealed the presence of mutant transthyretin protein in FAP HLC supernatants. In comparison to healthy control iPS cells, we demonstrated the formation of transthyretin amyloid fibril-like structures in FAP HLC supernatants using the amyloid-specific dyes Congo red and thioflavin T. These dyes were also applicable for the quantitative determination of in vitro formed transthyretin fibril-like structures. Moreover, we confirmed the inhibition of fibril formation by the TTR kinetic stabiliser diclofenac. Thioflavin T fluorescence intensity measurements even allowed the quantification of amyloid fibril-like structures in 96-well plate formats as a prerequisite for patient-specific drug screening approaches.

Efficient programming of human eye conjunctiva-derived induced pluripotent stem (ECiPS) cells into definitive endoderm-like cells.

PubMed

Massumi, Mohammad; Hoveizi, Elham; Baktash, Parvaneh; Hooti, Abdollah; Ghazizadeh, Leili; Nadri, Samad; Pourasgari, Farzaneh; Hajarizadeh, Athena; Soleimani, Masoud; Nabiuni, Mohammad; Khorramizadeh, Mohammad R

2014-03-10

Due to pluripotency of induced pluripotent stem (iPS) cells, and the lack of immunological incompatibility and ethical issues, iPS cells have been considered as an invaluable cell source for future cell replacement therapy. This study was aimed first at establishment of novel iPS cells, ECiPS, which directly reprogrammed from human Eye Conjunctiva-derived Mesenchymal Stem Cells (EC-MSCs); second, comparing the inductive effects of Wnt3a/Activin A biomolecules to IDE1 small molecule in derivation of definitive endoderm (DE) from the ECiPS cells. To that end, first, the EC-MSCs were transduced by SOKM-expressing lentiviruses and characterized for endogenous expression of embryonic markers Then the established ECiPS cells were induced to DE formation by Wnt3a/Activin A or IDE1. Quantification of GSC, Sox17 and Foxa2 expression, as DE-specific markers, in both mRNA and protein levels revealed that induction of ECiPS cells by either Wnt3a/Activin A or IDE1 could enhance the expression level of the genes; however the levels of increase were higher in Wnt3a/Activin A induced ECiPS-EBs than IDE1 induced cells. Furthermore, the flow cytometry analyses showed no synergistic effect between Activin A and Wnt3a to derive DE-like cells from ECiPS cells. The comparative findings suggest that although both Wnt3a/Activin A signaling and IDE1 molecule could be used for differentiation of iPS into DE cells, the DE-inducing effect of Wnt3a/Activin A was statistically higher than IDE1. Copyright © 2014 Elsevier Inc. All rights reserved.

Soy Biodiesel and Petrodiesel Emissions Differ in Size, Chemical Composition and Stimulation of Inflammatory Responses in Cells and Animals

PubMed Central

Fukagawa, Naomi K.; Li, Muyao; Poynter, Matthew E.; Palmer, Brian C.; Parker, Erin; Kasumba, John; Holmén, Britt A.

2013-01-01

Debate about the biological effects of biodiesel exhaust emissions exists due to variation in methods of exhaust generation and biological models used to assess responses. Because studies in cells do not necessarily reflect the integrated response of a whole animal, experiments were conducted in two human cell lines representing bronchial epithelial cells and macrophages and female mice using identical particle suspensions of raw exhaust generated by a Volkswagen light-duty diesel engine using petrodiesel (B0) and a biodiesel blend (B20: 20% soy biodiesel/80% B0 by volume). Tailpipe particle emissions measurement showed B0 generated two times more particle mass, larger ultrafine particle number distribution modes, and particles of more nonpolar organic composition than the B20 fuel. Biological assays (inflammatory mediators, oxidative stress biomarkers) demonstrated that particulate matter (PM) generated by combustion of the two fuels induced different responses in in vitro and in vivo models. Concentrations of inflammatory mediators (Interleukin-6, IL-6; Interferon-gamma-induced Protein 10, IP-10; Granulocyte-stimulating factor, G-CSF) in the medium of B20-treated cells and in bronchoalveolar lavage fluid of mice exposed to B20 were ~20–30% higher than control or B0 PM, suggesting that addition of biodiesel to diesel fuels will reduce PM emissions but not necessarily adverse health outcomes. PMID:24053625

Detailed study of spontaneous rotation generation in diverted H-mode plasma using the full-f gyrokinetic code XGC1

NASA Astrophysics Data System (ADS)

Seo, Janghoon; Chang, C. S.; Ku, S.; Kwon, J. M.; Yoon, E. S.

2013-10-01

The Full-f gyrokinetic code XGC1 is used to study the details of toroidal momentum generation in H-mode plasma. Diverted DIII-D geometry is used, with Monte Carlo neutral particles that are recycled at the limiter wall. Nonlinear Coulomb collisions conserve particle, momentum, and energy. Gyrokinetic ions and adiabatic electrons are used in the present simulation to include the effects from ion gyrokinetic turbulence and neoclassical physics, under self-consistent radial electric field generation. Ion orbit loss physics is automatically included. Simulations show a strong co-Ip flow in the H-mode layer at outside midplane, similarly to the experimental observation from DIII-D and ASDEX-U. The co-Ip flow in the edge propagates inward into core. It is found that the strong co-Ip flow generation is mostly from neoclassical physics. On the other hand, the inward momentum transport is from turbulence physics, consistently with the theory of residual stress from symmetry breaking. Therefore, interaction between the neoclassical and turbulence physics is a key factor in the spontaneous momentum generation.

Melanopsin tristability for sustained and broadband phototransduction.

PubMed

Emanuel, Alan Joseph; Do, Michael Tri Hoang

2015-03-04

Mammals rely upon three ocular photoreceptors to sense light: rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs). Rods and cones resolve details in the visual scene. Conversely, ipRGCs integrate over time and space, primarily to support "non-image" vision. The integrative mechanisms of ipRGCs are enigmatic, particularly since these cells use a phototransduction motif that allows invertebrates like Drosophila to parse light with exceptional temporal resolution. Here, we provide evidence for a single mechanism that allows ipRGCs to integrate over both time and wavelength. Light distributes the visual pigment, melanopsin, across three states, two silent and one signaling. Photoequilibration among states maintains pigment availability for sustained signaling, stability of the signaling state permits minutes-long temporal summation, and modest spectral separation of the silent states promotes uniform activation across wavelengths. By broadening the tuning of ipRGCs in both temporal and chromatic domains, melanopsin tristability produces signal integration for physiology and behavior. Copyright © 2015 Elsevier Inc. All rights reserved.

Audio CAPTCHA for SIP-Based VoIP

NASA Astrophysics Data System (ADS)

Soupionis, Yannis; Tountas, George; Gritzalis, Dimitris

Voice over IP (VoIP) introduces new ways of communication, while utilizing existing data networks to provide inexpensive voice communications worldwide as a promising alternative to the traditional PSTN telephony. SPam over Internet Telephony (SPIT) is one potential source of future annoyance in VoIP. A common way to launch a SPIT attack is the use of an automated procedure (bot), which generates calls and produces audio advertisements. In this paper, our goal is to design appropriate CAPTCHA to fight such bots. We focus on and develop audio CAPTCHA, as the audio format is more suitable for VoIP environments and we implement it in a SIP-based VoIP environment. Furthermore, we suggest and evaluate the specific attributes that audio CAPTCHA should incorporate in order to be effective, and test it against an open source bot implementation.

Airpower In The Information Age: Embracing TCP/IP Within Airborne Networks

DTIC Science & Technology

2015-04-01

proved successful at seamlessly connecting large numbers of users to a vast array of applications. Airborne TCP/IP communication systems within DoD are...behind the development of next generation weapons systems. Admiral Fitzgerald recommends that the network architecture be developed and the...environment makes traditional ISR much more difficult and the military will need to rely on fifth- generation platforms like the F-35 that are able to

ABCG2/BCRP decreases the transfer of a food-born chemical carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in perfused term human placenta.

PubMed

Myllynen, Päivi; Kummu, Maria; Kangas, Tiina; Ilves, Mika; Immonen, Elina; Rysä, Jaana; Pirilä, Rauna; Lastumäki, Anni; Vähäkangas, Kirsi H

2008-10-15

We have studied the role of ATP binding cassette (ABC) transporters in fetal exposure to carcinogens using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) a known substrate for ABC transporters as a model compound. In perfusion of human term placenta, transfer of (14)C-PhIP (2 microM) through the placenta resulted in fetal-to-maternal concentration ratio (FM ratio) of 0.72+/-0.09 at 6 h. The specific ABCG2 inhibitor KO143 increased the transfer of (14)C-PhIP from maternal to fetal circulation (FM ratio 0.90+/-0.08 at 6 h, p<0.05) while the ABCC1/ABCC2 inhibitor probenecid had no effect (FM ratio at 6 h 0.75+/-0.10, p=0.84). There was a negative correlation between the expression of ABCG2 protein in perfused tissue and the FM ratio of (14)C-PhIP (R=-0.81, p<0.01) at the end of the perfusion. The expression of ABCC2 protein did not correlate with FM ratio of PhIP (R: -0.11, p=0.76). In addition, PhIP induced the expression of ABC transporters in BeWo cells at mRNA level. In conclusion, our data indicates that ABCG2 decreases placental transfer of (14)C-PhIP in perfused human placenta. Also, PhIP may modify ABC transporter expression in choriocarcinoma cells.

ABCG2/BCRP decreases the transfer of a food-born chemical carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in perfused term human placenta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Myllynen, Paeivi; Kummu, Maria; Kangas, Tiina

2008-10-15

We have studied the role of ATP binding cassette (ABC) transporters in fetal exposure to carcinogens using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) a known substrate for ABC transporters as a model compound. In perfusion of human term placenta, transfer of {sup 14}C-PhIP (2 {mu}M) through the placenta resulted in fetal-to-maternal concentration ratio (FM ratio) of 0.72 {+-} 0.09 at 6 h. The specific ABCG2 inhibitor KO143 increased the transfer of {sup 14}C-PhIP from maternal to fetal circulation (FM ratio 0.90 {+-} 0.08 at 6 h, p < 0.05) while the ABCC1/ABCC2 inhibitor probenecid had no effect (FM ratio at 6 h 0.75 {+-}more » 0.10, p = 0.84). There was a negative correlation between the expression of ABCG2 protein in perfused tissue and the FM ratio of {sup 14}C-PhIP (R = - 0.81, p < 0.01) at the end of the perfusion. The expression of ABCC2 protein did not correlate with FM ratio of PhIP (R: - 0.11, p = 0.76). In addition, PhIP induced the expression of ABC transporters in BeWo cells at mRNA level. In conclusion, our data indicates that ABCG2 decreases placental transfer of {sup 14}C-PhIP in perfused human placenta. Also, PhIP may modify ABC transporter expression in choriocarinoma cells.« less

Transfer Error and Correction Approach in Mobile Network

NASA Astrophysics Data System (ADS)

Xiao-kai, Wu; Yong-jin, Shi; Da-jin, Chen; Bing-he, Ma; Qi-li, Zhou

With the development of information technology and social progress, human demand for information has become increasingly diverse, wherever and whenever people want to be able to easily, quickly and flexibly via voice, data, images and video and other means to communicate. Visual information to the people direct and vivid image, image / video transmission also been widespread attention. Although the third generation mobile communication systems and the emergence and rapid development of IP networks, making video communications is becoming the main business of the wireless communications, however, the actual wireless and IP channel will lead to error generation, such as: wireless channel multi- fading channels generated error and blocking IP packet loss and so on. Due to channel bandwidth limitations, the video communication compression coding of data is often beyond the data, and compress data after the error is very sensitive to error conditions caused a serious decline in image quality.

Mutation and repair induced by the carcinogen 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) in the dihydrofolate reductase gene of Chinese hamster ovary cells and conformational modeling of the dG-C8-PhIP adduct in DNA.

PubMed

Carothers, A M; Yuan, W; Hingerty, B E; Broyde, S; Grunberger, D; Snyderwine, E G

1994-01-01

Three experiments using 20 microM 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) were performed to induce mutations in the dihydrofolate reductase (DHFR) gene of a hemizygous Chinese hamster ovary (CHO) cell line (UA21). Metabolized forms of this chemical primarily bind at the C-8 position of guanine in DNA. In total, 21 independent induced mutants were isolated and 20 were characterized. DNA sequencing showed that the preferred mutation type found in 75% of the induced DHFR- clones was G.C-->T.A single and tandem double transversions. In addition to base substitutions, one mutant carried a-1 frameshift and another one had lost the entire locus by deletion. The induced changes affected purine targets on the nontranscribed strand of the gene in nearly all of the mutants sequenced (18/19). At the time that the first two experiments were performed, the initial adduct levels were quantitated in treated cells at the mutagenic dose by 32P-postlabeling. While the induced frequency of mutation was relatively low (approximately 5 x 10(-6), the adduct levels after a 1-h exposure of UA21 cells to 20 microM N-OH-PhIP were relatively high (13 adducts x 10(-6) nucleotides). This latter method was then employed to learn if the induced mutation frequency correlated with rapid overall genome repair of PhIP-DNA adducts. Total adduct levels, determined using DNA samples from treated cells collected after intervals of time, were reduced by about 50% after 6 h, and about 70% after 24 h. Since overall genome repair in CHO cells is relatively slow compared with preferential gene repair, the removal of dG-C8-PhIP adducts was apparently efficient. In order to better understand the mutational and repair results, we performed computational modeling to determine the lowest energy structure for the major dG-C8-PhIP adduct in a repetitively mutated duplex sequence opposite dA. Results of this analysis indicate that the PhIP-modified base resembles previous structural determinations of (deoxyguanosin-8-yl)-aminofluorene; the carcinogen is in the B-DNA minor groove and its adopts a syn conformation mispaired with an anti A. The implications of this conformational distortion in DNA structure for damage recognition by cellular repair enzymes are discussed.

Ischemic preconditioning modulates the expression of several genes, leading to the overproduction of IL-1Ra, iNOS, and Bcl-2 in a human model of liver ischemia-reperfusion.

PubMed

Barrier, Alain; Olaya, Natalia; Chiappini, Franck; Roser, François; Scatton, Olivier; Artus, Cédric; Franc, Brigitte; Dudoit, Sandrine; Flahault, Antoine; Debuire, Brigitte; Azoulay, Daniel; Lemoine, Antoinette

2005-10-01

Ischemia triggers an inflammatory response that precipitates cell death during reperfusion. Several studies have shown that tissues are protected by ischemic preconditioning (IP) consisting of 10 min of ischemia followed by 10 min of reperfusion just before ischemia. The molecular basis of this protective effect is poorly understood. We used cDNA arrays (20K) to compare global gene expression in liver biopsies from living human liver donors who underwent IP (n=7) or not (n=7) just before liver devascularization. Microarray data were analyzed using pairedt test with a type I error rate fixed at alpha = 2.5 10(6) (Bonferroni correction). We found that 60 genes were differentially expressed (36 over- and 24 underexpressed in preconditioning group). After IP, the most significantly overexpressed gene was IL-1Ra. This was confirmed by immunoblotting. Differentially expressed were genes involved in apoptosis (NOD2, ephrin-A1, and calpain) and in the carbohydrate metabolism. A significant increase in the amount of the anti-apoptotic protein Bcl-2 in preconditioned livers but no change in the cleavage of procaspase-3, -8, and -9 was observed. We also observed an increase in the amount in the inducible nitric oxide synthase. Therefore, the benefits of IP may be associated with the overproduction of IL-1Ra, Bcl-2, and NO countering the proinflammatory and proapoptotic effects generated during ischemia-reperfusion.

SraTailor: graphical user interface software for processing and visualizing ChIP-seq data.

PubMed

Oki, Shinya; Maehara, Kazumitsu; Ohkawa, Yasuyuki; Meno, Chikara

2014-12-01

Raw data from ChIP-seq (chromatin immunoprecipitation combined with massively parallel DNA sequencing) experiments are deposited in public databases as SRAs (Sequence Read Archives) that are publically available to all researchers. However, to graphically visualize ChIP-seq data of interest, the corresponding SRAs must be downloaded and converted into BigWig format, a process that involves complicated command-line processing. This task requires users to possess skill with script languages and sequence data processing, a requirement that prevents a wide range of biologists from exploiting SRAs. To address these challenges, we developed SraTailor, a GUI (Graphical User Interface) software package that automatically converts an SRA into a BigWig-formatted file. Simplicity of use is one of the most notable features of SraTailor: entering an accession number of an SRA and clicking the mouse are the only steps required to obtain BigWig-formatted files and to graphically visualize the extents of reads at given loci. SraTailor is also able to make peak calls, generate files of other formats, process users' own data, and accept various command-line-like options. Therefore, this software makes ChIP-seq data fully exploitable by a wide range of biologists. SraTailor is freely available at http://www.devbio.med.kyushu-u.ac.jp/sra_tailor/, and runs on both Mac and Windows machines. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

Cryopreservation of Human Stem Cells for Clinical Application: A Review

PubMed Central

Hunt, Charles J.

2011-01-01

Summary Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell. PMID:21566712

Cryopreservation of Human Stem Cells for Clinical Application: A Review.

PubMed

Hunt, Charles J

2011-01-01

SUMMARY: Stem cells have been used in a clinical setting for many years. Haematopoietic stem cells have been used for the treatment of both haematological and non-haematological disease; while more recently mesenchymal stem cells derived from bone marrow have been the subject of both laboratory and early clinical studies. Whilst these cells show both multipotency and expansion potential, they nonetheless do not form stable cell lines in culture which is likely to limit the breadth of their application in the field of regenerative medicine. Human embryonic stem cells are pluripotent cells, capable of forming stable cell lines which retain the capacity to differentiate into cells from all three germ layers. This makes them of special significance in both regenerative medicine and toxicology. Induced pluripotent stem (iPS) cells may also provide a similar breadth of utility without some of the confounding ethical issues surrounding embryonic stem cells. An essential pre-requisite to the commercial and clinical application of stem cells are suitable cryopreservation protocols for long-term storage. Whilst effective methods for cryopreservation and storage have been developed for haematopoietic and mesenchymal stem cells, embryonic cells and iPS cells have proved more refractory. This paper reviews the current state of cryopreservation as it pertains to stem cells and in particular the embryonic and iPS cell.

Machine Learning Approach to Automated Quality Identification of Human Induced Pluripotent Stem Cell Colony Images.

PubMed

Joutsijoki, Henry; Haponen, Markus; Rasku, Jyrki; Aalto-Setälä, Katriina; Juhola, Martti

2016-01-01

The focus of this research is on automated identification of the quality of human induced pluripotent stem cell (iPSC) colony images. iPS cell technology is a contemporary method by which the patient's cells are reprogrammed back to stem cells and are differentiated to any cell type wanted. iPS cell technology will be used in future to patient specific drug screening, disease modeling, and tissue repairing, for instance. However, there are technical challenges before iPS cell technology can be used in practice and one of them is quality control of growing iPSC colonies which is currently done manually but is unfeasible solution in large-scale cultures. The monitoring problem returns to image analysis and classification problem. In this paper, we tackle this problem using machine learning methods such as multiclass Support Vector Machines and several baseline methods together with Scaled Invariant Feature Transformation based features. We perform over 80 test arrangements and do a thorough parameter value search. The best accuracy (62.4%) for classification was obtained by using a k-NN classifier showing improved accuracy compared to earlier studies.

Polymyxin B Nephrotoxicity: From Organ to Cell Damage

PubMed Central

Pessoa, Edson Andrade

2016-01-01

Polymyxins have a long history of dose-limiting toxicity, but the underlying mechanism of polymyxin B-induced nephrotoxicity is unclear. This study investigated the link between the nephrotoxic effects of polymyxin B on renal metabolic functions and mitochondrial morphology in rats and on the structural integrity of LLC-PK1 cells. Fifteen Wistar rats were divided into two groups: Saline group, rats received 3 mL/kg of 0.9% NaCl intraperitoneally (i.p.) once a day for 5 days; Polymyxin B group, rats received 4 mg/kg/day of polymyxin B i.p. once a day for 5 days. Renal function, renal hemodynamics, oxidative stress, mitochondrial injury and histological characteristics were assessed. Cell membrane damage was evaluated via lactate dehydrogenase and nitric oxide levels, cell viability, and apoptosis in cells exposed to 12.5 μM, 75 μM and 375 μM polymyxin B. Polymyxin B was immunolocated using Lissamine rhodamine-polymyxin B in LLC-PK1 cells. Polymyxin B administration in rats reduced creatinine clearance and increased renal vascular resistance and oxidative damage. Mitochondrial damage was confirmed by electron microscopy and cytosolic localization of cytochrome c. Histological analysis revealed tubular dilatation and necrosis in the renal cortex. The reduction in cell viability and the increase in apoptosis, lactate dehydrogenase levels and nitric oxide levels confirmed the cytotoxicity of polymyxin B. The incubation of LLC-PK1 cells resulted in mitochondrial localization of polymyxin B. This study demonstrates that polymyxin B nephrotoxicity is characterized by mitochondrial dysfunction and free radical generation in both LLC-PK1 cells and rat kidneys. These data also provide support for clinical studies on the side effects of polymyxin B. PMID:27532263

Lymphoid follicle cells in chronic obstructive pulmonary disease overexpress the chemokine receptor CXCR3.

PubMed

Kelsen, Steven G; Aksoy, Mark O; Georgy, Mary; Hershman, Richard; Ji, Rong; Li, Xiuxia; Hurford, Matthew; Solomides, Charalambos; Chatila, Wissam; Kim, Victor

2009-05-01

The mechanisms underlying formation of lung lymphoid follicles (LF) in chronic obstructive pulmonary disease (COPD) are unknown. The chemokine receptor CXCR3 regulates immune responses in secondary lymphoid structures elsewhere in the body and is highly expressed by Th1 lymphocytes in the airway in COPD. Because chemokine receptors control inflammatory cell homing to inflamed tissue, we reasoned that CXCR3 may contribute to LF formation in COPD. We assessed the expression of CXCR3 and its ligands (IP-10/CXCL10, Mig/CXCL9, and ITAC/CXCL11) by LF cells in never-smokers, smokers without COPD, and subjects with COPD. CXCR3, IP-10, Mig, and ITAC expression were assessed in lung sections from 46 subjects (never-smokers, smokers without COPD [S], and subjects with COPD in GOLD stages 1-4) by immunohistochemistry. CXCR3-expressing T cells (CD8+ or CD4+) and B cells (CD20+) were topographically distributed at the follicle periphery and center, respectively. The percentage of immunohistochemically identified CXCR3+ cells increased progressively while proceeding from S through GOLD 3-4 (P < 0.01 for GOLD 3-4 vs. S). Moreover, the number of CXCR3+ follicular cells correlated inversely with FEV(1) (r = 0.60). The CXCR3 ligands IP-10 and Mig were expressed by several cell types in and around the follicle, including CD68+ dendritic cells/ macrophages, airway epithelial cells, endothelial cells, and T and B cells. These results suggest that LF form in the COPD lung by recruitment and/or retention of CXCR3-expressing T and B lymphocytes, which are attracted to the region through production of CXCR3 ligands IP-10 and Mig by lung structural and follicular cells.

Lymphoid Follicle Cells in Chronic Obstructive Pulmonary Disease Overexpress the Chemokine Receptor CXCR3

PubMed Central

Kelsen, Steven G.; Aksoy, Mark O.; Georgy, Mary; Hershman, Richard; Ji, Rong; Li, XiuXia; Hurford, Matthew; Solomides, Charalambos; Chatila, Wissam; Kim, Victor

2009-01-01

Rationale: The mechanisms underlying formation of lung lymphoid follicles (LF) in chronic obstructive pulmonary disease (COPD) are unknown. The chemokine receptor CXCR3 regulates immune responses in secondary lymphoid structures elsewhere in the body and is highly expressed by Th1 lymphocytes in the airway in COPD. Because chemokine receptors control inflammatory cell homing to inflamed tissue, we reasoned that CXCR3 may contribute to LF formation in COPD. Objectives: We assessed the expression of CXCR3 and its ligands (IP-10/CXCL10, Mig/CXCL9, and ITAC/CXCL11) by LF cells in never-smokers, smokers without COPD, and subjects with COPD. Methods: CXCR3, IP-10, Mig, and ITAC expression were assessed in lung sections from 46 subjects (never-smokers, smokers without COPD [S], and subjects with COPD in GOLD stages 1–4) by immunohistochemistry. Measurements and Main Results: CXCR3-expressing T cells (CD8+ or CD4+) and B cells (CD20+) were topographically distributed at the follicle periphery and center, respectively. The percentage of immunohistochemically identified CXCR3+ cells increased progressively while proceeding from S through GOLD 3–4 (P < 0.01 for GOLD 3–4 vs. S). Moreover, the number of CXCR3+ follicular cells correlated inversely with FEV1 (r = 0.60). The CXCR3 ligands IP-10 and Mig were expressed by several cell types in and around the follicle, including CD68+ dendritic cells/ macrophages, airway epithelial cells, endothelial cells, and T and B cells. Conclusions: These results suggest that LF form in the COPD lung by recruitment and/or retention of CXCR3-expressing T and B lymphocytes, which are attracted to the region through production of CXCR3 ligands IP-10 and Mig by lung structural and follicular cells. PMID:19218194

Rapid Recycling of Ca2+ between IP3-Sensitive Stores and Lysosomes

PubMed Central

López Sanjurjo, Cristina I.; Tovey, Stephen C.; Taylor, Colin W.

2014-01-01

Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways. PMID:25337829

Rapid recycling of Ca2+ between IP3-sensitive stores and lysosomes.

PubMed

López Sanjurjo, Cristina I; Tovey, Stephen C; Taylor, Colin W

2014-01-01

Inositol 1,4,5-trisphosphate (IP3) evokes release of Ca2+ from the endoplasmic reticulum (ER), but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK) cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH) evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM) to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

CD73-Generated Adenosine Is Critical for Immune Regulation during Toxoplasma gondii Infection

PubMed Central

Mahamed, Deeqa A.; Toussaint, Leon E.

2014-01-01

As an obligate intracellular pathogen, the apicomplexan parasite Toxoplasma gondii evades immune system-mediated clearance by undergoing stage differentiation to persist indefinitely in susceptible hosts. Previously, we found that mice deficient in the ectoenzyme CD73, which generates adenosine in the extracellular matrix, were resistant to chronic toxoplasmosis after oral infection with T. gondii. Resistance in CD73 knockout mice was due to a delay in parasite differentiation in the central nervous system (CNS). To further clarify the role of CD73 and extracellular adenosine in T. gondii pathogenesis, we infected wild-type (WT) and CD73−/− mice with T. gondii cysts systemically by the intraperitoneal (i.p.) route. In contrast to oral infection, i.p. infected CD73−/− mice were highly susceptible to immune-mediated pathology, with significantly increased infiltration of neutrophils and T cells into the peritoneal cavity. Administration of the broad-spectrum adenosine receptor agonist 5′-N-ethylcarboxamidoadenosine (NECA) protected CD73−/− mice against T. gondii-induced immunopathology, suggesting that the absence of CD73-generated adenosine led to the increased susceptibility in these mice. Peritoneal exudate cells from infected CD73−/− mice produced higher levels of the inflammatory mediators nitric oxide, tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β), without enhanced parasite killing or clearance. Bone marrow chimeras established that CD73 expression in both hematopoietic and nonhematopoietic compartments contributes to limiting T. gondii-induced immunopathology. In addition, mice deficient in the adenosine receptor A2A were more susceptible to immunopathology during intraperitoneal infection with T. gondii than WT mice. Thus, extracellular adenosine is a key immune regulator that limits collateral tissue damage due to an intracellular pathogen and promotes host survival. PMID:25452548

Arl6IP1 has the ability to shape the mammalian ER membrane in a reticulon-like fashion.

PubMed

Yamamoto, Yasunori; Yoshida, Asuka; Miyazaki, Naoyuki; Iwasaki, Kenji; Sakisaka, Toshiaki

2014-02-15

The ER (endoplasmic reticulum) consists of the nuclear envelope and a peripheral network of membrane sheets and tubules. Two classes of the evolutionarily conserved ER membrane proteins, reticulons and REEPs (receptor expression-enhancing proteins)/DP1 (deleted in polyposis locus 1)/Yop1 (YIP 1 partner), shape high-curvature ER tubules. In mammals, four members of the reticulon family and six members of the REEP family have been identified so far. In the present paper we report that Arl6IP1(ADP-ribosylation factor-like 6 interacting protein 1), an anti-apoptotic protein specific to multicellular organisms, is a potential player in shaping the ER tubules in mammalian cells. Arl6IP1, which does not share an overall primary sequence homology with reticulons, harbours reticulon-like short hairpin transmembrane domains and binds to atlastin, a GTPase that mediates the formation of the tubular ER network. Overexpression of Arl6IP1 induced extensive tubular structures of the ER and excluded a luminal protein. Furthermore, overexpression of Arl6IP1 stabilized the ER tubules, allowing the cells to maintain the ER tubules even in the absence of microtubules. Arl6IP1 constricted liposomes into tubules. The short hairpin structures of the transmembrane domains were required for the membrane-shaping activity of Arl6IP1. The results of the present study indicate that Arl6IP1 has the ability to shape high-curvature ER tubules in a reticulon-like fashion.

iPS-cell derived dendritic cells and macrophages for cancer therapy.

PubMed

Senju, Satoru

2016-08-01

Antibody-based anti-cancer immunotherapy was recently recognized as one of the truly effective therapies for cancer patients. Antibodies against cell surface cancer antigens, such as CD20, and also those against immune-inhibitory molecules called "immune checkpoint blockers", such as CTLA4 or PD1, have emerged. Large-scale clinical trials have confirmed that, in some cases, antibody-based drugs are superior to conventional chemotherapeutic agents. These antibody-based drugs are now being manufactured employing a mass-production system by pharmaceutical companies. Anti-cancer therapy by immune cells, i.e. cell-based immunotherapy, is expected to be more effective than antibody therapy, because immune cells can recognize, infiltrate, and act in cancer tissues more directly than antibodies. In order to achieve cell-based anti-cancer immunotherapy, it is necessary to develop manufacturing systems for mass-production of immune cells. Our group has been studying immunotherapy with myeloid cells derived from ES cells or iPS cells. These pluripotent stem cells can be readily propagated under constant culture conditions, with expansion into a large quantity. We consider these stem cells to be the most suitable cellular source for mass-production of immune cells. This review introduces our studies on anti-cancer therapy with iPS cell-derived dendritic cells and iPS cell-derived macrophages.

A role for human brain pericytes in neuroinflammation

PubMed Central

2014-01-01

Background Brain inflammation plays a key role in neurological disease. Although much research has been conducted investigating inflammatory events in animal models, potential differences in human brain versus rodent models makes it imperative that we also study these phenomena in human cells and tissue. Methods Primary human brain cell cultures were generated from biopsy tissue of patients undergoing surgery for drug-resistant epilepsy. Cells were treated with pro-inflammatory compounds IFNγ, TNFα, IL-1β, and LPS, and chemokines IP-10 and MCP-1 were measured by immunocytochemistry, western blot, and qRT-PCR. Microarray analysis was also performed on late passage cultures treated with vehicle or IFNγ and IL-1β. Results Early passage human brain cell cultures were a mixture of microglia, astrocytes, fibroblasts and pericytes. Later passage cultures contained proliferating fibroblasts and pericytes only. Under basal culture conditions all cell types showed cytoplasmic NFκB indicating that they were in a non-activated state. Expression of IP-10 and MCP-1 were significantly increased in response to pro-inflammatory stimuli. The two chemokines were expressed in mixed cultures as well as cultures of fibroblasts and pericytes only. The expression of IP-10 and MCP-1 were regulated at the mRNA and protein level, and both were secreted into cell culture media. NFκB nuclear translocation was also detected in response to pro-inflammatory cues (except IFNγ) in all cell types. Microarray analysis of brain pericytes also revealed widespread changes in gene expression in response to the combination of IFNγ and IL-1β treatment including interleukins, chemokines, cellular adhesion molecules and much more. Conclusions Adult human brain cells are sensitive to cytokine challenge. As expected ‘classical’ brain immune cells, such as microglia and astrocytes, responded to cytokine challenge but of even more interest, brain pericytes also responded to such challenge with a rich repertoire of gene expression. Immune activation of brain pericytes may play an important role in communicating inflammatory signals to and within the brain interior and may also be involved in blood brain barrier (BBB) disruption . Targeting brain pericytes, as well as microglia and astrocytes, may provide novel opportunities for reducing brain inflammation and maintaining BBB function and brain homeostasis in human brain disease. PMID:24920309

Vesicular glutamate transporter 2 (VGLUT2) is co-stored with PACAP in projections from the rat melanopsin-containing retinal ganglion cells.

PubMed

Engelund, Anna; Fahrenkrug, Jan; Harrison, Adrian; Hannibal, Jens

2010-05-01

The retinal ganglion cell layer of the eye comprises a subtype of cells characterized by their intrinsic photosensitivity and expression of melanopsin (ipRGCs). These cells regulate a variety of non-image-forming (NIF) functions such as light entrainment of circadian rhythms, acute suppression of locomotor activity (masking), and pupillary light reflex. Two neurotransmitters have been identified in ipRGCs, glutamate and pituitary adenylate cyclase-activating polypeptide (PACAP). To date, little is known about their release and interplay. Here, we describe the presence and co-localization of vesicular glutamate transporter 2 (VGLUT2; a marker of glutamate signaling) and PACAP in ipRGCs and their projections in the brain. Nine adult male Wistar rats were assigned to one of three groups; anterograde tracing (n = 3), eye enucleation (n = 3), and untreated (n = 3). Under anaesthesia, rats were transcardially perfusion-fixated, after which the brains and eyes were removed for double immunohistochemical staining using a polyclonal anti-VGLUT2 antibody and a mouse monoclonal anti-PACAP antibody. Results revealed that VGLUT2- and PACAP-immunoreactivity (-ir) were present in ipRGCs and co-localized in their projections in the suprachiasmatic nucleus, the intergeniculate leaflet, and the olivary pretectal nucleus. We conclude that there is evidence to support the use of glutamate and PACAP as neurotransmitters in NIF photoperception by rat ipRGCs, and that these neurotransmitters are co-stored and probably released from the same nerve terminals. Furthermore, we conclude that VGLUT2 is the preferred subtype of vesicular transporter used by these cells.

Protein-based human iPS cells efficiently generate functional dopamine neurons and can treat a rat model of Parkinson disease.

PubMed

Rhee, Yong-Hee; Ko, Ji-Yun; Chang, Mi-Yoon; Yi, Sang-Hoon; Kim, Dohoon; Kim, Chun-Hyung; Shim, Jae-Won; Jo, A-Young; Kim, Byung-Woo; Lee, Hyunsu; Lee, Suk-Ho; Suh, Wonhee; Park, Chang-Hwan; Koh, Hyun-Chul; Lee, Yong-Sung; Lanza, Robert; Kim, Kwang-Soo; Lee, Sang-Hun

2011-06-01

Parkinson disease (PD) involves the selective loss of midbrain dopamine (mDA) neurons and is a possible target disease for stem cell-based therapy. Human induced pluripotent stem cells (hiPSCs) are a potentially unlimited source of patient-specific cells for transplantation. However, it is critical to evaluate the safety of hiPSCs generated by different reprogramming methods. Here, we compared multiple hiPSC lines derived by virus- and protein-based reprogramming to human ES cells (hESCs). Neuronal precursor cells (NPCs) and dopamine (DA) neurons delivered from lentivirus-based hiPSCs exhibited residual expression of exogenous reprogramming genes, but those cells derived from retrovirus- and protein-based hiPSCs did not. Furthermore, NPCs derived from virus-based hiPSCs exhibited early senescence and apoptotic cell death during passaging, which was preceded by abrupt induction of p53. In contrast, NPCs derived from hESCs and protein-based hiPSCs were highly expandable without senescence. DA neurons derived from protein-based hiPSCs exhibited gene expression, physiological, and electrophysiological properties similar to those of mDA neurons. Transplantation of these cells into rats with striatal lesions, a model of PD, significantly rescued motor deficits. These data support the clinical potential of protein-based hiPSCs for personalized cell therapy of PD.

Combining multiple ChIP-seq peak detection systems using combinatorial fusion.

PubMed

Schweikert, Christina; Brown, Stuart; Tang, Zuojian; Smith, Phillip R; Hsu, D Frank

2012-01-01

Due to the recent rapid development in ChIP-seq technologies, which uses high-throughput next-generation DNA sequencing to identify the targets of Chromatin Immunoprecipitation, there is an increasing amount of sequencing data being generated that provides us with greater opportunity to analyze genome-wide protein-DNA interactions. In particular, we are interested in evaluating and enhancing computational and statistical techniques for locating protein binding sites. Many peak detection systems have been developed; in this study, we utilize the following six: CisGenome, MACS, PeakSeq, QuEST, SISSRs, and TRLocator. We define two methods to merge and rescore the regions of two peak detection systems and analyze the performance based on average precision and coverage of transcription start sites. The results indicate that ChIP-seq peak detection can be improved by fusion using score or rank combination. Our method of combination and fusion analysis would provide a means for generic assessment of available technologies and systems and assist researchers in choosing an appropriate system (or fusion method) for analyzing ChIP-seq data. This analysis offers an alternate approach for increasing true positive rates, while decreasing false positive rates and hence improving the ChIP-seq peak identification process.

Pseudo-random dynamic address configuration (PRDAC) algorithm for mobile ad hoc networks

NASA Astrophysics Data System (ADS)

Wu, Shaochuan; Tan, Xuezhi

2007-11-01

By analyzing all kinds of address configuration algorithms, this paper provides a new pseudo-random dynamic address configuration (PRDAC) algorithm for mobile ad hoc networks. Based on PRDAC, the first node that initials this network randomly chooses a nonlinear shift register that can generates an m-sequence. When another node joins this network, the initial node will act as an IP address configuration sever to compute an IP address according to this nonlinear shift register, and then allocates this address and tell the generator polynomial of this shift register to this new node. By this means, when other node joins this network, any node that has obtained an IP address can act as a server to allocate address to this new node. PRDAC can also efficiently avoid IP conflicts and deal with network partition and merge as same as prophet address (PA) allocation and dynamic configuration and distribution protocol (DCDP). Furthermore, PRDAC has less algorithm complexity, less computational complexity and more sufficient assumption than PA. In addition, PRDAC radically avoids address conflicts and maximizes the utilization rate of IP addresses. Analysis and simulation results show that PRDAC has rapid convergence, low overhead and immune from topological structures.

Disruption of dopamine D1/D2 receptor complex is involved in the function of haloperidol in cardiac H9c2 cells.

PubMed

Lencesova, L; Szadvari, I; Babula, P; Kubickova, J; Chovancova, B; Lopusna, K; Rezuchova, I; Novakova, Z; Krizanova, O; Novakova, M

2017-12-15

Haloperidol is an antipsychotic agent and acts as dopamine D2 receptor (D2R) antagonist, as a prototypical ligand of sigma1 receptors (Sig1R) and it increases expression of type 1 IP 3 receptors (IP 3 R1). However, precise mechanism of haloperidol action on cardiomyocytes through dopaminergic signaling was not described yet. This study investigated a role of dopamine receptors in haloperidol-induced increase in IP 3 R1 and Sig1R, and compared physiological effect of melperone and haloperidol on basic heart parameters in rats. We used differentiated NG-108 cells and H9c2 cells. Gene expression, Western blot and immunofluorescence were used to evaluate haloperidol-induced differences; proximity ligation assay (PLA) and immunoprecipitation to determine interactions of D1/D2 receptors. To evaluate cardiac parameters, Wistar albino male rats were used. We have shown that antagonism of D2R with either haloperidol or melperone results in upregulation of both, IP 3 R1 and Sig1R, which is associated with increased D2R, but reduced D1R expression. Immunofluorescence, immunoprecipitation and PLA support formation of heteromeric D1/D2 complexes in H9c2 cells. Treatment with haloperidol (but not melperone) caused decrease in systolic and diastolic blood pressure and significant increase in heart rate. Because D1R/D2R complexes can engage Gq-like signaling in other experimental systems, these results are consistent with the possibility that disruption of D1R/D2R complex in H9c2 cells might cause a decrease in IP 3 R1 activity, which in turn may account for the increase expression of IP 3 R and Sig1R. D2R is probably not responsible for changes in cardiac parameters, since melperone did not have any effect. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells.

PubMed

Abdel-Latif, A A; Ding, K H; Akhtar, R A; Yousufzai, S Y

1996-09-01

In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A2 (PLA2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol trisphosphate (IP3), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP3 production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC50 = 1.2 nM) than in activating PLC (EC50 = 1.5 nM) or PLA2 (EC50 = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA2 was stimulated first (t1/2 = 12 s), followed by PLC (t1/2 = 48 s) and lastly PLD (t1/2 = 106 s). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ETB receptor agonist, had no effect on the phospholipases, and indomethacin, a cyclooxygenase inhibitor, had no effect on the stimulatory effect of ET-1 on the phospholipases. These results suggest that in this smooth muscle cell line, ET-1 interacts with the ETA receptor subtype to activate, via G proteins, phospholipases A2, C and D in a temporal sequence.

Antiepileptic drugs and brain maturation: fetal exposure to lamotrigine generates cortical malformations in rats.

PubMed

Manent, Jean-Bernard; Jorquera, Isabel; Franco, Valentina; Ben-Ari, Yehezkel; Perucca, Emilio; Represa, Alfonso

2008-02-01

Intake of antiepileptic drugs (AEDs) during pregnancy can provoke severe and subtle fetal malformations associated with deleterious sequelae, reflecting the need for experimental investigations on the comparative teratogenic potential of these agents. We recently reported that prenatal exposure to vigabatrin and valproate, two AEDs which act through GABAergic mechanisms, induces hippocampal and cortical dysplasias in rodents. We have now investigated the effects of phenobarbital (PB, 30 mg/kg day) i.p.), a drug also endowed with GABAergic effects, and the new generation AEDs lamotrigine (LTG, 5-20mg/kg/day i.p.), topiramate (TPM, 10mg/kg/day i.p.), and levetiracetam (LEV, 50mg/kg/day i.p.) on brain development. Prenatal exposure to LTG induced hippocampal and cortical malformations in a dose-dependent manner, at maternal plasma concentrations within the clinically occurring range. These abnormalities were not observed after exposure to PB, TP and LEV. These observations raise concerns about potential clinical correlates and call for detailed comparative investigations on the consequences of AED use during pregnancy.

Mask pattern generator employing EPL technology

NASA Astrophysics Data System (ADS)

Yoshioka, Nobuyuki; Yamabe, Masaki; Wakamiya, Wataru; Endo, Nobuhiro

2003-08-01

Mask cost is one of crucial issues in device fabrication, especially in SoC (System on a Chip) with small-volume production. The cost mainly depends on productivity of mask manufacturing tools such as mask writers and defect inspection tools. EPL (Electron Projection Lithography) has been developing as a high-throughput electron beam exposure technology that will succeed optical lithography. The application of EPL technology to mask writing will result in high productivity and contribute to decrease the mask cost. The concept of a mask pattern generator employing EPL technology is proposed in this paper. It is very similar to EPL technology used for pattern printing on a wafer. The mask patterns on the glass substrate are exposed by projecting the basic circuit patterns formed on the mother EPL mask. One example of the mother EPL mask is a stencil type made with 200-mm Si wafer. The basic circuit patterns are IP patterns and logical primitive patterns such as cell libraries (AND, OR, Inverter, Flip-Flop and etc.) to express the SoC device patterns. Since the SoC patterns are exposed with its collective units such as IP and logical primitive patterns by using this method, the high throughput will be expected comparing with conventional mask E-beam writers. In this paper, the mask pattern generator with the EPL technology is proposed. The concept, its advantages and issues to be solved are discussed.

Interplanetary Scintillation Observations with a New Generation of Radio Telescopes: First results from the MWA

NASA Astrophysics Data System (ADS)

Morgan, J. S.; Macquart, J. P.; Ekers, R.; Bisi, M. M.; Jackson, B. V.; Tokumaru, M.; Manoharan, P. K.; Chhetri, R.

2016-12-01

Interplanetary scintillation (IPS) is a phenomenon which can be used to probe both the heliospheric plasma and the structure of compact astrophysical radio sources. It is a vital tool for near-real-time monitoring of space weather. Previous IPS studies have generally relied on single concentrated collecting areas (either phased arrays or dishes). The Murchison Widefield Array (MWA) by contrast is a new-generation instrument consisting of a 128-element interferometer with an extremely wide field of view, and outstanding instantaneous imaging capability. This enables IPS studies of 1000 sources simultaneously, increasing the number of daily measurements that can be made by a factor of two or more. Here we report on progress from an ongoing IPS survey with the MWA where observations are made simultaneously at 80MHz and 150MHz. Dual-frequency observations allow solar wind velocities to be determined even with a single station, more accurately than from the analyses of a single-frequency IPS spectrum alone. Furthermore, the different refractive indices at different wavelengths leads to a lag in the cross correlation of the two frequency bands. This allows the bulk density of the outer solar corona to be probed along multiple lines of sight. We will discuss recent results and how they might be integrated into international Space Weather Prediction efforts such as the Worldwide IPS Stations (WIPSS) Network.

Unusual Father-to-Daughter Transmission of Incontinentia Pigmenti Due to Mosaicism in IP Males.

PubMed

Fusco, Francesca; Conte, Matilde Immacolata; Diociaiuti, Andrea; Bigoni, Stefania; Branda, Maria Francesca; Ferlini, Alessandra; El Hachem, Maya; Ursini, Matilde Valeria

2017-09-01

Incontinentia pigmenti (IP; Online Mendelian Inheritance in Man catalog #308300) is an X-linked dominant ectodermal disorder caused by mutations of the inhibitor of κ polypeptide gene enchancer in B cells, kinase γ ( IKBKG )/ nuclear factor κB, essential modulator ( NEMO ) gene. Hemizygous IKBKG/NEMO loss-of-function (LoF) mutations are lethal in males, thus patients are female, and the disease is always transmitted from an IP-affected mother to her daughter. We present 2 families with father-to-daughter transmission of IP and provide for the first time molecular evidence that the combination of somatic and germ-line mosaicism for IKBKG/NEMO loss of function mutations in IP males resulted in the transmission of the disease to a female child. We searched for the IKBKG/NEMO mutant allele in blood, urine, skin, and sperm DNA and found that the 2 fathers were somatic and germ-line mosaics for the p.Gln132×mutation or the exon 4-10 deletion of IKBKG/NEMO , respectively. The highest level of IKBKG/NEMO mutant cells was detected in the sperm, which might explain the recurrence of the disease. We therefore recommend careful clinical evaluation in IP male cases and the genetic investigation in sperm DNA to ensure correct genetic counseling and prevent the risk of paternal transmission of IP. Copyright © 2017 by the American Academy of Pediatrics.

Impact of non-thermal plasma surface modification on porous calcium hydroxyapatite ceramics for bone regeneration

PubMed Central

Moriguchi, Yu; Lee, Dae-Sung; Thamina, Khair; Masuda, Kazuto; Itsuki, Dai; Yoshikawa, Hideki; Hamaguchi, Satoshi; Myoui, Akira

2018-01-01

In the physiochemical sciences, plasma is used to describe an ionized gas. Previous studies have implicated plasma surface treatment in the enhancement of hydrophilicity of implanted musculoskeletal reconstructive materials. Hydroxyapatite (HA) ceramics, widely used in bone tissue regeneration, have made great advancements to skeletal surgery. In the present study, we investigate the impact of low-pressure plasma on the interconnected porous calcium hydroxyapatite (IP-CHA) both in vitro and in vivo. Our results indicate that dielectric barrier discharge (DBD) plasma, when used with oxygen, can augment the hydrophilicity of non-porous HA surfaces and the osteoconductivity of the IP-CHA disc via increased water penetration of inner porous structures, as demonstrated through microfocus computed tomography (μCT) assay. In vivo implantation of plasma-treated IP-CHA displayed superior bone ingrowth than untreated IP-CHA. Though plasma-treated IP-CHA did not alter osteoblast cell proliferation, it accelerated osteogenic differentiation of seeded marrow mesenchymal stem cells. In vitro X-ray photoelectron spectroscopy (XPS) revealed that this plasma treatment increases levels of oxygen, rather than nitrogen, on the plasma-treated IP-CHA surface. These findings suggest that plasma treatment, an easy and simple processing, can significantly improve the osteoconductive potential of commonly used artificial bones such as IP-CHA. Further optimization of plasma treatment and longer-term follow-up of in vivo application are required toward its clinical application. PMID:29538457

Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses.

PubMed

Teste, Bruno; Champ, Jerome; Londono-Vallejo, Arturo; Descroix, Stéphanie; Malaquin, Laurent; Viovy, Jean-Louis; Draskovic, Irena; Mottet, Guillaume

2017-01-31

Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

ATM QoS Experiments Using TCP Applications: Performance of TCP/IP Over ATM in a Variety of Errored Links

NASA Technical Reports Server (NTRS)

Frantz, Brian D.; Ivancic, William D.

2001-01-01

Asynchronous Transfer Mode (ATM) Quality of Service (QoS) experiments using the Transmission Control Protocol/Internet Protocol (TCP/IP) were performed for various link delays. The link delay was set to emulate a Wide Area Network (WAN) and a Satellite Link. The purpose of these experiments was to evaluate the ATM QoS requirements for applications that utilize advance TCP/IP protocols implemented with large windows and Selective ACKnowledgements (SACK). The effects of cell error, cell loss, and random bit errors on throughput were reported. The detailed test plan and test results are presented herein.

Stem cells in pharmaceutical biotechnology.

PubMed

Zuba-Surma, Ewa K; Józkowicz, Alicja; Dulak, Józef

2011-11-01

Multiple populations of stem cells have been indicated to potentially participate in regeneration of injured organs. Especially, embryonic stem cells (ESC) and recently inducible pluripotent stem cells (iPS) receive a marked attention from scientists and clinicians for regenerative medicine because of their high proliferative and differentiation capacities. Despite that ESC and iPS cells are expected to give rise into multiple regenerative applications when their side effects are overcame during appropriate preparation procedures, in fact their most recent application of human ESC may, however, reside in their use as a tool in drug development and disease modeling. This review focuses on the applications of stem cells in pharmaceutical biotechnology. We discuss possible relevance of pluripotent cell stem populations in developing physiological models for any human tissue cell type useful for pharmacological, metabolic and toxicity evaluation necessary in the earliest steps of drug development. The present models applied for preclinical drug testing consist of primary cells or immortalized cell lines that show limitations in terms of accessibility or relevance to their in vivo counterparts. The availability of renewable human cells with functional similarities to their in vivo counterparts is the first landmark for a new generation of cell-based assays. We discuss the approaches for using stem cells as valuable physiological targets of drug activity which may increase the strength of target validation and efficacy potentially resulting in introducing new safer remedies into clinical trials and the marketplace. Moreover, we discuss the possible applications of stem cells for elucidating mechanisms of disease pathogenesis. The knowledge about the mechanisms governing the development and progression of multitude disorders which would come from the cellular models established based on stem cells, may give rise to new therapeutical strategies for such diseases. All together, the applications of various cell types derived from patient specific pluripotent stem cells may lead to targeted drug and cellular therapies for certain individuals.

Prostaglandin E2 Inhibits Histamine-Evoked Ca2+ Release in Human Aortic Smooth Muscle Cells through Hyperactive cAMP Signaling Junctions and Protein Kinase A

PubMed Central

Taylor, Emily J. A.; Pantazaka, Evangelia; Shelley, Kathryn L.

2017-01-01

In human aortic smooth muscle cells, prostaglandin E2 (PGE2) stimulates adenylyl cyclase (AC) and attenuates the increase in intracellular free Ca2+ concentration evoked by activation of histamine H1 receptors. The mechanisms are not resolved. We show that cAMP mediates inhibition of histamine-evoked Ca2+ signals by PGE2. Exchange proteins activated by cAMP were not required, but the effects were attenuated by inhibition of cAMP-dependent protein kinase (PKA). PGE2 had no effect on the Ca2+ signals evoked by protease-activated receptors, heterologously expressed muscarinic M3 receptors, or by direct activation of inositol 1,4,5-trisphosphate (IP3) receptors by photolysis of caged IP3. The rate of Ca2+ removal from the cytosol was unaffected by PGE2, but PGE2 attenuated histamine-evoked IP3 accumulation. Substantial inhibition of AC had no effect on the concentration-dependent inhibition of Ca2+ signals by PGE2 or butaprost (to activate EP2 receptors selectively), but it modestly attenuated responses to EP4 receptors, activation of which generated less cAMP than EP2 receptors. We conclude that inhibition of histamine-evoked Ca2+ signals by PGE2 occurs through “hyperactive signaling junctions,” wherein cAMP is locally delivered to PKA at supersaturating concentrations to cause uncoupling of H1 receptors from phospholipase C. This sequence allows digital signaling from PGE2 receptors, through cAMP and PKA, to histamine-evoked Ca2+ signals. PMID:28877931

Inositol 1,4,5-trisphosphate receptor regulates replication, differentiation, infectivity and virulence of the parasitic protist Trypanosoma cruzi.

PubMed

Hashimoto, Muneaki; Enomoto, Masahiro; Morales, Jorge; Kurebayashi, Nagomi; Sakurai, Takashi; Hashimoto, Tetsuo; Nara, Takeshi; Mikoshiba, Katsuhiko

2013-03-01

In animals, inositol 1,4,5-trisphosphate receptors (IP3 Rs) are ion channels that play a pivotal role in many biological processes by mediating Ca(2+) release from the endoplasmic reticulum. Here, we report the identification and characterization of a novel IP3 R in the parasitic protist, Trypanosoma cruzi, the pathogen responsible for Chagas disease. DT40 cells lacking endogenous IP3 R genes expressing T. cruzi IP3 R (TcIP3 R) exhibited IP3 -mediated Ca(2+) release from the ER, and demonstrated receptor binding to IP3 . TcIP3 R was expressed throughout the parasite life cycle but the expression level was much lower in bloodstream trypomastigotes than in intracellular amastigotes or epimastigotes. Disruption of two of the three TcIP3 R gene loci led to the death of the parasite, suggesting that IP3 R is essential for T. cruzi. Parasites expressing reduced or increased levels of TcIP3 R displayed defects in growth, transformation and infectivity, indicating that TcIP3 R is an important regulator of the parasite's life cycle. Furthermore, mice infected with T. cruzi expressing reduced levels of TcIP3 R exhibited a reduction of disease symptoms, indicating that TcIP3 R is an important virulence factor. Combined with the fact that the primary structure of TcIP3 R has low similarity to that of mammalian IP3 Rs, TcIP3 R is a promising drug target for Chagas disease. © 2013 Blackwell Publishing Ltd.

Carbachol-induced phosphoinositide turnover in NCB-20 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chuang, D.M.; Dillon-Carter, O.

NCB-20 cells (fetal Chinese hamster brain cell x neuroblastoma hybrids) have been shown to contain a variety of neurotransmitter receptors. The authors now report that this cloned cell line also contains acetylcholne receptors which are linked to phospholipase C. Confluent cell cultures were preincubated with /sup 3/H-myo-inositol to label endogenous phosphoinositide (PI) and the accumulation of a PI metabolite, inositol monophosphate (IP/sub 1/), was measured in the presence of LiCl. Carbachol increased IP/sub 1/), accumulation be more than 400% with a EC/sub 50/ of about 50 ..mu..M. Acetylcholine and muscarine were also effective, whereas oxotremorine and McN-A-343 were weak inmore » both potency and efficacy. The carbachol-induced IP/sub 1/ accumulation was completely blocked by atropine (Ki approx. 0.6 nM) and pirenzepine (Ki approx. 15 nM). The presence of KCl was not required for the carbachol-induced effect. The formation of inositol bis- and triphosphate was also increased carbachol; these increases occurred earlier but were of much smaller magnitude. Pretreatment of cells with 4 ..beta..-phorbol dibutyrate or 4 ..beta..-phorbol myristate acetate was found to attenuate the carbachol-induced formation of IP/sub 1/ (IC/sub 50/ in the low nanomolar concentration ranges), however 4 ..beta..-phorbol, the biologically inactive phorbol ester, was ineffective in causing this attenuation. These results suggest a feedback inhibition of PI turnover in NCB-20 cells through the action of protein kinase C.« less

Treatment of Diabetes Mellitus Using iPS Cells and Spice Polyphenols

PubMed Central

Chen, Liang

2017-01-01

Diabetes mellitus is a chronic disease that threatens human health. The disease is caused by a metabolic disorder of the endocrine system, and long-term illness can lead to tissue and organ damage to the cardiovascular, endocrine, nervous, and urinary systems. Currently, the disease prevalence is 11.4%, the treatment rate is 48.2%, and the mortality rate is 2.7% worldwide. Comprehensive and effective control of diabetes, as well as the use of insulin, requires further study to develop additional treatment options. Here, we reviewed the current reprogramming of somatic cells using specific factors to induced pluripotent stem (iPS) cells capable of repairing islet β cell damage in diabetes patients to treat patients with type 1 diabetes mellitus. We also discuss the shortcomings associated with clinical use of iPS cells. Additionally, certain polyphenols found in spices might improve glucose homeostasis and insulin resistance in diabetes patients, thereby constituting promising options for the treatment of type 2 diabetes. PMID:28758131

Treatment of Diabetes Mellitus Using iPS Cells and Spice Polyphenols.

PubMed

Ge, Qi; Chen, Liang; Chen, Keping

2017-01-01

Diabetes mellitus is a chronic disease that threatens human health. The disease is caused by a metabolic disorder of the endocrine system, and long-term illness can lead to tissue and organ damage to the cardiovascular, endocrine, nervous, and urinary systems. Currently, the disease prevalence is 11.4%, the treatment rate is 48.2%, and the mortality rate is 2.7% worldwide. Comprehensive and effective control of diabetes, as well as the use of insulin, requires further study to develop additional treatment options. Here, we reviewed the current reprogramming of somatic cells using specific factors to induced pluripotent stem (iPS) cells capable of repairing islet β cell damage in diabetes patients to treat patients with type 1 diabetes mellitus. We also discuss the shortcomings associated with clinical use of iPS cells. Additionally, certain polyphenols found in spices might improve glucose homeostasis and insulin resistance in diabetes patients, thereby constituting promising options for the treatment of type 2 diabetes.

Focal Activation of Cells by Plasmon Resonance Assisted Optical Injection of Signaling Molecules

PubMed Central

2015-01-01

Experimental methods for single cell intracellular delivery are essential for probing cell signaling dynamics within complex cellular networks, such as those making up the tumor microenvironment. Here, we show a quantitative and general method of interrogation of signaling pathways. We applied highly focused near-infrared laser light to optically inject gold-coated liposomes encapsulating bioactive molecules into single cells for focal activation of cell signaling. For this demonstration, we encapsulated either inositol trisphosphate (IP3), an endogenous cell signaling second messenger, or adenophostin A (AdA), a potent analogue of IP, within 100 nm gold-coated liposomes, and injected these gold-coated liposomes and their contents into the cytosol of single ovarian carcinoma cells to initiate calcium (Ca2+) release from intracellular stores. Upon optical injection of IP3 or AdA at doses above the activation threshold, we observed increases in cytosolic Ca2+ concentration within the injected cell initiating the propagation of a Ca2+ wave throughout nearby cells. As confirmed by octanol-induced inhibition, the intercellular Ca2+ wave traveled via gap junctions. Optical injection of gold-coated liposomes represents a quantitative method of focal activation of signaling cascades of broad interest in biomedical research. PMID:24877558

Nuclease-mediated double-strand break (DSB) enhancement of small fragment homologous recombination (SFHR) gene modification in human-induced pluripotent stem cells (hiPSCs).

PubMed

Sargent, R Geoffrey; Suzuki, Shingo; Gruenert, Dieter C

2014-01-01

Recent developments in methods to specifically modify genomic DNA using sequence-specific endonucleases and donor DNA have opened the door to a new therapeutic paradigm for cell and gene therapy of inherited diseases. Sequence-specific endonucleases, in particular transcription activator-like (TAL) effector nucleases (TALENs), have been coupled with polynucleotide small/short DNA fragments (SDFs) to correct the most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, a 3-base-pair deletion at codon 508 (delF508), in induced pluripotent stem (iPS) cells. The studies presented here describe the generation of candidate TALENs and their co-transfection with wild-type (wt) CFTR-SDFs into CF-iPS cells homozygous for the delF508 mutation. Using an allele-specific PCR (AS-PCR)-based cyclic enrichment protocol, clonal populations of corrected CF-iPS cells were isolated and expanded.

Long-term maintenance of human induced pluripotent stem cells by automated cell culture system.

PubMed

Konagaya, Shuhei; Ando, Takeshi; Yamauchi, Toshiaki; Suemori, Hirofumi; Iwata, Hiroo

2015-11-17

Pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem (iPS) cells, are regarded as new sources for cell replacement therapy. These cells can unlimitedly expand under undifferentiated conditions and be differentiated into multiple cell types. Automated culture systems enable the large-scale production of cells. In addition to reducing the time and effort of researchers, an automated culture system improves the reproducibility of cell cultures. In the present study, we newly designed a fully automated cell culture system for human iPS maintenance. Using an automated culture system, hiPS cells maintained their undifferentiated state for 60 days. Automatically prepared hiPS cells had a potency of differentiation into three germ layer cells including dopaminergic neurons and pancreatic cells.

Lactocepin secreted by Lactobacillus exerts anti-inflammatory effects by selectively degrading proinflammatory chemokines.

PubMed

von Schillde, Marie-Anne; Hörmannsperger, Gabriele; Weiher, Monika; Alpert, Carl-Alfred; Hahne, Hannes; Bäuerl, Christine; van Huynegem, Karolien; Steidler, Lothar; Hrncir, Tomas; Pérez-Martínez, Gaspar; Kuster, Bernhard; Haller, Dirk

2012-04-19

The intestinal microbiota has been linked to inflammatory bowel diseases (IBD), and oral treatment with specific bacteria can ameliorate IBD. One bacterial mixture, VSL#3, containing Lactobacillus, Bifidobacterium, and Streptococcus, was clinically shown to reduce inflammation in IBD patients and normalize intestinal levels of IP-10, a lymphocyte-recruiting chemokine, in a murine colitis model. We identified Lactobacillus paracasei prtP-encoded lactocepin as a protease that selectively degrades secreted, cell-associated, and tissue-distributed IP-10, resulting in significantly reduced lymphocyte recruitment after intraperitoneal injection in an ileitis model. A human Lactobacillus casei isolate was also found to encode lactocepin and degrade IP-10. L. casei feeding studies in a murine colitis model (T cell transferred Rag2(-/-) mice) revealed that a prtP-disruption mutant was significantly less potent in reducing IP-10 levels, T cell infiltration and inflammation in cecal tissue compared to the isogenic wild-type strain. Thus, lactocepin-based therapies may be effective treatments for chemokine-mediated diseases like IBD. Copyright © 2012 Elsevier Inc. All rights reserved.

Androgen Deprivation Enhances PLZF-Repressed Cistrome that Promotes the Castration-Resistant Phenotype

DTIC Science & Technology

2014-10-01

Briefly, 10 million cells were used per IP. Cells were fixed with 1% formaldehyde solution. DNA was sonicated and subjected to immunoprecipitation with... formaldehyde solution. Total chromatin and RNAs were sonicated and subjected to immunoprecipitation with the same AR and Med1 antibodies used in ChIP...were fixed with 1% formaldehyde . Cell pellets were lysed and resuspended in restriction buffer for BstY1 and 0.1% SDS for 10 min at 65 °C. Triton X

Testing Brain Overgrowth and Synaptic Models of Autism Using NPC’s and Neurons from Patient-Derived IPS Cells

DTIC Science & Technology

2014-10-01

Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Autism and autism spectrum disorders (ASD) are complex neurodevelopmental ...1. INTRODUCTION: Autism and autism spectrum disorders (ASD) are complex neurodevelopmental diseases that affect about 1% of children in the...and neurons. 2. KEYWORDS: Autism spectrum disorder, ASD, neurodevelopmental disease, disease modeling, induced pluripotent stem cell, iPS

Human cord blood applications in cell therapy: looking back and look ahead.

PubMed

Zhou, Hongyan; Chang, Stephen; Rao, Mahendra

2012-08-01

Human umbilical cord blood (UCB) has been used as a reliable source of stem cells for blood-borne diseases and disorders. Recent advances in cell reprogramming technology to produce induced pluripotent stem (iPS) cells, which can be differentiated to multiple adult cell types, has further expanded the potential of cord blood cell therapy for treatment of non-blood-borne diseases. However, in order to harness this breakthrough technology and to provide clinical-grade cells for the patient, standardization of iPS production and differentiation, and good manufacturing practice (GMP) need to be employed. UCB is an ethical source of stem cells and has been used to treat diseases including leukemia, cancer and blood disorders. The development of iPS cell technology could potentially greatly increase the application of cord blood cells as a treatment for a broader range of diseases, UCB-iPS banks could, therefore, be a valuable complementary source of clinical-grade cells for cell therapy. The current applicability of GMP to UCB and UCB-iPS cell-based cell therapy will be discussed. Although cord blood stem cell therapies have been practiced for decades, UCB-iPS cell therapies are a new innovation currently in development. Successful clinical applications of such novel cell therapies will depend on the production of GMP-compliant cells and the establishment of cell banks.

In vivo anticancer activity of vanillin semicarbazone

PubMed Central

Ali, Shaikh M Mohsin; Azad, M Abul Kalam; Jesmin, Mele; Ahsan, Shamim; Rahman, M Mijanur; Khanam, Jahan Ara; Islam, M Nazrul; Shahriar, Sha M Shahan

2012-01-01

Objective To evaluate the anticancer activity of vanillin semicarbazone (VSC) against Ehrlich ascites carcinoma (EAC) cells in Swiss albino mice. Methods The compound VSC at three doses (5, 7.5 and 10 mg/kg i.p.) was administered into the intraperitoneal cavity of the EAC inoculated mice to observe its efficiency by studying the cell growth inhibition, reduction of tumour weight, enhancement of survival time as well as the changes in depleted hematological parameters. All such parameters were also studied with a known standard drug bleomycin at the dose of 0.3 mg/kg (i.p.). Results Among the doses studied, 10 mg/kg (i.p.) was found to be quite comparable in potency to that of bleomycin at the dose of 0.3 mg/kg (i.p.). The host toxic effects of VSC was found to be negligible. Conclusions It can be concluded that VSC can therefore be considered as potent anticancer agent. PMID:23569946

Mesenchymal stem cells display hepato-protective activity in lymphoma bearing xenografts.

PubMed

Secchiero, Paola; Corallini, Federica; Zavan, Barbara; Tripodo, Claudio; Vindigni, Vincenzo; Zauli, Giorgio

2012-04-01

A disseminated model of non-Hodgkin's lymphoma with prevalent liver metastasis was generated by intraperitoneal (i.p.) injection of EBV(+) B lymphoblastoid SKW6.4 in nude-SCID mice. The survival of SKW6.4 xenografts (median survival = 27 days) was significantly improved when hyaluronan scaffolds embedded with mesenchimal stem cells (MSC) were implanted in the abdominal area 4 days after SKW6.4 injection (median survival = 39.5 days). Mice implanted with MSC showed a significant improvement of hepatic functionality in lymphoma xenografts, as demonstrated by measurement of serum ALT/AST levels. Co-culture of MSC with lymphoma cells enhanced the release of hepatocyte growth factor (HGF) by MSC. These data suggest that hyaluronan-embedded MSC exert anti-lymphoma activity by ameliorating hepatic functionality.

Subtype-selective regulation of IP(3) receptors by thimerosal via cysteine residues within the IP(3)-binding core and suppressor domain.

PubMed

Khan, Samir A; Rossi, Ana M; Riley, Andrew M; Potter, Barry V L; Taylor, Colin W

2013-04-15

IP(3)R (IP(3) [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca(2+) channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP(3)R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP(3)-evoked Ca(2+) release via IP(3)R1 and IP(3)R2, but inhibited IP(3)R3. Activation of IP(3)R is initiated by IP(3) binding to the IBC (IP(3)-binding core; residues 224-604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1-223). Thimerosal (100 μM) stimulated IP(3) binding to the isolated NT (N-terminal; residues 1-604) of IP(3)R1 and IP(3)R2, but not to that of IP(3)R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP(3)) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP(3)R activation. IP(3) binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP(3)R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP(3) binding to the chimaeric NT and IP(3)-evoked Ca(2+) release from the chimaeric IP(3)R. This is the first systematic analysis of the effects of a thiol reagent on each IP(3)R subtype. We conclude that thimerosal selectively sensitizes IP(3)R1 and IP(3)R2 to IP(3) by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor.

Migration of iron-labeled KHYG-1 natural killer cells to subcutaneous tumors in nude mice, as detected by magnetic resonance imaging.

PubMed

Mallett, Christiane L; McFadden, Catherine; Chen, Yuhua; Foster, Paula J

2012-07-01

A novel cell line of cytotoxic natural killer (NK) cells, KHYG-1, was examined in vivo for immunotherapy against prostate cancer. The feasibility of using magnetic resonance imaging (MRI) tracking to monitor the fate of injected NK cells following intravenous (i.v.), intraperitoneal (i.p.) and subcutaneous (s.c.) administration was assessed. PC-3M human prostate cancer cells were injected s.c. into the flank of nude mice (day 0). KHYG-1 NK cells were labeled with an iron oxide contrast agent and injected s.c., i.v. or i.p. on day 8. Mice were imaged by MRI on days 7, 9 and 12. Tumor sections were examined with fluorescence microscopy and immunohistologic staining for NK cells. NK cells were detected in the tumors by histology after all three administration routes. NK cells and fluorescence from the iron label were co-localized. Signal loss was seen in the areas around the tumors and between the tumor lobes in the s.c. group. We are the first to label this cell line of NK cells with an iron oxide contrast agent. Accumulation of NK cells was visualized by MRI after s.c. injection but not after i.v. and i.p. injection.

Phosphatidylcholine-specific phospholipase C inhibition reduces HER2-overexpression, cell proliferation and in vivo tumor growth in a highly tumorigenic ovarian cancer model

PubMed Central

Spadaro, Francesca; Abalsamo, Laura; Pisanu, Maria Elena; Ricci, Alessandro; Cecchetti, Serena; Altabella, Luisa; Buoncervello, Maria; Lozneanu, Ludmila; Bagnoli, Marina; Ramoni, Carlo; Canevari, Silvana; Mezzanzanica, Delia

2017-01-01

Antagonizing the oncogenic effects of human epidermal growth factor receptor 2 (HER2) with current anti-HER2 agents has not yet yielded major progress in the treatment of advanced HER2-positive epithelial ovarian cancer (EOC). Using preclinical models to explore alternative molecular mechanisms affecting HER2 overexpression and oncogenicity may lead to new strategies for EOC patient treatment. We previously reported that phosphatidylcholine-specific phospholipase C (PC-PLC) exerts a pivotal role in regulating HER2 overexpression in breast cancer cells. The present study, conducted on two human HER2-overexpressing EOC cell lines - SKOV3 and its in vivo-passaged SKOV3.ip cell variant characterized by enhanced in vivo tumorigenicity - and on SKOV3.ip xenografts implanted in SCID mice, showed: a) about 2-fold higher PC-PLC and HER2 protein expression levels in SKOV3.ip compared to SKOV3 cells; b) physical association of PC-PLC with HER2 in non-raft domains; c) HER2 internalization and ca. 50% reduction of HER2 mRNA and protein expression levels in SKOV3.ip cells exposed to the PC-PLC inhibitor tricyclodecan-9-yl-potassium xanthate (D609); d) differential effects of D609 and trastuzumab on HER2 protein expression and cell proliferation; e) decreased in vivo tumor growth in SKOV3.ip xenografts during in vivo treatment with D609; f) potential use of in vivo magnetic resonance spectroscopy (MRS) and imaging (MRI) parameters as biomarkers of EOC response to PC-PLC inhibition. Overall, these findings support the view that PC-PLC inhibition may represent an effective means to target the tumorigenic effects of HER2 overexpression in EOC and that in vivo MR approaches can efficiently monitor its effects. PMID:28903399

Characterization of the hollow fiber assay for the determination of microtubule disruption in vivo.

PubMed

Suggitt, Marie; Swaine, David J; Pettit, George R; Bibby, Michael C

2004-10-01

The hollow fiber assay is used successfully as a routine in vivo screening model to quantitatively define anticancer activity by the National Cancer Institute. This study investigates whether the hollow fiber assay can be used as a short-term in vivo model to demonstrate specific pharmacodynamic end points, namely microtubule and cell cycle disruption. The growth of A549 cells was characterized within hollow fibers over 5 days in vivo at both subcutaneous (s.c.) and intraperitoneal (i.p.) sites. Drugs were administered on day 4 (i.p.). At 24 hours, cells were retrieved from fibers at both i.p. and s.c. sites of paclitaxel-treated (20 mg/kg) and combretastatin A1 phosphate-treated (150 mg/kg) mice. Cell cycle analysis after paclitaxel treatment revealed a mean G(2)-M phase population of 48.04% (i.p.) and 25.76% (s.c.) compared with vehicle group mice (6.78 and 5.56%, respectively; P = <0.001 and 0.005, respectively). Tumor cells retrieved from combretastatin A1 phosphate-treated mice had a mean G2-M phase population of 36.3% (i.p.) and 29.36% (s.c.) compared with cells retrieved from vehicle group mice (5.58 and 5.49%, respectively; P = <0.001). Using fluorescence and laser-confocal microscopy, paclitaxel was revealed to induce the formation of spindle asters and tubulin polymerization. Combretastatin A1 phosphate was shown to hold cells in mitosis. Changes in nuclear morphology were also observed. These data demonstrate that the hollow fiber assay can be used as a short-term in vivo model for studying the pharmacodynamic effects of both standard and novel compounds on microtubules. Evidence has also been provided to support the routine use of the in vivo hollow fiber assay for demonstrating the mechanism of action of a drug.

Immunopotentiator from Pantoea agglomerans Prevents Atopic Dermatitis Induced by Dermatophagoides farinae Extract in NC/Nga Mouse.

PubMed

Wakame, Koji; Komatsu, Ken-Ichi; Inagawa, Hiroyuki; Nishizawa, Takashi

2015-08-01

Pantoea agglomerans LPS (immunopotentiator from Pantoea agglomerans 1: IP-PA1) has been reported to have anti-inflammatory effects in in vitro and in vivo models. The aim of the present study was to investigate the effects of orally-administered IP-PA1 on atopic dermatitis (AD) symptoms induced by Dermatophagoides farinae body extract (DFE) in NC/Nga mice. Using the NC/Nga AD murine model, mice were orally administered 0.1% (High) or 0.01% (Low) water-containing IP-PA1. Skin lesion assessment and blood collection from the caudal vein was performed on days 0, 7, 21 and 31. On day 31, all mice were sacrificed and blood, skin, spleen, as well as intestine samples, were obtained. Assessment score of the skin lesion and serum immunoglobulin E (IgE) level of both IP-PA1 groups were significantly lower than that of the DFE group on days 14 and 21. The serum periostin and thymus and activation-regulated chemokine (TARC) level of IP-PA1-Low group was significantly lower than that of the DFE group on day 31. On histological examination of the skin, hyperplasia of epidermal and dermal layers and infiltration of inflammatory cells were suppressed by IP-PA1 administration. Deposition of periostin was observed in the DFE group skin tissue. Moreover, the CD4(+)/CD8(+) ratio of splenic T-cells increased by IP-PA1 administration. IP-PA1 administration may have an inhibitory effect on AD skin lesions. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Genetic correction of β-thalassemia patient-specific iPS cells and its use in improving hemoglobin production in irradiated SCID mice.

PubMed

Wang, Yixuan; Zheng, Chen-Guang; Jiang, Yonghua; Zhang, Jiqin; Chen, Jiayu; Yao, Chao; Zhao, Qingguo; Liu, Sheng; Chen, Ke; Du, Juan; Yang, Ze; Gao, Shaorong

2012-04-01

The generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells by over-expression of several transcription factors has the potential to cure many genetic and degenerative diseases currently recalcitrant to traditional clinical approaches. One such genetic disease is β-thalassemia major (Cooley's anemia). This disease is caused by either a point mutation or the deletion of several nucleotides in the β-globin gene, and it threatens the lives of millions of people in China. In the present study, we successfully generated iPSCs from fibroblasts collected from a 2-year-old patient who was diagnosed with a homozygous 41/42 deletion in his β-globin gene. More importantly, we successfully corrected this genetic mutation in the β-thalassemia iPSCs by homologous recombination. Furthermore, transplantation of the genetically corrected iPSCs-derived hematopoietic progenitors into sub-lethally irradiated immune deficient SCID mice showed improved hemoglobin production compared with the uncorrected iPSCs. Moreover, the generation of human β-globin could be detected in the mice transplanted with corrected iPSCs-derived hematopietic progenitors. Our study provides strong evidence that iPSCs generated from a patient with a genetic disease can be corrected by homologous recombination and that the corrected iPSCs have potential clinical uses.

The role of PIP2 and the IP3/DAG pathway in intracellular calcium release and cell survival during nanosecond electric pulse exposures

NASA Astrophysics Data System (ADS)

Steelman, Zachary A.; Tolstykh, Gleb P.; Estlack, Larry E.; Roth, Caleb C.; Ibey, Bennett L.

2015-03-01

Phosphatidylinositol4,5-biphosphate (PIP2) is a membrane phospholipid of particular importance in cell-signaling pathways. Hydrolysis of PIP2 releases inositol-1,4,5-triphosphate (IP3) from the membrane, activating IP3 receptors on the smooth endoplasmic reticulum (ER) and facilitating a release of intracellular calcium stores and activation of protein kinase C (PKC). Recent studies suggest that nanosecond pulsed electric fields (nsPEF) cause depletion of PIP2 in the cellular membrane, activating the IP3 signaling pathway. However, the exact mechanism(s) causing this observed depletion of PIP2 are unknown. Complicating the matter, nsPEF create nanopores in the plasma membrane, allowing calcium to enter the cell and thus causing an increase in intracellular calcium. While elevated intracellular calcium can cause activation of phospholipase C (PLC) (a known catalyst of PIP2 hydrolysis), PIP2 depletion has been shown to occur in the absence of both extracellular and intracellular calcium. These observations have led to the hypothesis that the high electric field itself may be playing a direct role in the hydrolysis of PIP2 from the plasma membrane. To support this hypothesis, we used edelfosine to block PLC and prevent activation of the IP3/DAG pathway in Chinese Hamster Ovarian (CHO) cells prior to applying nsPEF. Fluorescence microscopy was used to monitor intracellular calcium bursts during nsPEF, while MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) survivability assays were utilized to determine whether edelfosine improved cell survival during nsPEF exposure. This work is critical to refine the role of PIP2 in the cellular response to nsPEF, and also to determine the fundamental biological effects of high electric field exposures.

Quantitative analysis of ChIP-seq data uncovers dynamic and sustained H3K4me3 and H3K27me3 modulation in cancer cells under hypoxia.

PubMed

Adriaens, Michiel E; Prickaerts, Peggy; Chan-Seng-Yue, Michelle; van den Beucken, Twan; Dahlmans, Vivian E H; Eijssen, Lars M; Beck, Timothy; Wouters, Bradly G; Voncken, Jan Willem; Evelo, Chris T A

2016-01-01

A comprehensive assessment of the epigenetic dynamics in cancer cells is the key to understanding the molecular mechanisms underlying cancer and to improving cancer diagnostics, prognostics and treatment. By combining genome-wide ChIP-seq epigenomics and microarray transcriptomics, we studied the effects of oxygen deprivation and subsequent reoxygenation on histone 3 trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) in a breast cancer cell line, serving as a model for abnormal oxygenation in solid tumors. A priori, epigenetic markings and gene expression levels not only are expected to vary greatly between hypoxic and normoxic conditions, but also display a large degree of heterogeneity across the cell population. Where traditionally ChIP-seq data are often treated as dichotomous data, the model and experiment here necessitate a quantitative, data-driven analysis of both datasets. We first identified genomic regions with sustained epigenetic markings, which provided a sample-specific reference enabling quantitative ChIP-seq data analysis. Sustained H3K27me3 marking was located around centromeres and intergenic regions, while sustained H3K4me3 marking is associated with genes involved in RNA binding, translation and protein transport and localization. Dynamic marking with both H3K4me3 and H3K27me3 (hypoxia-induced bivalency) was found in CpG-rich regions at loci encoding factors that control developmental processes, congruent with observations in embryonic stem cells. In silico -identified epigenetically sustained and dynamic genomic regions were confirmed through ChIP-PCR in vitro, and obtained results are corroborated by published data and current insights regarding epigenetic regulation.

Suppressive effects of metformin on T-helper 1-related chemokines expression in the human monocytic leukemia cell line THP-1.

PubMed

Chen, Yen-Chun; Kuo, Chang-Hung; Tsai, Ying-Ming; Lin, Yi-Ching; Hsiao, Hui-Pin; Chen, Bai-Hsiun; Chen, Yi-Ting; Wang, Shih-Ling; Hung, Chih-Hsing

2018-04-09

Type 1 and type 2 diabetes mellitus (DM) are chronic T-cell-mediated inflammatory diseases. Metformin is a widely used drug for type 2 DM that reduces the need for insulin in type 1 DM. However, whether metformin has an anti-inflammatory effect for treating DM is unknown. We investigated the anti-inflammatory mechanism of metformin in the human monocytic leukemia cell line THP-1. The human monocytic leukemia cell line THP-1 was pretreated with metformin and stimulated with lipopolysaccharide (LPS). The production of T-helper (Th)-1-related chemokines including interferon-γ-induced protein-10 (IP-10) and monocyte chemoattractant protein-1 (MCP-1), Th2-related chemokine macrophage-derived chemokine, and the proinflammatory chemokine tumor necrosis factor-α was measured using enzyme-linked immunosorbent assay. Intracellular signaling pathways were investigated using Western blot analysis and chromatin immunoprecipitation assay. Metformin suppressed LPS-induced IP-10 and MCP-1 production as well as LPS-induced phosphorylation of c-Jun N-terminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK), and nuclear factor-kappa B (NF-κB). Moreover, metformin suppressed LPS-induced acetylation of histones H3 and H4 at the IP-10 promoter. Metformin suppressed the production of Th1-related chemokines IP-10 and MCP-1 in THP-1 cells. Suppressive effects of metformin on IP-10 production might be attributed at least partially to the JNK, p38, ERK, and NF-κB pathways as well as to epigenetic regulation through the acetylation of histones H3 and H4. These results indicated the therapeutic anti-inflammatory potential of metformin.

Rapid desensitization of vasopressin-stimulated phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine hydrolysis questions the role of these pathways in sustained diacylglycerol formation in A10 vascular-smooth-muscle cells.

PubMed

Plevin, R; Wakelam, M J

1992-08-01

The kinetics of vasopressin-stimulated PtdIns(4,5)P2 and phosphatidylcholine (PtdCho) hydrolysis in relation to sustained diacylglycerol (DAG) formation was investigated in A10 vascular-smooth-muscle cells in culture. Vasopressin stimulated a transient increase in Ins(1,4,5)P3 mass formation, which was mirrored by a decrease in PtdIns(4,5)P2 mass levels. Vasopressin stimulated sustained accumulation of total [3H]inositol phosphates ([3H]IP) in the presence of Li+; however, this was significantly decreased by adding a vasopressin-receptor antagonist at different times after initial stimulation. Vasopressin-stimulated phospholipase D (PLD) activity was found to be a transient phenomenon lasting approx. 2 min. Experiments involving agonist preincubation with subsequent addition of butanol confirmed that vasopressin-stimulated PLD activity was desensitized. Vasopressin stimulated an increase in formation of choline, but not of phosphocholine, suggesting that PLD was the major catalytic route of PtdCho hydrolysis in this cell line. The roles of choline and inositol phospholipid hydrolysis in the prolonged phase of DAG formation was examined by comparing vasopressin-stimulated changes in DAG levels in the presence of butanol, the protein kinase C inhibitor Ro-31-8220 or a V1a-receptor antagonist. Vasopressin-stimulated DAG formation was decreased by 40-50% in the presence of butanol between 1 and 10 min; however, during more prolonged stimulation butanol was without significant effect. In cells pretreated with Ro-31-8220, vasopressin-stimulated DAG formation was decreased by approx. 30% at 2 min, but was significantly potentiated at later times. This coincided with an enhancement of vasopressin-stimulated [3H]IP accumulation. In cells exposed to the V1a-receptor antagonist 5 min after addition of vasopressin, subsequent DAG formation was significantly decreased, indicating that sustained formation of DAG, like [3H]IP accumulation, was dependent on continual agonist receptor activation. The results are discussed in terms of different phospholipid-hydrolytic pathways providing DAG generation.

Negative regulation of DAB2IP by Akt and SCFFbw7 pathways.

PubMed

Dai, Xiangping; North, Brian J; Inuzuka, Hiroyuki

2014-05-30

Deletion of ovarian carcinoma 2/disabled homolog 2 (DOC-2/DAB2) interacting protein (DAB2IP), is a tumor suppressor that serves as a scaffold protein involved in coordinately regulating cell proliferation, survival and apoptotic pathways. DAB2IP is epigenetically down-regulated in a variety of tumors through the action of the histone methyltransferase EZH2. Although DAB2IP is transcriptionally down-regulated in a variety of tumors, it remains unclear if other mechanisms contribute to functional inactivation of DAB2IP. Here we demonstrate that DAB2IP can be functionally down-regulated by two independent mechanisms. First, we identified that Akt1 can phosphorylate DAB2IP on S847, which regulates the interaction between DAB2IP and its effector molecules H-Ras and TRAF2. Second, we demonstrated that DAB2IP can be degraded in part through ubiquitin-proteasome pathway by SCF(Fbw7). DAB2IP harbors two Fbw7 phosho-degron motifs, which can be regulated by the kinase, CK1δ. Our data hence indicate that in addition to epigenetic down-regulation, two additional pathways can functional inactivate DAB2IP. Given that DAB2IP has previously been identified to possess direct causal role in tumorigenesis and metastasis, our data indicate that a variety of pathways may pass through DAB2IP to govern cancer development, and therefore highlight DAB2IP agonists as potential therapeutic approaches for future anti-cancer drug development.

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo

PubMed Central

Roux, Clémence; Saviane, Gaëlle; Pini, Jonathan; Belaïd, Nourhène; Dhib, Gihen; Voha, Christine; Ibáñez, Lidia; Boutin, Antoine; Mazure, Nathalie M.; Wakkach, Abdelilah; Blin-Wakkach, Claudine; Rouleau, Matthieu

2018-01-01

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs), and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4+ FoxP3+ regulatory T (Treg) cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3+-Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo. They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance. PMID:29422893

Immunosuppressive Mesenchymal Stromal Cells Derived from Human-Induced Pluripotent Stem Cells Induce Human Regulatory T Cells In Vitro and In Vivo.

PubMed

Roux, Clémence; Saviane, Gaëlle; Pini, Jonathan; Belaïd, Nourhène; Dhib, Gihen; Voha, Christine; Ibáñez, Lidia; Boutin, Antoine; Mazure, Nathalie M; Wakkach, Abdelilah; Blin-Wakkach, Claudine; Rouleau, Matthieu

2017-01-01

Despite mesenchymal stromal cells (MSCs) are considered as a promising source of cells to modulate immune functions on cells from innate and adaptive immune systems, their clinical use remains restricted (few number, limited in vitro expansion, absence of a full phenotypic characterization, few insights on their in vivo fate). Standardized MSCs derived in vitro from human-induced pluripotent stem (huIPS) cells, remediating part of these issues, are considered as well as a valuable tool for therapeutic approaches, but their functions remained to be fully characterized. We generated multipotent MSCs derived from huiPS cells (huiPS-MSCs), and focusing on their immunosuppressive activity, we showed that human T-cell activation in coculture with huiPS-MSCs was significantly reduced. We also observed the generation of functional CD4 + FoxP3 + regulatory T (Treg) cells. Further tested in vivo in a model of human T-cell expansion in immune-deficient NSG mice, huiPS-MSCs immunosuppressive activity prevented the circulation and the accumulation of activated human T cells. Intracytoplasmic labeling of cytokines produced by the recovered T cells showed reduced percentages of human-differentiated T cells producing Th1 inflammatory cytokines. By contrast, T cells producing IL-10 and FoxP3 + -Treg cells, absent in non-treated animals, were detected in huiPS-MSCs treated mice. For the first time, these results highlight the immunosuppressive activity of the huiPS-MSCs on human T-cell stimulation with a concomitant generation of human Treg cells in vivo . They may favor the development of new tools and strategies based on the use of huiPS cells and their derivatives for the induction of immune tolerance.

Changes in inositol phosphates in wild carrot cells upon initiation of cell wall digestion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rincon, M.; Boss, W.F.

1987-04-01

Previous studies have shown that inositol trisphosphate (IP/sub 3/) stimulated /sup 45/Ca/sup +2/ efflux from fusogenic carrot protoplasts and it was suggested that IP/sub 3/ may serve as a second messenger for the mobilization of intracellular Ca/sup +2/ in higher plant cells. To determine whether or not inositol phosphate metabolism changes in response to external stimuli, the cells were labeled with myo-(2-/sup 3/H) inositol for 18 h and exposed to cell wall digestion enzymes, Driselase. The inositol phosphates were extracted with ice cold 10% TCA and separated by anion exchange chromatography. The radioactivity of the fraction that contained IP/sub 3/more » increased 2-3.8 fold and that which contained inositol bisphosphate increased 1.9-2.6 fold within 1.5 min of exposure to Driselase. After 6 min, the radioactivity of both fractions increased 6-7.7 fold and an increase in inositol monophosphate was observed. These data indicate that inositol phosphate metabolism is stimulated by Driselase and suggest polyphosphoinositide hydrolysis occurs upon initiation of cell wall digestion.« less

Securing TCP/IP and Dial-up Access to Administrative Data.

ERIC Educational Resources Information Center

Conrad, L. Dean

1992-01-01

This article describes Arizona State University's solution to security risk inherent in general access systems such as TCP/IP (Transmission Control Protocol/INTERNET Protocol). Advantages and disadvantages of various options are compared, and the process of selecting a log-on authentication approach involving generation of a different password at…

EQuIP-ped for Success

ERIC Educational Resources Information Center

Ewing, Molly

2015-01-01

The Educators Evaluating the Quality of Instructional Products (EQuIP) Rubric for science is a new tool for science educators that provides criteria by which to examine the alignment and overall quality of lessons and units with respect to the "Next Generation Science Standards" ("NGSS"). The rubric criteria are divided into…

78 FR 76097 - Request for Comment on Petition Filed by Sprint Corporation for Reconsideration of Certain Rules...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-16

... for Internet Protocol Captioned Telephone Service AGENCY: Federal Communications Commission. ACTION... Internet Protocol Captioned Telephone Service (IP CTS) in the IP CTS Reform Order. The Commission seeks... from the Interstate Telecommunications Relay Service Fund (TRS Fund) for minutes of use generated by...

Autocrine CCL2, CXCL4, CXCL9 and CXCL10 signal in retinal endothelial cells and are enhanced in diabetic retinopathy.

PubMed

Nawaz, M I; Van Raemdonck, K; Mohammad, G; Kangave, D; Van Damme, J; Abu El-Asrar, A M; Struyf, S

2013-04-01

This study aimed at examining the presence and role of chemokines (angiogenic CCL2/MCP-1 and angiostatic CXCL4/PF-4, CXCL9/Mig, CXCL10/IP-10) in proliferative diabetic retinopathy (PDR). Regulated chemokine production in human retinal microvascular cells (HRMEC) and chemokine levels in vitreous samples from 40 PDR and 29 non-diabetic patients were analyzed. MCP-1, PF-4, Mig, IP-10 and VEGF levels in vitreous fluid from PDR patients were significantly higher than in controls. Except for IP-10, cytokine levels were significantly higher in PDR with active neovascularization and PDR without traction retinal detachment (TRD) than those in inactive PDR, PDR with TRD and control subjects. Exploratory regression analysis identified associations between higher levels of IP-10 and inactive PDR and PDR with TRD. VEGF levels correlated positively with MCP-1 and IP-10. Significant positive correlations were observed between MCP-1 and IP-10 levels. In line with these clinical findings Western blot analysis revealed increased PF-4 expression in diabetic rat retinas. HRMEC produced MCP-1, Mig and IP-10 after stimulation with IFN-γ, IL-1β or lipopolysaccharide. IFN-γ synergistically enhanced Mig and IP-10 production in response to IL-1β or lipopolysaccharide. MCP-1 was produced by HRMEC in response to VEGF treatment and activated HRMEC via the ERK and Akt/PKB pathway. On the other hand, phosphorylation of ERK induced by VEGF and MCP-1 was inhibited by PF-4, Mig and IP-10. In accordance with inhibition of angiogenic signal transduction pathways, PF-4 inhibited in vitro migration of HRMEC. Thus, regulatory roles for chemokines in PDR were demonstrated. In particular, IP-10 might be associated with the resolution of active PDR and the development of TRD. Copyright © 2013 Elsevier Ltd. All rights reserved.

[UTP regulates spontaneous transient outward currents in porcine coronary artery smooth muscle cells through PLC-IP(3) signaling pathway].

PubMed

Li, Peng-Yun; Zeng, Xiao-Rong; Yang, Yan; Cai, Fang; Li, Miao-Ling; Liu, Zhi-Fei; Pei, Jie; Zhou, Wen

2008-02-25

The aim of the present study was to investigate the effects of inositol 1,4,5-trisphosphate (IP(3))-generating agonist UTP on spontaneous transient outward currents (STOCs), and explore the role of intracellular Ca(2+) release in the current response mediated by IP(3) in porcine coronary artery smooth muscle cells (CASMCs). The coronary artery was excised from the fresh porcine heart and cut into small segments (2 mm × 5 mm) and then transferred to enzymatic dissociation solution for incubation. Single CASMCs were obtained by two-step enzyme digestion at 37 °C. STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration in freshly isolated porcine CASMCs. The currents were amplified and filtered by patch-clamp amplifier (Axopatch 200B), and then the digitized data were recorded by pClamp 9.0 software and further analyzed by MiniAnalysis 6.0 program. The results were as follows: (1) UTP led to conspicuous increases in STOC amplitude by (57.54±5.34)% and in frequency by (77.46±8.42)% (P<0.01, n=38). (2) The specific blocker of phospholipase C (PLC) - U73122 (5 μmol/L) remarkably reduced STOC amplitude by (31.04±7.46)% and frequency by (41.65±16.59)%, respectively (P<0.05, n=10). In the presence of U73122, UTP failed to reactivate STOCs (n=7). (3) Verapamil (20 μmol/L) and CdCl2 (200 μmol/L), two blockers of L-type voltage-dependent Ca(2+) channels, had little effects on STOCs initiated by UTP (n=8). (4) 1 μmol/L bisindolylmaleimide I (BisI), a potent blocker of protein kinase C (PKC), significantly increased STOC amplitude by (65.44±24.66)% and frequency by (61.35±21.47)% (P<0.01, n=12); UTP (40 μmol/L), applied in the presence of 1 μmol/L BisI, could further increase STOC activity (P<0.05, P<0.01, n=12). Subsequent application of ryanodine (50 μmol/L) abolished STOC activity. (5) In the presence of UTP (40 μmol/L), inhibition of IP(3) receptors (IP(3)Rs) by 2-aminoethoxydiphenyl borate (2-APB, 40 μmol/L) reduced STOC amplitude by (24.08±3.97)% (P<0.05, n=8), but had little effect on STOC frequency (n=8). While application of 2-APB (80 μmol/L) significantly reduced STOC amplitude by (31.43±6.34)% and frequency by (40.59±19.01)%, respectively (P<0.05, P<0.01, n=6). Subsequent application of ryanodine (50 μmol/L) completely blocked STOC activity. Pretreatment of cells with 2-APB (40 μmol/L) or ryanodine (50 μmol/L), UTP (40 μmol/L) failed to reactivate STOCs. The results suggest that UTP activates STOCs mainly via PLC and IP(3)-dependent mechanisms. Complex Ca(2+)-mobilization pathways are involved in UTP-mediated STOC activation in porcine CASMCs.

Reprogramming of the MHC-I and its regulation by NFκB in human-induced pluripotent stem cells.

PubMed

Pick, Marjorie; Ronen, Daniel; Yanuka, Ofra; Benvenisty, Nissim

2012-12-01

The immunogenicity of human pluripotent stem cells plays a major role in their potential use in the clinic. We show that, during their reprogramming, human-induced pluripotent stem (iPS) cells downregulate expression of human leukocyte antigen (HLA)-A/B/C and β2 microglobulin (β2M), the two components of major histocompatibility complex-I (MHC-I). MHC-I expression in iPS cells can be restored by differentiation or treatment with interferon-gamma (IFNγ). To analyze the molecular mechanisms that regulate the expression of the MHC-I molecules in human iPS cells, we searched for correlation between the expression of HLA-A/B/C and β2M and the expression of transcription factors that bind to the promoter of these genes. Our results show a significant positive correlation between MHC-I expression and expression of the nuclear factors, nuclear factor kappa B 1 (NFκB1) and RelA, at the levels of RNA, protein and was confirmed by chromatin binding. Concordantly, we detected robust levels of NFκB1 and RelA proteins in the nucleus of somatic cells but not in the iPS cell derived from them. Overexpression of NFκB1 and RelA in undifferentiated pluripotent stem cells led to induction in expression of MHC-I, whereas silencing NFκB1 and RelA by small hairpin RNA decreased the expression of β2M after IFNγ treatment. Our data point to the critical role of NFκB proteins in regulating the MHC-I expression in human pluripotent stem cells. Copyright © 2012 AlphaMed Press.

Protein kinase C (PKC) phosphorylates human platelet inositol trisphosphate 5/sup +/-/-phosphomonoesterase (IP/sub 3/ 5'-p'tase) increasing phosphatase activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Connolly, T.M.; Majerus, P.W.

1986-05-01

Phosphoinositide breakdown in response to thrombin stimulation of human platelets generates messenger molecules that activate PKC (diglyceride) and mobilize Ca/sup + +/ (inositol tris-phosphates). The water soluble products of phospholipase C-mediated metabolism of phosphatidylinositol 4,5-diphosphate are inositol 1,4,5 P/sub 3/ (IP/sub 3/) and inositol 1:2-cyclic 4,5 P/sub 3/ (cIP/sub 3/). A specific phosphatase, IP/sub 3/ 5'-p'tase, cleaves the 5 phosphate from IP/sub 3/ or cIP/sub 3/ to form IP/sub 2/ or cIP/sub 2/ and P/sub i/, none of which mobilizes Ca/sup + +/. Thus, the IP/sub 3/ 5'-p'tase may regulate cellular responses to IP/sub 3/ or cIP/sub 3/. The authorsmore » find that IP/sub 3/ 5'-p'tase isolated from human platelets is phosphorylated by rat brain PKC, resulting in a 4-fold increase in IP/sub 3/ 5'-p'tase activity. The authors phosphorylated IP/sub 3/ 5'-p'tase using ..gamma.. /sup 32/P-ATP and found that the labeled enzyme comigrated on SDS-PAGE with the previously described 40K protein phosphorylated in response to thrombin stimulation of platelets. The similarity of the PKC-phosphorylated IP/sub 3/ 5'-p'tase observed in vitro and the thrombin-stimulated phosphorylated 40K protein known to be phosphorylated by PKC in vivo, suggests that these proteins may be the same. These results suggest that platelet Ca/sup + +/ mobilization maybe regulated by PKC phosphorylation of the IP/sub 3/ 5'-p'tase and can explain the observation that phorbol ester treatment of intact human platelets results in decreased production of IP/sub 3/ and decreased Ca/sup + +/ mobilization upon subsequent thrombin addition.« less

The metastasis suppressor RARRES3 as an endogenous inhibitor of the immunoproteasome expression in breast cancer cells

NASA Astrophysics Data System (ADS)

Anderson, Alison M.; Kalimutho, Murugan; Harten, Sarah; Nanayakkara, Devathri M.; Khanna, Kum Kum; Ragan, Mark A.

2017-01-01

In breast cancer metastasis, the dynamic continuum involving pro- and anti-inflammatory regulators can become compromised. Over 600 genes have been implicated in metastasis to bone, lung or brain but how these genes might contribute to perturbation of immune function is poorly understood. To gain insight, we adopted a gene co-expression network approach that draws on the functional parallels between naturally occurring bone marrow-derived mesenchymal stem cells (BM-MSCs) and cancer stem cells (CSCs). Our network analyses indicate a key role for metastasis suppressor RARRES3, including potential to regulate the immunoproteasome (IP), a specialized proteasome induced under inflammatory conditions. Knockdown of RARRES3 in near-normal mammary epithelial and breast cancer cell lines increases overall transcript and protein levels of the IP subunits, but not of their constitutively expressed counterparts. RARRES3 mRNA expression is controlled by interferon regulatory factor IRF1, an inducer of the IP, and is sensitive to depletion of the retinoid-related receptor RORA that regulates various physiological processes including immunity through modulation of gene expression. Collectively, these findings identify a novel regulatory role for RARRES3 as an endogenous inhibitor of IP expression, and contribute to our evolving understanding of potential pathways underlying breast cancer driven immune modulation.

The ChIP-Seq tools and web server: a resource for analyzing ChIP-seq and other types of genomic data.

PubMed

Ambrosini, Giovanna; Dreos, René; Kumar, Sunil; Bucher, Philipp

2016-11-18

ChIP-seq and related high-throughput chromatin profilig assays generate ever increasing volumes of highly valuable biological data. To make sense out of it, biologists need versatile, efficient and user-friendly tools for access, visualization and itegrative analysis of such data. Here we present the ChIP-Seq command line tools and web server, implementing basic algorithms for ChIP-seq data analysis starting with a read alignment file. The tools are optimized for memory-efficiency and speed thus allowing for processing of large data volumes on inexpensive hardware. The web interface provides access to a large database of public data. The ChIP-Seq tools have a modular and interoperable design in that the output from one application can serve as input to another one. Complex and innovative tasks can thus be achieved by running several tools in a cascade. The various ChIP-Seq command line tools and web services either complement or compare favorably to related bioinformatics resources in terms of computational efficiency, ease of access to public data and interoperability with other web-based tools. The ChIP-Seq server is accessible at http://ccg.vital-it.ch/chipseq/ .

Alteration of Epithelial Structure and Function Associated with PtdIns(4,5)P2 Degradation by a Bacterial Phosphatase

PubMed Central

Mason, David; Mallo, Gustavo V.; Terebiznik, Mauricio R.; Payrastre, Bernard; Finlay, B. Brett; Brumell, John H.; Rameh, Lucia; Grinstein, Sergio

2007-01-01

Elucidation of the role of PtdIns(4,5)P2 in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this phosphoinositide. Here we report that SigD, a phosphatase derived from Salmonella, can effectively hydrolyze PtdIns(4,5)P2, generating PtdIns(5)P. When expressed by microinjecting cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a phosphatase-deficient SigD mutant (C462S). Depletion of PtdIns(4,5)P2 in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cδ, used as a probe for the phosphoinositide by conjugation to green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na+/H+ exchange activity of epithelia was markedly inhibited by depletion of PtdIns(4,5)P2. Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P2 was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P2-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on phosphoinositides, SigD has been shown to convert inositol 1,3,4,5,6-pentakisphosphate (IP5) into inositol 1,4,5,6-tetrakisphosphate (IP4), and the latter has been postulated to mediate the diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP4. In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P2 with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na+/H+ exchange caused by PtdIns(4,5)P2 hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea. PMID:17389247

GnRH signalling pathways and GnRH-induced homologous desensitization in a gonadotrope cell line (alphaT3-1).

PubMed

Poulin, B; Rich, N; Mas, J L; Kordon, C; Enjalbert, A; Drouva, S V

1998-07-25

Exposure of the gonadotrope cells to gonadotropin-releasing hormone (GnRH) reduces their responsiveness to a new GnRH stimulation (homologous desensitization). The time frame as well as the mechanisms underlying this phenomenon are yet unclear. We studied in a gonadotrope cell line (alphaT3-1) the effects of short as well as long term GnRH pretreatments on the GnRH-induced phospholipases-C (PLC), -A2 (PLA2) and -D (PLD) activities, by measuring the production of IP3, total inositol phosphates (IPs), arachidonic acid (AA) and phosphatidylethanol (PEt) respectively. We demonstrated that although rapid desensitization of GnRH-induced IP3 formation did not occur in these cells, persistent stimulation of cells with GnRH or its analogue resulted in a time-dependent attenuation of GnRH-elicited IPs formation. GnRH-induced IPs desensitization was potentiated after direct activation of PKC by the phorbol ester TPA, suggesting the involvement of distinct mechanisms in the uncoupling exerted by either GnRH or TPA on GnRH-stimulated PI hydrolysis. The levels of individual phosphoinositides remained unchanged under any desensitization condition applied. Interestingly, while the GnRH-induced PLA2 activity was rapidly desensitized (2.5 min) after GnRH pretreatments, the neuropeptide-evoked PLD activation was affected at later times, indicating an important time-dependent contribution of these enzymatic activities in the sequential events underlying the GnRH-induced homologous desensitization processes in the gonadotropes. Under GnRH desensitization conditions, TPA was still able to induce PLD activation and to further potentiate the GnRH-evoked PLD activity. AlphaT3-1 cells possess several PKC isoforms which, except PKCzeta, were differentially down-regulated by TPA (PKCalpha, betaII, delta, epsilon, eta) or GnRH (PKCbetaII, delta, epsilon, eta). In spite of the presence of PKC inhibitors or down-regulation of PKC isoforms by TPA, the desensitizing effect of the neuropeptide on GnRH-induced IPs, AA and PEt formation remained unchanged. In conclusion, in alphaT3-1 cells the GnRH-induced homologous desensitization affects the GnRH coupling with PLC, PLA2 and PLD by mechanism(s) which do not implicate TPA-sensitive PKC isoforms, but likely reflect time-dependent modification(s) on the activation processes of the enzymes.

A five-primary photostimulator suitable for studying intrinsically photosensitive retinal ganglion cell functions in humans

PubMed Central

Cao, Dingcai; Nicandro, Nathaniel; Barrionuevo, Pablo A.

2015-01-01

Intrinsically photosensitive retinal ganglion cells (ipRGCs) can respond to light directly through self-contained photopigment, melanopsin. IpRGCs also receive synaptic inputs from rods and cones. Thus, studying ipRGC functions requires a novel photostimulating method that can account for all of the photoreceptor inputs. Here, we introduced an inexpensive LED-based five-primary photostimulator that can control the excitations of rods, S-, M-, L-cones, and melanopsin-containing ipRGCs in humans at constant background photoreceptor excitation levels, a critical requirement for studying the adaptation behavior of ipRGCs with rod, cone, or melanopsin input. We described the theory and technical aspects (including optics, electronics, software, and calibration) of the five-primary photostimulator. Then we presented two preliminary studies using the photostimulator we have implemented to measure melanopsin-mediated pupil responses and temporal contrast sensitivity function (TCSF). The results showed that the S-cone input to pupil responses was antagonistic to the L-, M- or melanopsin inputs, consistent with an S-OFF and (L + M)-ON response property of primate ipRGCs (Dacey et al., 2005). In addition, the melanopsin-mediated TCSF had a distinctive pattern compared with L + M or S-cone mediated TCSF. Other than controlling individual photoreceptor excitation independently, the five-primary photostimulator has the flexibility in presenting stimuli modulating any combination of photoreceptor excitations, which allows researchers to study the mechanisms by which ipRGCs combine various photoreceptor inputs. PMID:25624466

KChIP2 genotype dependence of transient outward current (Ito) properties in cardiomyocytes isolated from male and female mice

PubMed Central

Waldschmidt, Lara; Junkereit, Vera; Bähring, Robert

2017-01-01

The transient outward current (Ito) in cardiomyocytes is largely mediated by Kv4 channels associated with Kv Channel Interacting Protein 2 (KChIP2). A knockout model has documented the critical role of KChIP2 in Ito expression. The present study was conducted to characterize in both sexes the dependence of Ito properties, including current magnitude, inactivation kinetics, recovery from inactivation and voltage dependence of inactivation, on the number of functional KChIP2 alleles. For this purpose we performed whole-cell patch-clamp experiments on isolated left ventricular cardiomyocytes from male and female mice which had different KChIP2 genotypes; i.e., wild-type (KChIP2+/+), heterozygous knockout (KChIP2+/-) or complete knockout of KChIP2 (KChIP2-/-). We found in both sexes a KChIP2 gene dosage effect (i.e., a proportionality between number of alleles and phenotype) on Ito magnitude, however, concerning other Ito properties, KChIP2+/- resembled KChIP2+/+. Only in the total absence of KChIP2 (KChIP2-/-) we observed a slowing of Ito kinetics, a slowing of recovery from inactivation and a negative shift of a portion of the voltage dependence of inactivation. In a minor fraction of KChIP2-/- myocytes Ito was completely lost. The distinct KChIP2 genotype dependences of Ito magnitude and inactivation kinetics, respectively, seen in cardiomyocytes were reproduced with two-electrode voltage-clamp experiments on Xenopus oocytes expressing Kv4.2 and different amounts of KChIP2. Our results corroborate the critical role of KChIP2 in controlling Ito properties. They demonstrate that the Kv4.2/KChIP2 interaction in cardiomyocytes is highly dynamic, with a clear KChIP2 gene dosage effect on Kv4 channel surface expression but not on inactivation gating. PMID:28141821

KChIP2 genotype dependence of transient outward current (Ito) properties in cardiomyocytes isolated from male and female mice.

PubMed

Waldschmidt, Lara; Junkereit, Vera; Bähring, Robert

2017-01-01

The transient outward current (Ito) in cardiomyocytes is largely mediated by Kv4 channels associated with Kv Channel Interacting Protein 2 (KChIP2). A knockout model has documented the critical role of KChIP2 in Ito expression. The present study was conducted to characterize in both sexes the dependence of Ito properties, including current magnitude, inactivation kinetics, recovery from inactivation and voltage dependence of inactivation, on the number of functional KChIP2 alleles. For this purpose we performed whole-cell patch-clamp experiments on isolated left ventricular cardiomyocytes from male and female mice which had different KChIP2 genotypes; i.e., wild-type (KChIP2+/+), heterozygous knockout (KChIP2+/-) or complete knockout of KChIP2 (KChIP2-/-). We found in both sexes a KChIP2 gene dosage effect (i.e., a proportionality between number of alleles and phenotype) on Ito magnitude, however, concerning other Ito properties, KChIP2+/- resembled KChIP2+/+. Only in the total absence of KChIP2 (KChIP2-/-) we observed a slowing of Ito kinetics, a slowing of recovery from inactivation and a negative shift of a portion of the voltage dependence of inactivation. In a minor fraction of KChIP2-/- myocytes Ito was completely lost. The distinct KChIP2 genotype dependences of Ito magnitude and inactivation kinetics, respectively, seen in cardiomyocytes were reproduced with two-electrode voltage-clamp experiments on Xenopus oocytes expressing Kv4.2 and different amounts of KChIP2. Our results corroborate the critical role of KChIP2 in controlling Ito properties. They demonstrate that the Kv4.2/KChIP2 interaction in cardiomyocytes is highly dynamic, with a clear KChIP2 gene dosage effect on Kv4 channel surface expression but not on inactivation gating.

Size resolved infrared spectroscopy of Na(CH3OH)n (n = 4-7) clusters in the OH stretching region: unravelling the interaction of methanol clusters with a sodium atom and the emergence of the solvated electron.

PubMed

Forck, Richard M; Pradzynski, Christoph C; Wolff, Sabine; Ončák, Milan; Slavíček, Petr; Zeuch, Thomas

2012-03-07

Size resolved IR action spectra of neutral sodium doped methanol clusters have been measured using IR excitation modulated photoionisation mass spectroscopy. The Na(CH(3)OH)(n) clusters were generated in a supersonic He seeded expansion of methanol by subsequent Na doping in a pick-up cell. A combined analysis of IR action spectra, IP evolutions and harmonic predictions of IR spectra (using density functional theory) of the most stable structures revealed that for n = 4, 5 structures with an exterior Na atom showing high ionisation potentials (IPs) of ~4 eV dominate, while for n = 6, 7 clusters with lower IPs (~3.2 eV) featuring fully solvated Na atoms and solvated electrons emerge and dominate the IR action spectra. For n = 4 simulations of photoionisation spectra using an ab initio MD approach confirm the dominance of exterior structures and explain the previously reported appearance IP of 3.48 eV by small fractions of clusters with partly solvated Na atoms. Only for this cluster size a shift in the isomer composition with cluster temperature has been observed, which may be related to kinetic stabilisation of less Na solvated clusters at low temperatures. Features of slow fragmentation dynamics of cationic Na(+)(CH(3)OH)(6) clusters have been observed for the photoionisation near the adiabatic limit. This finding points to the relevance of previously proposed non-vertical photoionisation dynamics of this system.

Induced pluripotent stem (iPS) cells from human fetal stem cells.

PubMed

Guillot, Pascale V

2016-02-01

Pluripotency defines the ability of stem cells to differentiate into all the lineages of the three germ layers and self-renew indefinitely. Somatic cells can regain the developmental potential of embryonic stem cells following ectopic expression of a set of transcription factors or, in certain circumstances, via modulation of culture conditions and supplementation with small molecule, that is, induced pluripotent stem (iPS) cells. Here, we discuss the use of fetal tissues for reprogramming, focusing in particular on stem cells derived from human amniotic fluid, and the development of chemical reprogramming. We next address the advantages and disadvantages of deriving pluripotent cells from fetal tissues and the potential clinical applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Reciprocal sensitivity of diffuse large B-cell lymphoma cells to Bcl-2 inhibitors BIRD-2 versus venetoclax

PubMed Central

Vervloessem, Tamara; Akl, Haidar; Tousseyn, Thomas; De Smedt, Humbert; Parys, Jan B.; Bultynck, Geert

2017-01-01

Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2’s hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca2+ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP3R). The cell-permeable Bcl-2/IP3R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2’s BH4 domain, unleashing pro-apoptotic Ca2+-release events. We compared eight “primed to death” diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP3R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP3R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca2+ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca2+-dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca2+-signaling events via its BH4 domain. PMID:29340082

Reciprocal sensitivity of diffuse large B-cell lymphoma cells to Bcl-2 inhibitors BIRD-2 versus venetoclax.

PubMed

Vervloessem, Tamara; Akl, Haidar; Tousseyn, Thomas; De Smedt, Humbert; Parys, Jan B; Bultynck, Geert

2017-12-19

Bcl-2 is often upregulated in cancers to neutralize the BH3-only protein Bim at the mitochondria. BH3 mimetics (e.g. ABT-199 (venetoclax)) kill cancer cells by targeting Bcl-2's hydrophobic cleft and disrupting Bcl-2/Bim complexes. Some cancers with elevated Bcl-2 display poor responses towards BH3 mimetics, suggesting an additional function for anti-apoptotic Bcl-2 in these cancers. Indeed, Bcl-2 via its BH4 domain prevents cytotoxic Ca 2+ release from the endoplasmic reticulum (ER) by directly inhibiting the inositol 1,4,5-trisphosphate receptor (IP 3 R). The cell-permeable Bcl-2/IP 3 R disruptor-2 (BIRD-2) peptide can kill these Bcl-2-dependent cancers by targeting Bcl-2's BH4 domain, unleashing pro-apoptotic Ca 2+ -release events. We compared eight "primed to death" diffuse large B-cell lymphoma cell lines (DLBCL) for their apoptotic sensitivity towards BIRD-2 and venetoclax. By determining their IC 50 using cytometric cell-death analysis, we discovered a reciprocal sensitivity towards venetoclax versus BIRD-2. Using immunoblotting, we quantified the expression levels of IP 3 R2 and Bim in DLBCL cell lysates, revealing that BIRD-2 sensitivity correlated with IP 3 R2 levels but not with Bim levels. Moreover, the requirement of intracellular Ca 2+ for BIRD-2- versus venetoclax-induced cell death was different. Indeed, BAPTA-AM suppressed BIRD-2-induced cell death, but promoted venetoclax-induced cell death in DLBCL cells. Finally, compared to single-agent treatments, combining BIRD-2 with venetoclax synergistically enhanced cell-death induction, correlating with a Ca 2+ -dependent upregulation of Bim after BIRD-2 treatment. Our findings suggest that some cancer cells require Bcl-2 proteins at the mitochondria, preventing Bax activation via its hydrophobic cleft, while others require Bcl-2 proteins at the ER, preventing cytotoxic Ca 2+ -signaling events via its BH4 domain.

Idiopathic paraproteinaemia. IV. The role of genetic factors in the development of monoclonal B cell proliferative disorders--a study in the ageing C57BL/KaLwRij and CBA/BrARij mouse radiation chimeras.

PubMed Central

Radl, J; Heidt, P J; Knaan-Shanzer, S; van Zwieten, M J

1984-01-01

Mouse radiation chimeras, employing strains with a low (CBA/BrARij) and a high (C57BL/KaLwRij) frequency of idiopathic paraproteinaemia (IP), were used in a study on genetic influences in the development of IP, a benign B cell monoclonal proliferative disorder. Taking advantage of the different Igh1 allotypic markers between the two strains, the development of IP with increasing age was investigated by agar electrophoresis, immunoelectrophoresis and immunofixation. Four of 18 CBA recipients transplanted with C57BL bone marrow cells were shown to develop IP of the IgG2a isotype and the Igh1b (donor) allotype during their life. In contrast, none of the 23 C57BL recipients of CBA bone marrow developed an IgG2a paraprotein of the Igh1a allotype. However, in three of these 23 chimeras, an IgG2a and Igh1b (recipient) allotype paraprotein appeared with age; two of these mice proved to be reversals at 12 months and one at 15 months of age. The frequencies of homogeneous immunoglobulins of the donor type in the chimeras corresponded roughly to those of normal mice of the donor strain. Histopathological examination excluded a malignant origin of these monoclonal proliferations. These findings support the view that intrinsic cellular genetic factors are of major importance in the development of IP, a benign B cell neoplasia. PMID:6383667

Inositol 1,4,5-trisphosphate receptor 1 mutation perturbs glucose homeostasis and enhances susceptibility to diet-induced diabetes.

PubMed

Ye, Risheng; Ni, Min; Wang, Miao; Luo, Shengzhan; Zhu, Genyuan; Chow, Robert H; Lee, Amy S

2011-08-01

The inositol 1,4,5-trisphosphate receptors (IP3Rs) as ligand-gated Ca(2)(+) channels are key modulators of cellular processes. Despite advances in understanding their critical role in regulating neuronal function and cell death, how this family of proteins impact cell metabolism is just emerging. Unexpectedly, a transgenic mouse line (D2D) exhibited progressive glucose intolerance as a result of transgene insertion. Inverse PCR was used to identify the gene disruption in the D2D mice. This led to the discovery that Itpr1 is among the ten loci disrupted in chromosome 6. Itpr1 encodes for IP3R1, the most abundant IP3R isoform in mouse brain and also highly expressed in pancreatic β-cells. To study IP3R1 function in glucose metabolism, we used the Itpr1 heterozygous mutant mice, opt/+. Glucose homeostasis in male mice cohorts was examined by multiple approaches of metabolic phenotyping. Under regular diet, the opt/+ mice developed glucose intolerance but no insulin resistance. Decrease in second-phase glucose-stimulated blood insulin level was observed in opt/+ mice, accompanied by reduced β-cell mass and insulin content. Strikingly, when fed with high-fat diet, the opt/+ mice were more susceptible to the development of hyperglycemia, glucose intolerance, and insulin resistance. Collectively, our studies identify the gene Itpr1 being interrupted in the D2D mice and uncover a novel role of IP3R1 in regulation of in vivo glucose homeostasis and development of diet-induced diabetes.

Methemoglobinemia Hemotoxicity of Some Antimalarial 8-Aminoquinoline Analogues and Their Hydroxylated Derivatives: Density Functional Theory Computation of Ionization Potentials.

PubMed

Ding, Yuanqing; Liu, Haining; Tekwani, Babu L; Nanayakkara, N P Dhammika; Khan, Ikhlas A; Walker, Larry A; Doerksen, Robert J

2016-07-18

The administration of primaquine (PQ), an essential drug for the treatment and radical cure of malaria, can lead to methemoglobin formation and life-threatening hemolysis for glucose-6-phosphate dehydrogenase deficient patients. The ionization potential (IP, a quantitative measure of the ability to lose an electron) of the metabolites generated by antimalarial 8-aminoquinoline (8-AQ) drugs like PQ has been believed to be correlated in part to this methemoglobinemia hemotoxicity: the lower the IP of an 8-AQ derivative, the higher the concentration of methemoglobin generated. In this work, demethoxylated primaquine (AQ02) was employed as a model, by intensive computation at the B3LYP-SCRF(PCM)/6-311++G**//B3LYP/6-31G** level in water, to study the effects of hydroxylation at various positions on the ionization potential. Compared to the parent AQ02, the IPs of AQ02's metabolites hydroxylated at N1', C5, and C7 were lower by 61, 30, and 19 kJ/mol, respectively, while differences in the IP relative to PQ were small for hydroxylation at all other positions. The C6 position, at which the IP of the hydroxylated metabolite was greater than that of AQ02, by 2 kJ/mol, was found to be unique. Several literature and proposed 8-AQ analogues were studied to evaluate substituent effects on their potential to generate methemoglobin, with the finding that hydroxylations at N1' and C5 contribute the most to the potential hemotoxicity of PQ-based antimalarials, whereas hydroxylation at C7 has little effect. Phenoxylation at C5 in PQ-based 8-AQs can block the hydroxylation at C5 and reduce the potential for methemoglobin generation, while -CF3 and chlorines attached to the phenolic ring can further reduce the risk. The H-shift at N1' during the cationization of hydroxylated metabolites of 8-AQs sharply decreased their IPs, but this effect can be significantly reduced by the introduction of an electron-withdrawing group to the quinoline core. The results and this approach may be utilized for the design of safer antimalarial 8-AQ analogues.

Endothelial progenitor cells from human dental pulp-derived iPS cells as a therapeutic target for ischemic vascular diseases.

PubMed

Yoo, Chae Hwa; Na, Hee-Jun; Lee, Dong-Seol; Heo, Soon Chul; An, Yuri; Cha, Junghwa; Choi, Chulhee; Kim, Jae Ho; Park, Joo-Cheol; Cho, Yee Sook

2013-11-01

Human dental pulp cells (hDPCs) are a valuable source for the generation of patient-specific human induced pluripotent stem cells (hiPSCs). An advanced strategy for the safe and efficient reprogramming of hDPCs and subsequent lineage-specific differentiation is a critical step toward clinical application. In present research, we successfully generated hDPC-iPSCs using only two non-oncogenic factors: Oct4 and Sox2 (2F hDPC-hiPSCs) and evaluated the feasibility of hDPC-iPSCs as substrates for endothelial progenitor cells (EPCs), contributing to EPC-based therapies. Under conventional differentiation conditions, 2F hDPC-hiPSCs showed higher differentiation efficiency, compared to hiPSCs from other cell types, into multipotent CD34(+) EPCs (2F-hEPCs) capable to differentiate into functional endothelial and smooth muscle cells. The angiogenic and neovasculogenic activities of 2F-hEPCs were confirmed using a Matrigel plug assay in mice. In addition, the therapeutic effects of 2F-hEPC transplantation were confirmed in mouse models of hind-limb ischemia and myocardial infarction. Importantly, 2F-EPCs effectively integrated into newly formed vascular structures and enhanced neovascularization via likely both direct and indirect paracrine mechanisms. 2F hDPC-hiPSCs have a robust capability for the generation of angiogenic and vasculogenic EPCs, representing a strategy for patient-specific EPC therapies and disease modeling, particularly for ischemic vascular diseases. Copyright © 2013 Elsevier Ltd. All rights reserved.

Library construction for next-generation sequencing: Overviews and challenges

PubMed Central

Head, Steven R.; Komori, H. Kiyomi; LaMere, Sarah A.; Whisenant, Thomas; Van Nieuwerburgh, Filip; Salomon, Daniel R.; Ordoukhanian, Phillip

2014-01-01

High-throughput sequencing, also known as next-generation sequencing (NGS), has revolutionized genomic research. In recent years, NGS technology has steadily improved, with costs dropping and the number and range of sequencing applications increasing exponentially. Here, we examine the critical role of sequencing library quality and consider important challenges when preparing NGS libraries from DNA and RNA sources. Factors such as the quantity and physical characteristics of the RNA or DNA source material as well as the desired application (i.e., genome sequencing, targeted sequencing, RNA-seq, ChIP-seq, RIP-seq, and methylation) are addressed in the context of preparing high quality sequencing libraries. In addition, the current methods for preparing NGS libraries from single cells are also discussed. PMID:24502796

Can Stem Cells be Used to Generate New Lungs? Ex Vivo Lung Bioengineering with Decellularized Whole Lung Scaffolds

PubMed Central

Wagner, Darcy E.; Bonvillain, Ryan W.; Jensen, Todd J.; Girard, Eric D.; Bunnell, Bruce A.; Finck, Christine M.; Hoffman, Andrew M.; Weiss, Daniel J.

2013-01-01

For patients with end-stage lung diseases, lung transplantation is the only available therapeutic option. However, the number of suitable donor lungs is insufficient and lung transplants are complicated by significant graft failure and complications of immunosuppressive regimens. An alternative to classic organ replacement is desperately needed. Engineering of bioartificial organs using either natural or synthetic scaffolds is an exciting new potential option for generation of functional pulmonary tissue for human clinical application. Natural organ scaffolds can be generated by decellularization of native tissues; these acellular scaffolds retain the native organ ultrastructure and can be seeded with autologous cells toward the goal of regenerating functional tissues. Several decellularization strategies have been employed for lung, however, there is no consensus on the optimal approach. A variety of cell types have been investigated as potential candidates for effective recellularization of acellular lung scaffolds. Candidate cells that might be best utilized are those which can be easily and reproducibly isolated, expanded in vitro, seeded onto decellularized matrices, induced to differentiate into pulmonary lineage cells, and which survive to functional maturity. Whole lung cell suspensions, endogenous progenitor cells, embryonic and adult stem cells, and induced pluripotent stem (iPS) cells have been investigated for their applicability to repopulate acellular lung matrices. Ideally, patient-derived autologous cells would be used for lung recellularization as they have the potential to reduce the need for post-transplant immunosuppression. Several studies have performed transplantation of rudimentary bioengineered lung scaffolds in animal models with limited, short-term functionality but much further study is needed. PMID:23614471

Spatially-Selective Membrane Permeabilization Induced by Cell-Solution Electrode Atmospheric Pressure Plasma Irradiation

NASA Astrophysics Data System (ADS)

Sasaki, Shota; Hokari, Yutaro; Kanzaki, Makoto; Kaneko, Toshiro

2015-09-01

Gene transfection, which is the process of deliberately introducing nucleic acids into cells, is expected to play an important role in medical treatment because the process is necessary for gene therapy and creation of induced pluripotent stem (iPS) cells. However, the conventional transfection methods have some problems, so we focus attention on promising transfection methods by atmospheric pressure plasma (APP). We have previously reported that the cell membrane permeability, which is closely related with gene transfection, is improved using a cell-solution electrode for generating He-APP. He-APP is irradiated to the solution containing the adherent cells and delivery materials such as fluorescent dyes (YOYO-1) and plasmid DNA (GFP). In case of YOYO-1 delivery, more than 80% of cells can be transferred only in the plasma-irradiated area and the spatially-selective membrane permeabilization is realized by the plasma irradiation. In addition, it is confirmed that plasmid DNA is transfected and the GFP genes are expressed using same APP irradiation system with no obvious cellular damage.

ATF7IP-Mediated Stabilization of the Histone Methyltransferase SETDB1 Is Essential for Heterochromatin Formation by the HUSH Complex.

PubMed

Timms, Richard T; Tchasovnikarova, Iva A; Antrobus, Robin; Dougan, Gordon; Lehner, Paul J

2016-10-11

The histone methyltransferase SETDB1 plays a central role in repressive chromatin processes, but the functional requirement for its binding partner ATF7IP has remained enigmatic. Here, we show that ATF7IP is essential for SETDB1 stability: nuclear SETDB1 protein is degraded by the proteasome upon ablation of ATF7IP. As a result, ATF7IP is critical for repression that requires H3K9 trimethylation by SETDB1, including transgene silencing by the HUSH complex. Furthermore, we show that loss of ATF7IP phenocopies loss of SETDB1 in genome-wide assays. ATF7IP and SETDB1 knockout cells exhibit near-identical defects in the global deposition of H3K9me3, which results in similar dysregulation of the transcriptome. Overall, these data identify a critical functional role for ATF7IP in heterochromatin formation by regulating SETDB1 abundance in the nucleus. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia.

PubMed

Landt, Stephen G; Marinov, Georgi K; Kundaje, Anshul; Kheradpour, Pouya; Pauli, Florencia; Batzoglou, Serafim; Bernstein, Bradley E; Bickel, Peter; Brown, James B; Cayting, Philip; Chen, Yiwen; DeSalvo, Gilberto; Epstein, Charles; Fisher-Aylor, Katherine I; Euskirchen, Ghia; Gerstein, Mark; Gertz, Jason; Hartemink, Alexander J; Hoffman, Michael M; Iyer, Vishwanath R; Jung, Youngsook L; Karmakar, Subhradip; Kellis, Manolis; Kharchenko, Peter V; Li, Qunhua; Liu, Tao; Liu, X Shirley; Ma, Lijia; Milosavljevic, Aleksandar; Myers, Richard M; Park, Peter J; Pazin, Michael J; Perry, Marc D; Raha, Debasish; Reddy, Timothy E; Rozowsky, Joel; Shoresh, Noam; Sidow, Arend; Slattery, Matthew; Stamatoyannopoulos, John A; Tolstorukov, Michael Y; White, Kevin P; Xi, Simon; Farnham, Peggy J; Lieb, Jason D; Wold, Barbara J; Snyder, Michael

2012-09-01

Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals.

ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia

PubMed Central

Landt, Stephen G.; Marinov, Georgi K.; Kundaje, Anshul; Kheradpour, Pouya; Pauli, Florencia; Batzoglou, Serafim; Bernstein, Bradley E.; Bickel, Peter; Brown, James B.; Cayting, Philip; Chen, Yiwen; DeSalvo, Gilberto; Epstein, Charles; Fisher-Aylor, Katherine I.; Euskirchen, Ghia; Gerstein, Mark; Gertz, Jason; Hartemink, Alexander J.; Hoffman, Michael M.; Iyer, Vishwanath R.; Jung, Youngsook L.; Karmakar, Subhradip; Kellis, Manolis; Kharchenko, Peter V.; Li, Qunhua; Liu, Tao; Liu, X. Shirley; Ma, Lijia; Milosavljevic, Aleksandar; Myers, Richard M.; Park, Peter J.; Pazin, Michael J.; Perry, Marc D.; Raha, Debasish; Reddy, Timothy E.; Rozowsky, Joel; Shoresh, Noam; Sidow, Arend; Slattery, Matthew; Stamatoyannopoulos, John A.; Tolstorukov, Michael Y.; White, Kevin P.; Xi, Simon; Farnham, Peggy J.; Lieb, Jason D.; Wold, Barbara J.; Snyder, Michael

2012-01-01

Chromatin immunoprecipitation (ChIP) followed by high-throughput DNA sequencing (ChIP-seq) has become a valuable and widely used approach for mapping the genomic location of transcription-factor binding and histone modifications in living cells. Despite its widespread use, there are considerable differences in how these experiments are conducted, how the results are scored and evaluated for quality, and how the data and metadata are archived for public use. These practices affect the quality and utility of any global ChIP experiment. Through our experience in performing ChIP-seq experiments, the ENCODE and modENCODE consortia have developed a set of working standards and guidelines for ChIP experiments that are updated routinely. The current guidelines address antibody validation, experimental replication, sequencing depth, data and metadata reporting, and data quality assessment. We discuss how ChIP quality, assessed in these ways, affects different uses of ChIP-seq data. All data sets used in the analysis have been deposited for public viewing and downloading at the ENCODE (http://encodeproject.org/ENCODE/) and modENCODE (http://www.modencode.org/) portals. PMID:22955991

Control of IP3-mediated Ca2+ puffs in Xenopus laevis oocytes by the Ca2+-binding protein parvalbumin

PubMed Central

John, Linu M; Mosquera-Caro, Monica; Camacho, Patricia; Lechleiter, James D

2001-01-01

Elementary events of Ca2+ release (Ca2+ puffs) can be elicited from discrete clusters of inositol 1,4,5 trisphosphate receptors (IP3Rs) at low concentrations of IP3. Ca2+ puffs have rarely been observed unless elicited by either hormone treatment or introduction of IP3 into the cell. However, cells appear to have sufficient concentrations of IP3 (0.1-3.0 μM) to induce Ca2+ release under resting conditions. Here, we investigated Ca2+ puff activity in non-stimulated Xenopus oocytes using confocal microscopy. The fluorescent Ca2+ dye indicators Calcium Green 1 and Oregon Green 488 BAPTA-2 were injected into oocytes to monitor basal Ca2+ activity. In this preparation, injection or overexpression of parvalbumin, an EF-hand Ca2+-binding protein (CaBP), induced Ca2+ puffs in resting Xenopus oocytes. This activity was inhibited by heparin, an IP3R channel blocker, and by mutation of the Ca2+-binding sites in parvalbumin. Ca2+ puff activity was also evoked by injection of low concentrations of the Ca2+ chelator EGTA, but not by calbindin D28k, another member of the EF-hand CaBP superfamily. BAPTA and the Ca2+ indicator dye Oregon Green 488 BAPTA-1 evoked Ca2+ puff activity, while the dextran conjugate of Oregon Green 488 BAPTA-1 did not. These data indicate that a Ca2+ buffer must be mobile in order to increase Ca2+ puff activity. Together, the data indicate that some IP3Rs spontaneously release Ca2+ under resting concentrations of IP3. These elementary Ca2+ events appear to be below the level of detection of current imaging techniques. We suggest that parvalbumin evokes Ca2+ puffs by coordinating the activity of elementary IP3R channel openings. We conclude that Ca2+ release can be evoked not only by hormone-induced increases in IP3, but also by expression of mobile cytosolic CaBPs under resting concentrations of IP3. PMID:11507154

Genome wide approaches to identify protein-DNA interactions.

PubMed

Ma, Tao; Ye, Zhenqing; Wang, Liguo

2018-05-29

Transcription factors are DNA-binding proteins that play key roles in many fundamental biological processes. Unraveling their interactions with DNA is essential to identify their target genes and understand the regulatory network. Genome-wide identification of their binding sites became feasible thanks to recent progress in experimental and computational approaches. ChIP-chip, ChIP-seq, and ChIP-exo are three widely used techniques to demarcate genome-wide transcription factor binding sites. This review aims to provide an overview of these three techniques including their experiment procedures, computational approaches, and popular analytic tools. ChIP-chip, ChIP-seq, and ChIP-exo have been the major techniques to study genome-wide in vivo protein-DNA interaction. Due to the rapid development of next-generation sequencing technology, array-based ChIP-chip is deprecated and ChIP-seq has become the most widely used technique to identify transcription factor binding sites in genome-wide. The newly developed ChIP-exo further improves the spatial resolution to single nucleotide. Numerous tools have been developed to analyze ChIP-chip, ChIP-seq and ChIP-exo data. However, different programs may employ different mechanisms or underlying algorithms thus each will inherently include its own set of statistical assumption and bias. So choosing the most appropriate analytic program for a given experiment needs careful considerations. Moreover, most programs only have command line interface so their installation and usage will require basic computation expertise in Unix/Linux. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Sampled control stability of the ESA instrument pointing system

NASA Astrophysics Data System (ADS)

Thieme, G.; Rogers, P.; Sciacovelli, D.

Stability analysis and simulation results are presented for the ESA Instrument Pointing System (IPS) that is to be used in Spacelab's second launch. Of the two IPS plant dynamic models used in the ESA and NASA activities, one is based on six interconnected rigid bodies that represent the IPS and plant dynamic models used in the ESA and NASA activities, one is based on six interconnected rigid bodies that represent the IPS and its payload, while the other follows the NASA practice of defining an IPS-Spacelab 2 plant configuration through a structural finite element model, which is then used to generate modal data for various pointing directions. In both cases, the IPS dynamic plant model is truncated, then discretized at the sampling frequency and interfaces to a PID-based control law. A stability analysis has been carried out in discrete domain for various instrument pointing directions, taking into account suitable parameter variation ranges. A number of time simulations are presented.

Considerations on Experimental Design and Data Analysis of Chromatin Immunoprecipitation Experiments.

PubMed

Jordán-Pla, Antonio; Visa, Neus

2018-01-01

Arguably one of the most valuable techniques to study chromatin organization, ChIP is the method of choice to map the contacts established between proteins and genomic DNA. Ever since its inception, more than 30 years ago, ChIP has been constantly evolving, improving, and expanding its capabilities and reach. Despite its widespread use by many laboratories across a wide variety of disciplines, ChIP assays can be sometimes challenging to design, and are often sensitive to variations in practical implementation.In this chapter, we provide a general overview of the ChIP method and its most common variations, with a special focus on ChIP-seq. We try to address some of the most important aspects that need to be taken into account in order to design and perform experiments that generate the most reproducible, high-quality data. Some of the main topics covered include the use of properly characterized antibodies, alternatives to chromatin preparation, the need for proper controls, and some recommendations about ChIP-seq data analysis.

Generation of safe and therapeutically effective human induced pluripotent stem cell‐derived hepatocyte‐like cells for regenerative medicine

PubMed Central

Takayama, Kazuo; Akita, Naoki; Mimura, Natsumi; Akahira, Rina; Taniguchi, Yukimasa; Ikeda, Makoto; Sakurai, Fuminori; Ohara, Osamu; Morio, Tomohiro

2017-01-01

Hepatocyte‐like cells (HLCs) differentiated from human induced pluripotent stem (iPS) cells are expected to be applied for regenerative medicine. In this study, we attempted to generate safe and therapeutically effective human iPS‐HLCs for hepatocyte transplantation. First, human iPS‐HLCs were generated from a human leukocyte antigen‐homozygous donor on the assumption that the allogenic transplantation might be carried out. Highly efficient hepatocyte differentiation was performed under a feeder‐free condition using human recombinant laminin 111, laminin 511, and type IV collagen. The percentage of asialoglycoprotein receptor 1‐positive cells was greater than 80%, while the percentage of residual undifferentiated cells was approximately 0.003%. In addition, no teratoma formation was observed even at 16 weeks after human iPS‐HLC transplantation. Furthermore, harmful genetic somatic single‐nucleotide substitutions were not observed during the hepatocyte differentiation process. We also developed a cryopreservation protocol for hepatoblast‐like cells without negatively affecting their hepatocyte differentiation potential by programming the freezing temperature. To evaluate the therapeutic potential of human iPS‐HLCs, these cells (1 × 106 cells/mouse) were intrasplenically transplanted into acute liver injury mice treated with 3 mL/kg CCl4 only once and chronic liver injury mice treated with 0.6 mL/kg CCl4 twice weekly for 8 weeks. By human iPS‐HLC transplantation, the survival rate of the acute liver injury mice was significantly increased and the liver fibrosis level of chronic liver injury mice was significantly decreased. Conclusion: We were able to generate safe and therapeutically effective human iPS‐HLCs for hepatocyte transplantation. (Hepatology Communications 2017;1:1058–1069) PMID:29404442

Subtype-selective regulation of IP3 receptors by thimerosal via cysteine residues within the IP3-binding core and suppressor domain

PubMed Central

Khan, Samir A.; Rossi, Ana M.; Riley, Andrew M.; Potter, Barry V. L.; Taylor, Colin W.

2013-01-01

IP3R (IP3 [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca2+ channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP3R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP3-evoked Ca2+ release via IP3R1 and IP3R2, but inhibited IP3R3. Activation of IP3R is initiated by IP3 binding to the IBC (IP3-binding core; residues 224–604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1–223). Thimerosal (100 μM) stimulated IP3 binding to the isolated NT (N-terminal; residues 1–604) of IP3R1 and IP3R2, but not to that of IP3R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP3) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP3R activation. IP3 binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP3R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP3 binding to the chimaeric NT and IP3-evoked Ca2+ release from the chimaeric IP3R. This is the first systematic analysis of the effects of a thiol reagent on each IP3R subtype. We conclude that thimerosal selectively sensitizes IP3R1 and IP3R2 to IP3 by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor. PMID:23282150

Induced pluripotent stem cells with a pathological mitochondrial DNA deletion

PubMed Central

Cherry, Anne B. C.; Gagne, Katelyn E.; McLoughlin, Erin M.; Baccei, Anna; Gorman, Bryan; Hartung, Odelya; Miller, Justine D.; Zhang, Jin; Zon, Rebecca L.; Ince, Tan A.; Neufeld, Ellis J.; Lerou, Paul H.; Fleming, Mark D.; Daley, George Q.; Agarwal, Suneet

2013-01-01

In congenital mitochondrial DNA (mtDNA) disorders, a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues, which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown, and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders, as cytoplasmic genetic material is retained during direct reprogramming. Here we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage, we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth, mitochondrial function, and hematopoietic phenotype when differentiated in vitro, compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. PMID:23400930

Functional Significance of VEGFR-2 on Ovarian Cancer Cells

PubMed Central

Spannuth, Whitney A.; Nick, Alpa M.; Jennings, Nicholas B.; Armaiz-Pena, Guillermo N.; Mangala, Lingegowda S.; Danes, Christopher G.; Lin, Yvonne G.; Merritt, William M.; Thaker, Premal H.; Kamat, Aparna A.; Han, Liz Y.; Tonra, James R.; Coleman, Robert L.; Ellis, Lee M.; Sood, Anil K.

2009-01-01

Vascular endothelial growth factor receptor (VEGFR) has recently been discovered on ovarian cancer cells, but its functional significance is unknown and is the focus of the current study. By protein analysis, A2780-par and HeyA8 ovarian cancer cell lines expressed VEGFR-1 and HeyA8 and SKOV3ip1 expressed VEGFR-2. By in situ hybridization (ISH), 85% of human ovarian cancer specimens showed moderate to high VEGFR-2 expression while only 15% showed moderate to high VEGFR-1 expression. By immunofluorescence, little or no VEGFR-2 was detected in normal ovarian surface epithelial cells, whereas expression was detected in 75% of invasive ovarian cancer specimens. To differentiate between the effects of tumor versus host expression of VEGFR, nude mice were injected with SKOV3ip1 cells and treated with either human VEGFR-2 specific antibody (1121B), murine VEGFR-2 specific antibody (DC101), or the combination. Treatment with 1121B reduced SKOV3ip1 cell migration by 68% (p < 0.01) and invasion by 72% (p < 0.01), but exposure to VEGFR-1 antibody had no effect. Treatment with 1121B effectively blocked VEGF-induced phosphorylation of p130Cas. In vivo, treatment with either DC101 or 1121B significantly reduced tumor growth alone and in combination in the SKOV3ip1 and A2774 models. Decreased tumor burden after treatment with DC101 or 1121B correlated with increased tumor cell apoptosis, decreased proliferative index, and decreased microvessel density. These effects were significantly greater in the combination group (p<0.001). We show functionally active VEGFR-2 is present on most ovarian cancer cells. The observed anti-tumor activity of VEGF-targeted therapies may be mediated by both anti-angiogenic and direct anti-tumor effects. PMID:19058181

Myo-Inositol trisphosphate mobilizes calcium from fusogenic carrot (Daucus carota L. ) protoplasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rincon, M.; Boss, W.F.

1987-02-01

To determine whether or not inositol trisphosphate (IP/sub 3/) mobilizes calcium in higher plant cells; they investigated the effect of IP/sub 3/ on Ca/sup 2 +/ fluxes in fusogenic carrot (Daucus carota L.) protoplasts. The protoplasts were incubated in /sup 45/Ca/sup 2 +/-containing medium and the /sup 45/Ca/sup 2 +/ associated with the protoplasts was monitored with time. Addition of IP/sub 3/ (20 micromolar) caused a 17% net loss of the accumulated /sup 45/Ca/sup 2 +/ within 4 minutes. There was a reuptake of /sup 45/Ca/sup 2 +/ and the protoplasts recovered to their initial value by 10 minutes. Phyticmore » acid (IP/sub 6/), also stimulated /sup 45/Ca/sup 2 +/ efflux from the protoplasts. Both the IP/sub 3/- and the IP/sub 6/-induced /sup 45/Ca/sup 2 +/ efflux were inhibited by the calmodulin antagonist, trifluoperazine.« less

All-IP-Ethernet architecture for real-time sensor-fusion processing

NASA Astrophysics Data System (ADS)

Hiraki, Kei; Inaba, Mary; Tezuka, Hiroshi; Tomari, Hisanobu; Koizumi, Kenichi; Kondo, Shuya

2016-03-01

Serendipter is a device that distinguishes and selects very rare particles and cells from huge amount of population. We are currently designing and constructing information processing system for a Serendipter. The information processing system for Serendipter is a kind of sensor-fusion system but with much more difficulties: To fulfill these requirements, we adopt All IP based architecture: All IP-Ethernet based data processing system consists of (1) sensor/detector directly output data as IP-Ethernet packet stream, (2) single Ethernet/TCP/IP streams by a L2 100Gbps Ethernet switch, (3) An FPGA board with 100Gbps Ethernet I/F connected to the switch and a Xeon based server. Circuits in the FPGA include 100Gbps Ethernet MAC, buffers and preprocessing, and real-time Deep learning circuits using multi-layer neural networks. Proposed All-IP architecture solves existing problem to construct large-scale sensor-fusion systems.

Mini-dystrophin Expression Down-regulates Overactivation of G Protein–mediated IP3 Signaling Pathway in Dystrophin-deficient Muscle Cells

PubMed Central

Balghi, Haouaria; Sebille, Stéphane; Constantin, Bruno; Patri, Sylvie; Thoreau, Vincent; Mondin, Ludivine; Mok, Elise; Kitzis, Alain; Raymond, Guy; Cognard, Christian

2006-01-01

We present here evidence for the enhancement of an inositol 1,4,5-trisphosphate (IP3) mediated calcium signaling pathway in myotubes from dystrophin-deficient cell lines (SolC1(−)) as compared to a cell line from the same origin but transfected with mini-dystrophin (SolD(+)). With confocal microscopy, we demonstrated that calcium rise, induced by the perifusion of a solution containing a high potassium concentration, was higher in SolC1(−) than in SolD(+) myotubes. The analysis of amplitude and kinetics of the calcium increase in SolC1(−) and in SolD(+) myotubes during the exposure with SR Ca2+ channel inhibitors (ryanodine and 2-APB) suggested the presence of two mechanisms of SR calcium release: (1) a fast SR calcium release that depended on ryanodine receptors and (2) a slow SR calcium release mediated by IP3 receptors. Detection analyses of mRNAs (reverse transcriptase [RT]-PCR) and proteins (Western blot and immunolocalization) demonstrated the presence of the three known isoforms of IP3 receptors in both SolC1(−) and SolD(+) myotubes. Furthermore, analysis of the kinetics of the rise in calcium revealed that the slow IP3-dependent release may be increased in the SolC1(−) as compared to the SolD(+), suggesting an inhibitory effect of mini-dystrophin in this signaling pathway. Upon incubation with pertussis toxin (PTX), an inhibitory effect similar to that of the IP3R inhibitor (2-APB) was observed on K+-evoked calcium release. This result suggests the involvement of a Gi protein upstream of the IP3 pathway in these stimulation conditions. A hypothetical model is depicted in which both Gi protein and IP3 production could be involved in K+-evoked calcium release as well as a possible interaction with mini-dystrophin. Our findings demonstrate the existence of a potential relationship between mini-dystrophin and SR calcium release as well as a regulatory role of mini-dystrophin on intracellular signaling. PMID:16446505

Downstream targets of HOXB4 in a cell line model of primitive hematopoietic progenitor cells.

PubMed

Lee, Han M; Zhang, Hui; Schulz, Vincent; Tuck, David P; Forget, Bernard G

2010-08-05

Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell self-renewal and expansion ex vivo and in vivo. To investigate the downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line EML. Two genome-wide analytical techniques were used: RNA expression profiling using microarrays and chromatin immunoprecipitation (ChIP)-chip. RNA expression profiling revealed that 465 gene transcripts were differentially expressed in KLS (c-Kit(+), Lin(-), Sca-1(+))-EML cells that overexpressed HOXB4 (KLS-EML-HOXB4) compared with control KLS-EML cells that were transduced with vector alone. In particular, erythroid-specific gene transcripts were observed to be highly down-regulated in KLS-EML-HOXB4 cells. ChIP-chip analysis revealed that the promoter region for 1910 genes, such as CD34, Sox4, and B220, were occupied by HOXB4 in KLS-EML-HOXB4 cells. Side-by-side comparison of the ChIP-chip and RNA expression profiling datasets provided correlative information and identified Gp49a and Laptm4b as candidate "stemness-related" genes. Both genes were highly ranked in both dataset lists and have been previously shown to be preferentially expressed in hematopoietic stem cells and down-regulated in mature hematopoietic cells, thus making them attractive candidates for future functional studies in hematopoietic cells.

Advanced Models and Algorithms for Self-Similar IP Network Traffic Simulation and Performance Analysis

NASA Astrophysics Data System (ADS)

Radev, Dimitar; Lokshina, Izabella

2010-11-01

The paper examines self-similar (or fractal) properties of real communication network traffic data over a wide range of time scales. These self-similar properties are very different from the properties of traditional models based on Poisson and Markov-modulated Poisson processes. Advanced fractal models of sequentional generators and fixed-length sequence generators, and efficient algorithms that are used to simulate self-similar behavior of IP network traffic data are developed and applied. Numerical examples are provided; and simulation results are obtained and analyzed.

Blood-derived integration-free iPS cell line UKBi011-A from a diagnosed male Alzheimer's disease patient with APOE ɛ4/ɛ4 genotype.

PubMed

Peitz, Michael; Bechler, Tamara; Thiele, Catrin Cornelia; Veltel, Monika; Bloschies, Melanie; Fliessbach, Klaus; Ramirez, Alfredo; Brüstle, Oliver

2018-04-23

Alzheimer's disease (AD) is most the frequent neurodegenerative disease, and the APOE ε4 allele is the most prominent risk factor for late-onset AD. Here, we present an iPSC line generated from peripheral blood cells of a male AD patient employing Sendai virus vectors encoding the transcription factors OCT4, SOX2, KLF4 and c-MYC. The characterized iPSC line expresses typical human pluripotency markers and shows differentiation into all three germ layers, complete reprogramming vector clearance, a normal SNP genotype and maintenance of the APOE ε4/ε4 allele. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Chromatin immunoprecipitation of mouse embryos.

PubMed

Voss, Anne K; Dixon, Mathew P; McLennan, Tamara; Kueh, Andrew J; Thomas, Tim

2012-01-01

During prenatal development, a large number of different cell types are formed, the vast majority of which contain identical genetic material. The basis of the great variety in cell phenotype and function is the differential expression of the approximately 25,000 genes in the mammalian genome. Transcriptional activity is regulated at many levels by proteins, including members of the basal transcriptional apparatus, DNA-binding transcription factors, and chromatin-binding proteins. Importantly, chromatin structure dictates the availability of a specific genomic locus for transcriptional activation as well as the efficiency, with which transcription can occur. Chromatin immunoprecipitation (ChIP) is a method to assess if chromatin modifications or proteins are present at a specific locus. ChIP involves the cross linking of DNA and associated proteins and immunoprecipitation using specific antibodies to DNA-associated proteins followed by examination of the co-precipitated DNA sequences or proteins. In the last few years, ChIP has become an essential technique for scientists studying transcriptional regulation and chromatin structure. Using ChIP on mouse embryos, we can document the presence or absence of specific proteins and chromatin modifications at genomic loci in vivo during mammalian development. Here, we describe a ChIP technique adapted for mouse embryos.

Modulation of A-type potassium channels by a family of calcium sensors.

PubMed

An, W F; Bowlby, M R; Betty, M; Cao, J; Ling, H P; Mendoza, G; Hinson, J W; Mattsson, K I; Strassle, B W; Trimmer, J S; Rhodes, K J

2000-02-03

In the brain and heart, rapidly inactivating (A-type) voltage-gated potassium (Kv) currents operate at subthreshold membrane potentials to control the excitability of neurons and cardiac myocytes. Although pore-forming alpha-subunits of the Kv4, or Shal-related, channel family form A-type currents in heterologous cells, these differ significantly from native A-type currents. Here we describe three Kv channel-interacting proteins (KChIPs) that bind to the cytoplasmic amino termini of Kv4 alpha-subunits. We find that expression of KChIP and Kv4 together reconstitutes several features of native A-type currents by modulating the density, inactivation kinetics and rate of recovery from inactivation of Kv4 channels in heterologous cells. All three KChIPs co-localize and co-immunoprecipitate with brain Kv4 alpha-subunits, and are thus integral components of native Kv4 channel complexes. The KChIPs have four EF-hand-like domains and bind calcium ions. As the activity and density of neuronal A-type currents tightly control responses to excitatory synaptic inputs, these KChIPs may regulate A-type currents, and hence neuronal excitability, in response to changes in intracellular calcium.

De novo prediction of human chromosome structures: Epigenetic marking patterns encode genome architecture.

PubMed

Di Pierro, Michele; Cheng, Ryan R; Lieberman Aiden, Erez; Wolynes, Peter G; Onuchic, José N

2017-11-14

Inside the cell nucleus, genomes fold into organized structures that are characteristic of cell type. Here, we show that this chromatin architecture can be predicted de novo using epigenetic data derived from chromatin immunoprecipitation-sequencing (ChIP-Seq). We exploit the idea that chromosomes encode a 1D sequence of chromatin structural types. Interactions between these chromatin types determine the 3D structural ensemble of chromosomes through a process similar to phase separation. First, a neural network is used to infer the relation between the epigenetic marks present at a locus, as assayed by ChIP-Seq, and the genomic compartment in which those loci reside, as measured by DNA-DNA proximity ligation (Hi-C). Next, types inferred from this neural network are used as an input to an energy landscape model for chromatin organization [Minimal Chromatin Model (MiChroM)] to generate an ensemble of 3D chromosome conformations at a resolution of 50 kilobases (kb). After training the model, dubbed Maximum Entropy Genomic Annotation from Biomarkers Associated to Structural Ensembles (MEGABASE), on odd-numbered chromosomes, we predict the sequences of chromatin types and the subsequent 3D conformational ensembles for the even chromosomes. We validate these structural ensembles by using ChIP-Seq tracks alone to predict Hi-C maps, as well as distances measured using 3D fluorescence in situ hybridization (FISH) experiments. Both sets of experiments support the hypothesis of phase separation being the driving process behind compartmentalization. These findings strongly suggest that epigenetic marking patterns encode sufficient information to determine the global architecture of chromosomes and that de novo structure prediction for whole genomes may be increasingly possible. Copyright © 2017 the Author(s). Published by PNAS.