Genome-wide DNA methylation drives human embryonic stem cell erythropoiesis by remodeling gene expression dynamics.

PubMed

Liu, Zhijing; Feng, Qiang; Sun, Pengpeng; Lu, Yan; Yang, Minlan; Zhang, Xiaowei; Jin, Xiangshu; Li, Yulin; Lu, Shi-Jiang; Quan, Chengshi

2017-12-01

To investigate the role of DNA methylation during erythrocyte production by human embryonic stem cells (hESCs). We employed an erythroid differentiation model from hESCs, and then tracked the genome-wide DNA methylation maps and gene expression patterns through an Infinium HumanMethylation450K BeadChip and an Ilumina Human HT-12 v4 Expression Beadchip, respectively. A negative correlation between DNA methylation and gene expression was substantially enriched during the later differentiation stage and was present in both the promoter and the gene body. Moreover, erythropoietic genes with differentially methylated CpG sites that were primarily enriched in nonisland regions were upregulated, and demethylation of their gene bodies was associated with the presence of enhancers and DNase I hypersensitive sites. Finally, the components of JAK-STAT-NF-κB signaling were DNA hypomethylated and upregulated, which targets the key genes for erythropoiesis. Erythroid lineage commitment by hESCs requires genome-wide DNA methylation modifications to remodel gene expression dynamics.

Phenotype discovery by gene expression profiling: mapping of biological processes linked to BMP-2-mediated osteoblast differentiation.

PubMed

Balint, Eva; Lapointe, David; Drissi, Hicham; van der Meijden, Caroline; Young, Daniel W; van Wijnen, Andre J; Stein, Janet L; Stein, Gary S; Lian, Jane B

2003-05-15

Understanding physiological control of osteoblast differentiation necessitates characterization of the regulatory signals that initiate the events directing a cell to lineage commitment and establishing competency for bone formation. The bone morphogenetic protein, BMP-2, a member of the TGFbeta superfamily, induces osteoblast differentiation and functions through the Smad signal transduction pathway during in vivo bone formation. However, the molecular targets of BMP-mediated gene transcription during the process of osteoblast differentiation have not been comprehensively identified. In the present study, BMP-2 responsive factors involved in the early stages of commitment and differentiation to the osteoblast phenotype were analyzed by microarray gene expression profiling in samples ranging from 1 to 24 h following BMP-2 dependent differentiation of C2C12 premyoblasts into the osteogenic lineage. A total of 1,800 genes were responsive to BMP-2 and expression was modulated from 3- to 14-fold for less than 100 genes during the time course. Approximately 50% of these 100 genes are either up- or downregulated. Major events associated with phenotypic changes towards the osteogenic lineage were identified from hierarchical and functional clustering analyses. BMP-2 immediately responsive genes (1-4 h), which exhibited either transient or sustained expression, reflect activation and repression of non-osseous BMP-2 developmental systems. This initial response was followed by waves of expression of nuclear proteins and developmental regulatory factors including inhibitors of DNA binding, Runx2, C/EBP, Zn finger binding proteins, forkhead, and numerous homeobox proteins (e.g., CDP/cut, paired, distaless, Hox) which are expressed at characterized stages during osteoblast differentiation. A sequential profile of genes mediating changes in cell morphology, cell growth, and basement membrane formation is observed as a secondary transient early response (2-8 h). Commitment to the osteogenic phenotype is recognized by 8 h, reflected by downregulation of most myogenic-related genes and induction of a spectrum of signaling proteins and enzymes facilitating synthesis and assembly of an extracellular skeletal environment. These genes included collagens Type I and VI and the small leucine rich repeat family of proteoglycans (e.g., decorin, biglycan, osteomodulin, fibromodulin, and osteoadherin/osteoglycin) that reached peak expression at 24 h. With extracellular matrix development, the bone phenotype was further established from 16 to 24 h by induction of genes for cell adhesion and communication and enzymes that organize the bone ECM. Our microarray analysis resulted in the discovery of a class of genes, initially described in relation to differentiation of astrocytes and oligodendrocytes that are functionally coupled to signals for cellular extensions. They include nexin, neuropilin, latexin, neuroglian, neuron specific gene 1, and Ulip; suggesting novel roles for these genes in the bone microenvironment. This global analysis identified a multistage molecular and cellular cascade that supports BMP-2-mediated osteoblast differentiation. Copyright 2003 Wiley-Liss, Inc.

The heterocyst regulatory protein HetP and its homologs modulate heterocyst commitment in Anabaena sp. strain PCC 7120.

PubMed

Videau, Patrick; Rivers, Orion S; Hurd, Kathryn; Ushijima, Blake; Oshiro, Reid T; Ende, Rachel J; O'Hanlon, Samantha M; Cozy, Loralyn M

2016-10-24

The commitment of differentiating cells to a specialized fate is fundamental to the correct assembly of tissues within a multicellular organism. Because commitment is often irreversible, entry into and progression through this phase of development must be tightly regulated. Under nitrogen-limiting conditions, the multicellular cyanobacterium Anabaena sp. strain PCC 7120 terminally commits ∼10% of its cells to become specialized nitrogen-fixing heterocysts. Although commitment is known to occur 9-14 h after the induction of differentiation, the factors that regulate the initiation and duration of this phase have yet to be elucidated. Here, we report the identification of four genes that share a functional domain and modulate heterocyst commitment: hetP (alr2818), asl1930, alr2902, and alr3234 Epistatic relationships between all four genes relating to commitment were revealed by deleting them individually and in combination; asl1930 and alr3234 acted most upstream to delay commitment, alr2902 acted next in the pathway to inhibit development, and hetP acted most downstream to drive commitment forward. Possible protein-protein interactions between HetP, its homologs, and the heterocyst master regulator, HetR, were assessed, and interaction partners were defined. Finally, patterns of gene expression for each homolog, as determined by promoter fusions to gfp and reverse transcription-quantitative PCR, were distinct from that of hetP in both spatiotemporal organization and regulation. We posit that a dynamic succession of protein-protein interactions modulates the timing and efficiency of the commitment phase of development and note that this work highlights the utility of a multicellular cyanobacterium as a model for the study of developmental processes.

Different stress-related gene expression in depression and suicide.

PubMed

Zhao, J; Qi, X-R; Gao, S-F; Lu, J; van Wamelen, D J; Kamphuis, W; Bao, A-M; Swaab, D F

2015-09-01

Suicide occurs in some, but not all depressed patients. So far, it remains unknown whether the studied stress-related candidate genes change in depression, suicide or both. The prefrontal cortex (PFC) is involved in, among other things, impulse control and inhibitory behavior and plays an important role in both suicide and depression. We have employed qPCR to study 124 anterior cingulate cortex (ACC) and dorsolateral PFC (DLPFC) brain samples, obtained from two brain banks, from: i) young depressed patients (average age 43 years) who committed suicide (MDD-S) and depressed patients who died from causes other than suicide (MDD-NS) and from ii) elderly depressed patients (average age 75 years) who did not commit suicide (DEP). Both cohorts were individually matched with non-psychiatric non-suicide control subjects. We determined the transcript levels of hypothalamic-pituitary-adrenal axis-regulating molecules (corticotropin-releasing hormone (CRH), CRH receptors, CRH binding protein, mineralocorticoid receptor/glucocorticoid receptor), transcription factors that regulate CRH expression, CRH-stimulating cytokines, chaperone proteins, retinoid signaling, brain-derived neurotrophic factor and tropomyosin-related kinase B, cytochrome proteins, nitric oxide synthase (NOS) and monoamines. In the MDD-S group, expression levels of CRH and neuronal NOS-interacting DHHC domain-containing protein with dendritic mRNA (NIDD) were increased. Other changes were only present in the DEP group, i.e. decreased NIDD, and increased and 5-hydroxytryptamine receptor 1A (5-HT1A) expression levels. Changes were found to be more pronounced in the anterior cingulate cortex than in the dorsolateral PFC. Depressed patients who committed suicide have different gene expression patterns than depressed patients who died of causes other than suicide. Copyright © 2015 Elsevier Ltd. All rights reserved.

Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment

PubMed Central

Moussy, Alice; Cosette, Jérémie; Parmentier, Romuald; da Silva, Cindy; Corre, Guillaume; Richard, Angélique; Gandrillon, Olivier; Stockholm, Daniel

2017-01-01

Individual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterising transcriptional changes in cord blood-derived CD34+ cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the 2 stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the 2 phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology, and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process (which is different from a simple binary switch between 2 options, as it is usually envisioned). PMID:28749943

Cell Fate Decision as High-Dimensional Critical State Transition

PubMed Central

Zhou, Joseph; Castaño, Ivan G.; Leong-Quong, Rebecca Y. Y.; Chang, Hannah; Trachana, Kalliopi; Giuliani, Alessandro; Huang, Sui

2016-01-01

Cell fate choice and commitment of multipotent progenitor cells to a differentiated lineage requires broad changes of their gene expression profile. But how progenitor cells overcome the stability of their gene expression configuration (attractor) to exit the attractor in one direction remains elusive. Here we show that commitment of blood progenitor cells to the erythroid or myeloid lineage is preceded by the destabilization of their high-dimensional attractor state, such that differentiating cells undergo a critical state transition. Single-cell resolution analysis of gene expression in populations of differentiating cells affords a new quantitative index for predicting critical transitions in a high-dimensional state space based on decrease of correlation between cells and concomitant increase of correlation between genes as cells approach a tipping point. The detection of “rebellious cells” that enter the fate opposite to the one intended corroborates the model of preceding destabilization of a progenitor attractor. Thus, early warning signals associated with critical transitions can be detected in statistical ensembles of high-dimensional systems, offering a formal theory-based approach for analyzing single-cell molecular profiles that goes beyond current computational pattern recognition, does not require knowledge of specific pathways, and could be used to predict impending major shifts in development and disease. PMID:28027308

VEGF-C and TGF-β reciprocally regulate mesenchymal stem cell commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes.

PubMed

Igarashi, Yasuyuki; Chosa, Naoyuki; Sawada, Shunsuke; Kondo, Hisatomo; Yaegashi, Takashi; Ishisaki, Akira

2016-04-01

The direction of mesenchymal stem cell (MSC) differentiation is regulated by stimulation with various growth factors and cytokines. We recently established MSC lines, [transforming growth factor-β (TGF-β)-responsive SG‑2 cells, bone morphogenetic protein (BMP)-responsive SG‑3 cells, and TGF-β/BMP-non-responsive SG‑5 cells], derived from the bone marrow of green fluorescent protein-transgenic mice. In this study, to compare gene expression profiles in these MSC lines, we used DNA microarray analysis to characterize the specific gene expression profiles observed in the TGF-β-responsive SG‑2 cells. Among the genes that were highly expressed in the SG‑2 cells, we focused on vascular endothelial growth factor (VEGF) receptor 3 (VEGFR3), the gene product of FMS-like tyrosine kinase 4 (Flt4). We found that VEGF-C, a specific ligand of VEGFR3, significantly induced the cell proliferative activity, migratory ability (as shown by Transwell migration assay), as well as the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the SG‑2 cells. Additionally, VEGF-C significantly increased the expression of prospero homeobox 1 (Prox1) and lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), which are lymphatic endothelial cell markers, and decreased the expression of osteogenic differentiation marker genes in these cells. By contrast, TGF-β significantly increased the expression of early-phase osteogenic differentiation marker genes in the SG‑2 cells and markedly decreased the expression of lymphatic endothelial cell markers. The findings of our study strongly suggest the following: i) that VEGF-C promotes the proliferative activity and migratory ability of MSCs; and ii) VEGF-C and TGF-β reciprocally regulate MSC commitment to differentiation into lymphatic endothelial or osteoblastic phenotypes, respectively. Our findings provide new insight into the molecular mechanisms underlying the regenerative ability of MSCs.

Fine regulation of RhoA and Rock is required for skeletal muscle differentiation.

PubMed

Castellani, Loriana; Salvati, Erica; Alemà, Stefano; Falcone, Germana

2006-06-02

The RhoA GTPase controls a variety of cell functions such as cell motility, cell growth, and gene expression. Previous studies suggested that RhoA mediates signaling inputs that promote skeletal myogenic differentiation. We show here that levels and activity of RhoA protein are down-regulated in both primary avian myoblasts and mouse satellite cells undergoing differentiation, suggesting that a fine regulation of this GTPase is required. In addition, ectopic expression of activated RhoA in primary quail myocytes, but not in mouse myocytes, inhibits accumulation of muscle-specific proteins and cell fusion. By disrupting RhoA signaling with specific inhibitors, we have shown that this GTPase, although required for cell identity in proliferating myoblasts, is not essential for commitment to terminal differentiation and muscle gene expression. Ectopic expression of an activated form of its downstream effector, Rock, impairs differentiation of both avian and mouse myoblasts. Conversely, Rock inhibition with specific inhibitors and small interfering RNA-mediated gene silencing leads to accelerated progression in the lineage and enhanced cell fusion, underscoring a negative regulatory function of Rock in myogenesis. Finally, we have reported that Rock acts independently from RhoA in preventing myoblast exit from the cell cycle and commitment to differentiation and may receive signaling inputs from Raf-1 kinase.

Stem cells isolated from adipose tissue of obese patients show changes in their transcriptomic profile that indicate loss in stemcellness and increased commitment to an adipocyte-like phenotype

PubMed Central

2013-01-01

Background The adipose tissue is an endocrine regulator and a risk factor for atherosclerosis and cardiovascular disease when by excessive accumulation induces obesity. Although the adipose tissue is also a reservoir for stem cells (ASC) their function and “stemcellness” has been questioned. Our aim was to investigate the mechanisms by which obesity affects subcutaneous white adipose tissue (WAT) stem cells. Results Transcriptomics, in silico analysis, real-time polymerase chain reaction (PCR) and western blots were performed on isolated stem cells from subcutaneous abdominal WAT of morbidly obese patients (ASCmo) and of non-obese individuals (ASCn). ASCmo and ASCn gene expression clustered separately from each other. ASCmo showed downregulation of “stemness” genes and upregulation of adipogenic and inflammatory genes with respect to ASCn. Moreover, the application of bioinformatics and Ingenuity Pathway Analysis (IPA) showed that the transcription factor Smad3 was tentatively affected in obese ASCmo. Validation of this target confirmed a significantly reduced Smad3 nuclear translocation in the isolated ASCmo. Conclusions The transcriptomic profile of the stem cells reservoir in obese subcutaneous WAT is highly modified with significant changes in genes regulating stemcellness, lineage commitment and inflammation. In addition to body mass index, cardiovascular risk factor clustering further affect the ASC transcriptomic profile inducing loss of multipotency and, hence, capacity for tissue repair. In summary, the stem cells in the subcutaneous WAT niche of obese patients are already committed to adipocyte differentiation and show an upregulated inflammatory gene expression associated to their loss of stemcellness. PMID:24040759

TLX activates MASH1 for induction of neuronal lineage commitment of adult hippocampal neuroprogenitors.

PubMed

Elmi, Muna; Matsumoto, Yoshiki; Zeng, Zhao-jun; Lakshminarasimhan, Pavithra; Yang, Weiwen; Uemura, Akiyoshi; Nishikawa, Shin-ichi; Moshiri, Alicia; Tajima, Nobuyoshi; Agren, Hans; Funa, Keiko

2010-10-01

The orphan nuclear receptor TLX has been proposed to act as a repressor of cell cycle inhibitors to maintain the neural stem cells in an undifferentiated state, and prevents commitment into astrocyte lineages. However, little is known about the mechanism of TLX in neuronal lineage commitment and differentiation. A majority of adult rat hippocampus-derived progenitors (AHPs) cultured in the presence of FGF express a high level of TLX and a fraction of these cells also express the proneural gene MASH1. Upon FGF withdrawal, TLX rapidly decreased, while MASH1 was intensely expressed within 1h, decreasing gradually to disappear at 24h. Adenoviral transduction of TLX in AHP cells in the absence of FGF transiently increased cell proliferation, however, later resulted in neuronal differentiation by inducing MASH1, Neurogenin1, DCX, and MAP2ab. Furthermore, TLX directly targets and activates the MASH1 promoter through interaction with Sp1, recruiting co-activators whereas dismissing the co-repressor HDAC4. Conversely, silencing of TLX in AHPs decreased beta-III tubulin and DCX expression and promoted glial differentiation. Our results thus suggest that TLX not only acts as a repressor of cell cycle and glial differentiation but also activates neuronal lineage commitment in AHPs. Copyright 2010 Elsevier Inc. All rights reserved.

Temporal and non-permanent division of labor during sorocarp formation in the social amoeba Acytostelium subglobosum.

PubMed

Mohri, Kurato; Kiyota, Yu; Kuwayama, Hidekazu; Urushihara, Hideko

2013-03-15

Somatic cell differentiation is crucial for the development of multicellular organisms. While the development of a fruiting body in Dictyostelium discoideum represents a simple model of this process with separation of stalk cells from the spore lineage, that of Acytostelium subglobosum is not accompanied by cell type separation. This species produces acellular stalks and, seemingly, all aggregated amoebae become spores; however, it possesses homologs for the stalk-cell marker genes of D. discoideum. In this study, we analyzed the spatio-temporal expression of A. subglobosum orthologs for D. discoideum stalk- or spore-lineage markers to clarify the developmental process of A. subglobosum. We first found that the prespore vesicles, which contained spore coat proteins, started to accumulate in the tip region and were observed in the entire sorogen throughout later development, confirming that all A. subglobosum cells became spores. The expression of a stalk-lineage gene ortholog, As-ecmA, started at the mound stage and was prominent in the protruding sorogen. Although two spore-lineage gene orthologs, As-cotD1 and -cotD2, were likewise detected shortly after cell aggregation and increased in intensity until tip formation, their expression diminished in the protruding sorogen. Double-fluorescence staining of these prestalk and prespore marker genes revealed that the expression of these marker genes was mutually exclusive and that expression switching occurred in the early tip. Our results indicate that A. subglobosum cells become committed to the spore lineage first, and then, while keeping this commitment intact, participate in stalk formation. Instead of the permanent division of labor observed in D. discoideum, A. subglobosum produces fruiting bodies by all cells contributing to the formation of the stalk as well as forming spores. Copyright © 2013 Elsevier Inc. All rights reserved.

Constraint of gene expression by the chromatin remodelling protein CHD4 facilitates lineage specification

PubMed Central

O'Shaughnessy-Kirwan, Aoife; Signolet, Jason; Costello, Ita; Gharbi, Sarah; Hendrich, Brian

2015-01-01

Chromatin remodelling proteins are essential for different aspects of metazoan biology, yet functional details of why these proteins are important are lacking. Although it is possible to describe the biochemistry of how they remodel chromatin, their chromatin-binding profiles in cell lines, and gene expression changes upon loss of a given protein, in very few cases can this easily translate into an understanding of how the function of that protein actually influences a developmental process. Here, we investigate how the chromatin remodelling protein CHD4 facilitates the first lineage decision in mammalian embryogenesis. Embryos lacking CHD4 can form a morphologically normal early blastocyst, but are unable to successfully complete the first lineage decision and form functional trophectoderm (TE). In the absence of a functional TE, Chd4 mutant blastocysts do not implant and are hence not viable. By measuring transcript levels in single cells from early embryos, we show that CHD4 influences the frequency at which unspecified cells in preimplantation stage embryos express lineage markers prior to the execution of this first lineage decision. In the absence of CHD4, this frequency is increased in 16-cell embryos, and by the blastocyst stage cells fail to properly adopt a TE gene expression programme. We propose that CHD4 allows cells to undertake lineage commitment in vivo by modulating the frequency with which lineage-specification genes are expressed. This provides novel insight into both how lineage decisions are made in mammalian cells, and how a chromatin remodelling protein functions to facilitate lineage commitment. PMID:26116663

Transcriptome analysis reveals determinant stages controlling human embryonic stem cell commitment to neuronal cells.

PubMed

Li, Yuanyuan; Wang, Ran; Qiao, Nan; Peng, Guangdun; Zhang, Ke; Tang, Ke; Han, Jing-Dong J; Jing, Naihe

2017-12-01

Proper neural commitment is essential for ensuring the appropriate development of the human brain and for preventing neurodevelopmental diseases such as autism spectrum disorders, schizophrenia, and intellectual disorders. However, the molecular mechanisms underlying the neural commitment in humans remain elusive. Here, we report the establishment of a neural differentiation system based on human embryonic stem cells (hESCs) and on comprehensive RNA sequencing analysis of transcriptome dynamics during early hESC differentiation. Using weighted gene co-expression network analysis, we reveal that the hESC neurodevelopmental trajectory has five stages: pluripotency (day 0); differentiation initiation (days 2, 4, and 6); neural commitment (days 8-10); neural progenitor cell proliferation (days 12, 14, and 16); and neuronal differentiation (days 18, 20, and 22). These stages were characterized by unique module genes, which may recapitulate the early human cortical development. Moreover, a comparison of our RNA-sequencing data with several other transcriptome profiling datasets from mice and humans indicated that Module 3 associated with the day 8-10 stage is a critical window of fate switch from the pluripotency to the neural lineage. Interestingly, at this stage, no key extrinsic signals were activated. In contrast, using CRISPR/Cas9-mediated gene knockouts, we also found that intrinsic hub transcription factors, including the schizophrenia-associated SIX3 gene and septo-optic dysplasia-related HESX1 gene, are required to program hESC neural determination. Our results improve the understanding of the mechanism of neural commitment in the human brain and may help elucidate the etiology of human mental disorders and advance therapies for managing these conditions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Androgens inhibit adipogenesis during human adipose stem cell commitment to predipocyte formation

PubMed Central

Chazenbalk, Gregorio; Singh, Prapti; Irge, Dana; Shah, Amy; Abbott, David H; Dumesic, Daniel A

2013-01-01

Androgens play a pivotal role in the regulation of body fat distribution. Adipogenesis is a process whereby multipotent adipose stem cells (ASCs) initially become preadipocytes (ASC commitment to preadipocytes) before differentiating into adipocytes. Androgens inhibit human (h) subcutaneous (SC) abdominal preadipocyte differentiation in both sexes, but their effects on hASC commitment to preadipocyte formation is unknown. We therefore examined whether androgen exposure to human (h) ASCs, isolated from SC abdominal adipose of nonobese women, impairs their commitment to preadipocyte formation and/or subsequent differentiation into adipocytes. For this, isolated hASCs from SC abdominal lipoaspirate were cultured in adipogenesis-inducing medium for 0.5–14 days in the presence of testosterone (T, 0–100 nM) or dihydrotestosterone (DHT, 0–50 nM). Adipogenesis was determined by immunofluorescence microscopy and by quantification of adipogenically relevant transcriptional factors, PPARγ, C/EBPα and C/EBPβ. We found that a 3-day exposure of hASCs to T (50 nM) or DHT (5 nM) in adipogenesis-inducing medium impaired lipid acquisition and decreased PPARγ, C/EBPα and C/EBPβ gene expression. The inhibitory effects of T and DHT at this early-stage of adipocyte differentiation, were partially and completely reversed by flutamide (F, 100 nM), respectively. The effect of androgens on hASC commitment to a preadipocyte phenotype was examined via activation of BMP4 signaling. T (50 nM) and DHT (5nM) significantly inhibited the stimulatory effect of BMP4-induced ASC commitment to the preadipocyte phenotype, as regards PPARγ and C/EBPα gene expression. Our findings indicate that androgens, in part through androgen receptor action, impair BMP4-induced commitment of SC hASCs to preadipocytes and also reduce early-stage adipocyte differentiation, perhaps limiting adipocyte numbers and fat storage in SC abdominal adipose. PMID:23707571

Brg1 modulates enhancer activation in mesoderm lineage commitment

DOE PAGES

Alexander, Jeffrey M.; Hota, Swetansu K.; He, Daniel; ...

2015-03-26

The interplay between different levels of gene regulation in modulating developmental transcriptional programs, such as histone modifications and chromatin remodeling, is not well understood. Here, we show that the chromatin remodeling factor Brg1 is required for enhancer activation in mesoderm induction. In an embryonic stem cell-based directed differentiation assay, the absence of Brg1 results in a failure of cardiomyocyte differentiation and broad deregulation of lineage-specific gene expression during mesoderm induction. We find that Brg1 co-localizes with H3K27ac at distal enhancers and is required for robust H3K27 acetylation at distal enhancers that are activated during mesoderm induction. Brg1 is also requiredmore » to maintain Polycomb-mediated repression of non-mesodermal developmental regulators, suggesting cooperativity between Brg1 and Polycomb complexes. Thus, Brg1 is essential for modulating active and repressive chromatin states during mesoderm lineage commitment, in particular the activation of developmentally important enhancers. In conclusion, these findings demonstrate interplay between chromatin remodeling complexes and histone modifications that, together, ensure robust and broad gene regulation during crucial lineage commitment decisions.« less

TGFβ1-Induced Differentiation of Human Bone Marrow-Derived MSCs Is Mediated by Changes to the Actin Cytoskeleton.

PubMed

Elsafadi, Mona; Manikandan, Muthurangan; Almalki, Sami; Mobarak, Mohammad; Atteya, Muhammad; Iqbal, Zafar; Hashmi, Jamil Amjad; Shaheen, Sameerah; Alajez, Nehad; Alfayez, Musaad; Kassem, Moustapha; Dawud, Raed Abu; Mahmood, Amer

2018-01-01

TGF β is a potent regulator of several biological functions in many cell types, but its role in the differentiation of human bone marrow-derived skeletal stem cells (hMSCs) is currently poorly understood. In the present study, we demonstrate that a single dose of TGF β 1 prior to induction of osteogenic or adipogenic differentiation results in increased mineralized matrix or increased numbers of lipid-filled mature adipocytes, respectively. To identify the mechanisms underlying this TGF β -mediated enhancement of lineage commitment, we compared the gene expression profiles of TGF β 1-treated hMSC cultures using DNA microarrays. In total, 1932 genes were upregulated, and 1298 genes were downregulated. Bioinformatics analysis revealed that TGF β l treatment was associated with an enrichment of genes in the skeletal and extracellular matrix categories and the regulation of the actin cytoskeleton. To investigate further, we examined the actin cytoskeleton following treatment with TGF β 1 and/or cytochalasin D. Interestingly, cytochalasin D treatment of hMSCs enhanced adipogenic differentiation but inhibited osteogenic differentiation. Global gene expression profiling revealed a significant enrichment of pathways related to osteogenesis and adipogenesis and of genes regulated by both TGF β 1 and cytochalasin D. Our study demonstrates that TGF β 1 enhances hMSC commitment to either the osteogenic or adipogenic lineages by reorganizing the actin cytoskeleton.

Biliary Tree Stem Cells, Precursors to Pancreatic Committed Progenitors: Evidence for Possible Life-long Pancreatic Organogenesis

PubMed Central

Wang, Yunfang; Lanzoni, Giacomo; Carpino, Guido; Cui, Cai-Bin; Dominguez-Bendala, Juan; Wauthier, Eliane; Cardinale, Vincenzo; Oikawa, Tsunekazu; Pileggi, Antonello; Gerber, David; Furth, Mark E.; Alvaro, Domenico; Gaudio, Eugenio; Inverardi, Luca; Reid, Lola M.

2013-01-01

Peribiliary glands (PBGs) in bile duct walls, and pancreatic duct glands (PDGs) associated with pancreatic ducts, in humans of all ages, contain a continuous, ramifying network of cells in overlapping maturational lineages. We show that proximal (PBGs)-to-distal (PDGs) maturational lineages start near the duodenum with cells expressing markers of pluripotency (NANOG,OCT4,SOX2), proliferation (Ki67), self-replication (SALL4), and early hepato-pancreatic commitment (SOX9,SOX17,PDX1,LGR5), transitioning to PDG cells with no expression of pluripotency or self-replication markers, maintenance of pancreatic genes (PDX1), and expression of markers of pancreatic endocrine maturation (NGN3,MUC6,insulin). Radial-axis lineages start in PBGs near the ducts’ fibromuscular layers with stem cells and end at the ducts’ lumens with cells devoid of stem cell traits and positive for pancreatic endocrine genes. Biliary tree-derived cells behaved as stem cells in culture under expansion conditions, culture plastic and serum-free Kubota’s Medium, proliferating for months as undifferentiated cells, whereas pancreas-derived cells underwent only ∼8-10 divisions, then partially differentiated towards an islet fate. Biliary tree-derived cells proved precursors of pancreas’ committed progenitors. Both could be driven by 3-dimensional conditions, islet-derived matrix components and a serum-free, hormonally defined medium for an islet fate (HDM-P), to form spheroids with ultrastructural, electrophysiological and functional characteristics of neoislets, including glucose regulatability. Implantation of these neoislets into epididymal fat pads of immuno-compromised mice, chemically rendered diabetic, resulted in secretion of human C-peptide, regulatable by glucose, and able to alleviate hyperglycemia in hosts. The biliary tree-derived stem cells and their connections to pancreatic committed progenitors constitute a biological framework for life-long pancreatic organogenesis. PMID:23847135

Requirement for sustained MAPK signaling in both CD4 and CD8 lineage commitment: a threshold model.

PubMed

Wilkinson, B; Kaye, J

2001-08-01

Although there is general agreement that the RAS/MAPK signaling pathway is required for positive selection of CD4 T cells in the thymus, the role of this pathway in CD8 lineage commitment remains controversial. We show here that the differentiation of isolated cultured thymocytes to the CD8 as well as CD4 T cell lineage is sensitive to MEK inhibition and that both CD4 and CD8 thymocyte differentiation requires sustained MEK signaling. However, CD4 lineage commitment is promoted by a stronger stimulus for longer duration than required for CD8 lineage commitment. Interestingly, CD4 lineage commitment is not irreversibly set even after 10 h of signaling, well past early changes in gene expression. These findings are presented in the context of a model of lineage commitment in which a default pathway of CD8 lineage commitment is altered to CD4 commitment if the thymocyte achieves a threshold level of active MAPK within a certain time frame. Copyright 2001 Academic Press.

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis

PubMed Central

Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K.

2012-01-01

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3′ untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior. PMID:22645327

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis.

PubMed

Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K

2012-06-12

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior.

ROLES OF CELL-INTRINSIC AND MICROENVIRONMENTAL FACTORS IN PHOTORECEPTOR CELL DIFFERENTIATION

PubMed Central

Bradford, Rebecca L.; Wang, Chenwei; Zack, Donald J.; Adler, Ruben

2005-01-01

Photoreceptor differentiation requires the coordinated expression of numerous genes. It is unknown whether those genes share common regulatory mechanisms or are independently regulated by distinct mechanisms. To distinguish between these scenarios, we have used in situ hybridization, RT-PCR and real time PCR to analyze the expression of visual pigments and other photoreceptor-specific genes during chick embryo retinal development in ovo, as well as in retinal cell cultures treated with molecules that regulate the expression of particular visual pigments. In ovo, onset of gene expression was asynchronous, becoming detectable at the time of photoreceptor generation (ED 5–8) for some photoreceptor genes, but only around the time of outer segment formation (ED 14–16) for others. Treatment of retinal cell cultures with activin, staurosporine or CNTF selectively induced or down-regulated specific visual pigment genes, but many cognate rod- or cone-specific genes were not affected by the treatments. These results indicate that many photoreceptor genes are independently regulated during development, are consistent with the existence of at least two distinct stages of gene expression during photoreceptor differentiation, suggest that intrinsic, coordinated regulation of a cascade of gene expression triggered by a commitment to the photoreceptor fate is not a general mechanism of photoreceptor differentiation, and imply that using a single photoreceptor-specific “marker” as a proxy to identify photoreceptor cell fate is problematic. PMID:16120439

Biased gene expression in early honeybee larval development

PubMed Central

2013-01-01

Background Female larvae of the honeybee (Apis mellifera) develop into either queens or workers depending on nutrition. This nutritional stimulus triggers different developmental trajectories, resulting in adults that differ from each other in physiology, behaviour and life span. Results To understand how these trajectories are established we have generated a comprehensive atlas of gene expression throughout larval development. We found substantial differences in gene expression between worker and queen-destined larvae at 6 hours after hatching. Some of these early changes in gene expression are maintained throughout larval development, indicating that caste-specific developmental trajectories are established much earlier than previously thought. Within our gene expression data we identified processes that potentially underlie caste differentiation. Queen-destined larvae have higher expression of genes involved in transcription, translation and protein folding early in development with a later switch to genes involved in energy generation. Using RNA interference, we were able to demonstrate that one of these genes, hexamerin 70b, has a role in caste differentiation. Both queen and worker developmental trajectories are associated with the expression of genes that have alternative splice variants, although only a single variant of a gene tends to be differentially expressed in a given caste. Conclusions Our data, based on the biases in gene expression early in development together with published data, supports the idea that caste development in the honeybee consists of two phases; an initial biased phase of development, where larvae can still switch to the other caste by differential feeding, followed by commitment to a particular developmental trajectory. PMID:24350621

Erasure and reestablishment of random allelic expression imbalance after epigenetic reprogramming

PubMed Central

Jeffries, Aaron Richard; Uwanogho, Dafe Aghogho; Cocks, Graham; Perfect, Leo William; Dempster, Emma; Mill, Jonathan; Price, Jack

2016-01-01

Clonal level random allelic expression imbalance and random monoallelic expression provides cellular heterogeneity within tissues by modulating allelic dosage. Although such expression patterns have been observed in multiple cell types, little is known about when in development these stochastic allelic choices are made. We examine allelic expression patterns in human neural progenitor cells before and after epigenetic reprogramming to induced pluripotency, observing that loci previously characterized by random allelic expression imbalance (0.63% of expressed genes) are generally reset to a biallelic state in induced pluripotent stem cells (iPSCs). We subsequently neuralized the iPSCs and profiled isolated clonal neural stem cells, observing that significant random allelic expression imbalance is reestablished at 0.65% of expressed genes, including novel loci not found to show allelic expression imbalance in the original parental neural progenitor cells. Allelic expression imbalance was associated with altered DNA methylation across promoter regulatory regions, with clones characterized by skewed allelic expression being hypermethylated compared to their biallelic sister clones. Our results suggest that random allelic expression imbalance is established during lineage commitment and is associated with increased DNA methylation at the gene promoter. PMID:27539784

Erasure and reestablishment of random allelic expression imbalance after epigenetic reprogramming.

PubMed

Jeffries, Aaron Richard; Uwanogho, Dafe Aghogho; Cocks, Graham; Perfect, Leo William; Dempster, Emma; Mill, Jonathan; Price, Jack

2016-10-01

Clonal level random allelic expression imbalance and random monoallelic expression provides cellular heterogeneity within tissues by modulating allelic dosage. Although such expression patterns have been observed in multiple cell types, little is known about when in development these stochastic allelic choices are made. We examine allelic expression patterns in human neural progenitor cells before and after epigenetic reprogramming to induced pluripotency, observing that loci previously characterized by random allelic expression imbalance (0.63% of expressed genes) are generally reset to a biallelic state in induced pluripotent stem cells (iPSCs). We subsequently neuralized the iPSCs and profiled isolated clonal neural stem cells, observing that significant random allelic expression imbalance is reestablished at 0.65% of expressed genes, including novel loci not found to show allelic expression imbalance in the original parental neural progenitor cells. Allelic expression imbalance was associated with altered DNA methylation across promoter regulatory regions, with clones characterized by skewed allelic expression being hypermethylated compared to their biallelic sister clones. Our results suggest that random allelic expression imbalance is established during lineage commitment and is associated with increased DNA methylation at the gene promoter. © 2016 Jeffries et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Developmental fate and lineage commitment of singled mouse blastomeres.

PubMed

Lorthongpanich, Chanchao; Doris, Tham Puay Yoke; Limviphuvadh, Vachiranee; Knowles, Barbara B; Solter, Davor

2012-10-01

The inside-outside model has been invoked to explain cell-fate specification of the pre-implantation mammalian embryo. Here, we investigate whether cell-cell interaction can influence the fate specification of embryonic blastomeres by sequentially separating the blastomeres in two-cell stage mouse embryos and continuing separation after each cell division throughout pre-implantation development. This procedure eliminates information provided by cell-cell interaction and cell positioning. Gene expression profiles, polarity protein localization and functional tests of these separated blastomeres reveal that cell interactions, through cell position, influence the fate of the blastomere. Blastomeres, in the absence of cell contact and inner-outer positional information, have a unique pattern of gene expression that is characteristic of neither inner cell mass nor trophectoderm, but overall they have a tendency towards a 'trophectoderm-like' gene expression pattern and preferentially contribute to the trophectoderm lineage.

MicroRNA changes through Müller glia dedifferentiation and early/late rod photoreceptor differentiation.

PubMed

Quintero, H; Gómez-Montalvo, A I; Lamas, M

2016-03-01

Cell-type determination is a complex process driven by the combinatorial effect of extrinsic signals and the expression of transcription factors and regulatory genes. MicroRNAs (miRNAs) are non-coding RNAs that, generally, inhibit the expression of target genes and have been involved, among other processes, in cell identity acquisition. To search for candidate miRNAs putatively involved in mice rod photoreceptor and Müller glia (MG) identity, we compared miRNA expression profiles between late-stage retinal progenitor cells (RPCs), CD73-immunopositive (CD73+) rods and postnatal MG. We found a close similarity between RPCs and CD73+ miRNA expression profiles but a divergence between CD73+ and MG miRNA signatures. We validated preferentially expressed miRNAs in the CD73+ subpopulation (miR-182, 183, 124a, 9(∗), 181c and 301b(∗)) or MG (miR-143, 145, 214, 199a-5p, 199b(∗), and 29a). Taking advantage of the unique capacity of MG to dedifferentiate into progenitor-like cells that can be differentiated to a rod phenotype in response to external cues, we evaluated changes of selected miRNAs in MG-derived progenitors (MGDP) during neuronal differentiation. We found decreased levels of miR-143 and 145, but increased levels of miR-29a in MGDP. In MGDPs committed to early neuronal lineages we found increased levels of miR-124a and upregulation of miR-124a, 9(∗) and 181c during MGDP acquisition of rod phenotypes. Furthermore, we demonstrated that ectopic miR-124 expression is sufficient to enhance early neuronal commitment of MGDP. Our data reveal a dynamic regulation of miRNAs in MGDP through early and late neuronal commitment and miRNAs that could be potential targets to exploit the silent neuronal differentiation capacity of MG in mammals. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

The mouse F3/contactin glycoprotein

PubMed Central

Bizzoca, Antonella; Corsi, Patrizia

2009-01-01

F3/Contactin is an immunoglobulin superfamily component expressed in the nervous tissue of several species. Here we focus on the structural and functional properties of its mouse relative, on the mechanisms driving its regulated expression and on its developmental role. F3/Contactin is differentially expressed in distinct populations of central and peripheral neurons and in some non-neuronal cells. Accordingly, the regulatory region of the underlying gene includes promoter elements undergoing differential activation, associated with an intricate splicing profile, indicating that transcriptional and posttranscriptional mechanisms contribute to its expression. Transgenic models allowed to follow F3/Contactin promoter activation in vivo and to modify F3/Contactin gene expression under a heterologous promoter, which resulted in morphological and functional phenotypes. Besides axonal growth and pathfinding, these concerned earlier events, including precursor proliferation and commitment. This wide role in neural ontogenesis is consistent with the recognized interaction of F3/Contactin with developmental control genes belonging to the Notch pathway. PMID:19372728

Sca-1(+) mesenchymal stromal cells inhibit splenic marginal zone B lymphocytes commitment through Caspase-3.

PubMed

Chen, Yaozhen; Yang, Jialei; Zhang, Hui-Jie; Fan, Hong; An, Ning; Xin, Jiajia; Li, Na; Xu, Jinmei; Yin, Wen; Wu, Zhongliang; Hu, Xingbin

2016-05-01

Mesenchymal stromal cells (MSCs) have been characterized as an important component of hematopoietic niche, which are capable of modulating the immune system through interaction with a wide range of immune cells. Marginal zone B cells, one main type of mature B lymphocytes, play a central role in eliciting antibody response against pathogens. However, how MSCs and its subpopulations regulate marginal zone B cells commitment is unknown yet. In this study, we assessed the contribution of Sca-1(+) MSCs on marginal zone B cells commitment. Our results showed that Sca-1(+) MSCs inhibit the commitment of marginal zone B lymphocytes. The inhibition was exerted through lowered Caspase-3 expression. Furthermore, we found marginal zone B lymphocytes in spleen of Caspase-3 knockout mice decreased and Caspase-3 knockout Sca-1(+) MSCs accounted for the MZB lymphocytes decrease. In conclusion, our investigation provided clues about Sca-1(+) MSCs regulation on the commitment of marginal zone B cells through Caspase-3 gene. © 2016 International Federation for Cell Biology.

The transcriptional landscape of αβ T cell differentiation

PubMed Central

Mingueneau, Michael; Kreslavsky, Taras; Gray, Daniel; Heng, Tracy; Cruse, Richard; Ericson, Jeffrey; Bendall, Sean; Spitzer, Matt; Nolan, Garry; Kobayashi, Koichi; von Boehmer, Harald; Mathis, Diane; Benoist, Christophe

2013-01-01

αβT cell differentiation from thymic precursors is a complex process, explored here with the breadth of ImmGen expression datasets, analyzing how differentiation of thymic precursors gives rise to transcriptomes. After surprisingly gradual changes though early T commitment, transit through the CD4+CD8+ stage involves a shutdown or rare breadth, and correlating tightly with MYC. MHC-driven selection promotes a large-scale transcriptional reactivation. We identify distinct signatures that mark cells destined for positive selection versus apoptotic deletion. Differential expression of surprisingly few genes accompany CD4 or CD8 commitment, a similarity that carries through to peripheral T cells and their activation, revealed by mass cytometry phosphoproteomics. The novel transcripts identified as candidate mediators of key transitions help define the “known unknown” of thymocyte differentiation. PMID:23644507

Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells

PubMed Central

Bargaje, Rhishikesh; Trachana, Kalliopi; Shelton, Martin N.; McGinnis, Christopher S.; Zhou, Joseph X.; Chadick, Cora; Cook, Savannah; Cavanaugh, Christopher; Huang, Sui; Hood, Leroy

2017-01-01

Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or “tipping point” at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations. PMID:28167799

Cell population structure prior to bifurcation predicts efficiency of directed differentiation in human induced pluripotent cells.

PubMed

Bargaje, Rhishikesh; Trachana, Kalliopi; Shelton, Martin N; McGinnis, Christopher S; Zhou, Joseph X; Chadick, Cora; Cook, Savannah; Cavanaugh, Christopher; Huang, Sui; Hood, Leroy

2017-02-28

Steering the differentiation of induced pluripotent stem cells (iPSCs) toward specific cell types is crucial for patient-specific disease modeling and drug testing. This effort requires the capacity to predict and control when and how multipotent progenitor cells commit to the desired cell fate. Cell fate commitment represents a critical state transition or "tipping point" at which complex systems undergo a sudden qualitative shift. To characterize such transitions during iPSC to cardiomyocyte differentiation, we analyzed the gene expression patterns of 96 developmental genes at single-cell resolution. We identified a bifurcation event early in the trajectory when a primitive streak-like cell population segregated into the mesodermal and endodermal lineages. Before this branching point, we could detect the signature of an imminent critical transition: increase in cell heterogeneity and coordination of gene expression. Correlation analysis of gene expression profiles at the tipping point indicates transcription factors that drive the state transition toward each alternative cell fate and their relationships with specific phenotypic readouts. The latter helps us to facilitate small molecule screening for differentiation efficiency. To this end, we set up an analysis of cell population structure at the tipping point after systematic variation of the protocol to bias the differentiation toward mesodermal or endodermal cell lineage. We were able to predict the proportion of cardiomyocytes many days before cells manifest the differentiated phenotype. The analysis of cell populations undergoing a critical state transition thus affords a tool to forecast cell fate outcomes and can be used to optimize differentiation protocols to obtain desired cell populations.

Gene expression profiling of bone marrow mesenchymal stem cells from Osteogenesis Imperfecta patients during osteoblast differentiation.

PubMed

Kaneto, Carla Martins; Pereira Lima, Patrícia S; Prata, Karen Lima; Dos Santos, Jane Lima; de Pina Neto, João Monteiro; Panepucci, Rodrigo Alexandre; Noushmehr, Houtan; Covas, Dimas Tadeu; de Paula, Francisco José Alburquerque; Silva, Wilson Araújo

2017-06-01

Mesenchymal stem cells (MSCs) are precursors present in adult bone marrow that are able to differentiate into osteoblasts, adipocytes and chondroblasts that have gained great importance as a source for cell therapy. Recently, a number of studies involving the analysis of gene expression of undifferentiated MSCs and of MSCs in the differentiation into multiple lineage processes were observed but there is no information concerning the gene expression of MSCs from Osteogenesis Imperfecta (OI) patients. Osteogenesis Imperfecta is characterized as a genetic disorder in which a generalized osteopenia leads to excessive bone fragility and severe bone deformities. The aim of this study was to analyze gene expression profile during osteogenic differentiation from BMMSCs (Bone Marrow Mesenchymal Stem Cells) obtained from patients with Osteogenesis Imperfecta and from control subjects. Bone marrow samples were collected from three normal subjects and five patients with OI. Mononuclear cells were isolated for obtaining mesenchymal cells that had been expanded until osteogenic differentiation was induced. RNA was harvested at seven time points during the osteogenic differentiation period (D0, D+1, D+2, D+7, D+12, D+17 and D+21). Gene expression analysis was performed by the microarray technique and identified several differentially expressed genes. Some important genes for osteoblast differentiation had lower expression in OI patients, suggesting a smaller commitment of these patient's MSCs with the osteogenic lineage. Other genes also had their differential expression confirmed by RT-qPCR. An increase in the expression of genes related to adipocytes was observed, suggesting an increase of adipogenic differentiation at the expense osteogenic differentiation. Copyright © 2017. Published by Elsevier Masson SAS.

Oscillatory fluid flow induces the osteogenic lineage commitment of mesenchymal stem cells: The effect of shear stress magnitude, frequency, and duration.

PubMed

Stavenschi, Elena; Labour, Marie-Noelle; Hoey, David A

2017-04-11

A potent regulator of bone anabolism is physical loading. However, it is currently unclear whether physical stimuli such as fluid shear within the marrow cavity is sufficient to directly drive the osteogenic lineage commitment of resident mesenchymal stem cells (MSC). Therefore, the objective of the study is to employ a systematic analysis of oscillatory fluid flow (OFF) parameters predicted to occur in vivo on early MSC osteogenic responses and late stage lineage commitment. MSCs were exposed to OFF of 1Pa, 2Pa and 5Pa magnitudes at frequencies of 0.5Hz, 1Hz and 2Hz for 1h, 2h and 4h of stimulation. Our findings demonstrate that OFF elicits a positive osteogenic response in MSCs in a shear stress magnitude, frequency, and duration dependent manner that is gene specific. Based on the mRNA expression of osteogenic markers Cox2, Runx2 and Opn after short-term fluid flow stimulation, we identified that a regime of 2Pa shear magnitude and 2Hz frequency induces the most robust and reliable upregulation in osteogenic gene expression. Furthermore, long-term mechanical stimulation utilising this regime, elicits a significant increase in collagen and mineral deposition when compared to static control demonstrating that mechanical stimuli predicted within the marrow is sufficient to directly drive osteogenesis. Copyright © 2017. Published by Elsevier Ltd.

Validation and Interrogation of Differentially Expressed and Alternatively Spliced Genes in African-American Prostate Cancer

DTIC Science & Technology

2015-10-01

Epigenetic Regulation of Tyrosine Receptor Kinases and Childhood Respiratory Disease (Murphy – Duke site PI) Time Commitment: 5% 0.6 calendar...children to the risk of childhood asthma and lower lung function when exposed to prenatal tobacco smoke. Specific Aims: Specific aims are: (1) to...epigenetic and/or genetic variation in the TAM genes are independently or jointly associated with childhood asthma and 24 with attained lung function at

The beetle Tribolium castaneum has a fushi tarazu homolog expressed in stripes during segmentation

NASA Technical Reports Server (NTRS)

Brown, S. J.; Hilgenfeld, R. B.; Denell, R. E.; Spooner, B. S. (Principal Investigator)

1994-01-01

The genetic control of embryonic organization is far better understood for the fruit fly Drosophila melanogaster than for any other metazoan. A gene hierarchy acts during oogenesis and embryogenesis to regulate the establishment of segmentation along the anterior-posterior axis, and homeotic selector genes define developmental commitments within each parasegmental unit delineated. One of the most intensively studied Drosophila segmentation genes is fushi tarazu (ftz), a pair-rule gene expressed in stripes that is important for the establishment of the parasegmental boundaries. Although ftz is flanked by homeotic selector genes conserved throughout the metazoa, there is no evidence that it was part of the ancestral homeotic complex, and it has been unclear when the gene arose and acquired a role in segmentation. We show here that the beetle Tribolium castaneum has a ftz homolog located in its Homeotic complex and expressed in a pair-rule fashion, albeit in a register differing from that of the fly gene. These and other observations demonstrate that a ftz gene preexisted the radiation of holometabolous insects and suggest that it has a role in beetle embryogenesis which differs somewhat from that described in flies.

Plasticity of mesenchymal stem cells under microgravity: from cytoskeletal reorganization to commitment shift

NASA Astrophysics Data System (ADS)

Buravkova, Ludmila

Mesenchymal stem cells (MSCs) can be used to examine osteogenesis of uncommitted cells maintaining the bone differentiation potential such as osteogenic gene expression, osteogenic markers, matrix maturation and mineralization. MSCs are therefore a good model for studying osteogenesis in the space environment. Recent investigations have demonstrated that MSCs change in response to microgravity and, consequently, can be involved in the development of osteopenia detected in space travelers. This is a factor that can limit human space missions due to potential risks of osteoporosis and its aftereffects during and after flight. Simulated microgravity inhibited MSC differentiation towards osteoblasts and accelerated adipocyte development due to cytoskeleton modifications, including its structure and regulation associated with signal transduction cascades. We identified transient changes in the actin cytoskeleton of non-committed human bone marrow MSCs in short-term RPM experiments. In addition, we detected transient changes in the expression of genes encoding actin cytoskeleton proteins and associated elements (ACTA1, ACTG, RHOA, CFL1, VCL). When discussing the microgravity effects on MSC osteogenic differentiation, it should be mentioned the inhibition of Runx2 and ALPL and stimulation of PPARg2 in the MSCs induced for osteogenesis. It is probable that the reciprocal regulation of the two transcription factors is a molecular mechanism underlying progenitor cell response to microgravity. It is very likely that these genes are involved in the universal circuits within which mechanical (or gravity ) signals are sensed by MSCs. Recently, the list of osteogenic markers was extended to include several new proteins as microgravity targets (proteoglycans, osteomodulin, osteoglycin). It can be believed that exposure to microgravity produces similar effects on mature bone cells (osteoblasts) and non-committed osteogenic cells (MSCs). This finds a support in the fact that terminal differentiation stages, i.e., bone matrix mineralization, are inhibited to the same extent in both osteoblasts and MSCs. When examining gravity-dependent molecular processes responsible for susceptibility and/or adaptation of progenitor cells to microgravity, it is important to concentrate not only on recognized pathways of signal transduction, such as MAPK-kinase and cytoskeleton kinase, but also on the expression pattern of genes, which are allegedly not directly involved in the MSC differentiation. Progenitor cells change their transcriptomic profile in the course of their growth, differentiation and maturation It is important to take into account the fact that MSCs can display their differentiation potential as a result of up- or down-regulation of associated or independent genes or their groups. Any interference in this process may cause significant changes in MSC metabolism and commitment. Although the number of relevant studies is much smaller than that of investigations into the typical markers of MSC differentiation in microgravity, there are publications suggesting that the pattern of MSC gene expression undergoes changes when exposed to microgravity. Our RPM experiments with human MSCs revealed significant changes in the so-called stem cell markers: up-regulation of genes associated with cell proliferation, adhesion and intracellular signaling and down-regulation of genes, most of which are involved in cell differentiation. In spite of significant progress achieved in our understanding of the cell gravitational biology, we, however, need to gain better insight into the specific molecular mechanisms underlying the susceptibility of MSCs and more committed osteogenic precursor cells to microgravity effects in vivo and in vitro. A comprehensive study of the biology of these cells is of particular importance in view of the fact that at present age- and drug-related osteoporosis has transformed into a major medical and social problem. This work was supported by grant NSc #371.2014.4

Functional specialization of one copy of glutamine phosphoribosyl pyrophosphate amidotransferase in ureide production from symbiotically fixed nitrogen in Phaseolus vulgaris.

PubMed

Coleto, Inmaculada; Trenas, Almudena T; Erban, Alexander; Kopka, Joachim; Pineda, Manuel; Alamillo, Josefa M

2016-08-01

Purines are essential molecules formed in a highly regulated pathway in all organisms. In tropical legumes, the nitrogen fixed in the nodules is used to generate ureides through the oxidation of de novo synthesized purines. Glutamine phosphoribosyl pyrophosphate amidotransferase (PRAT) catalyses the first committed step of de novo purine synthesis. In Phaseolus vulgaris there are three genes coding for PRAT. The three full-length sequences, which are intron-less genes, were cloned, and their expression levels were determined under conditions that affect the synthesis of purines. One of the three genes, PvPRAT3, is highly expressed in nodules and protein amount and enzymatic activity in these tissues correlate with nitrogen fixation activity. Inhibition of PvPRAT3 gene expression by RNAi-silencing and subsequent metabolomic analysis of the transformed roots shows that PvPRAT3 is essential for the synthesis of ureides in P. vulgaris nodules. © 2016 John Wiley & Sons Ltd.

EBF factors drive expression of multiple classes of target genes governing neuronal development.

PubMed

Green, Yangsook S; Vetter, Monica L

2011-04-30

Early B cell factor (EBF) family members are transcription factors known to have important roles in several aspects of vertebrate neurogenesis, including commitment, migration and differentiation. Knowledge of how EBF family members contribute to neurogenesis is limited by a lack of detailed understanding of genes that are transcriptionally regulated by these factors. We performed a microarray screen in Xenopus animal caps to search for targets of EBF transcriptional activity, and identified candidate targets with multiple roles, including transcription factors of several classes. We determined that, among the most upregulated candidate genes with expected neuronal functions, most require EBF activity for some or all of their expression, and most have overlapping expression with ebf genes. We also found that the candidate target genes that had the most strongly overlapping expression patterns with ebf genes were predicted to be direct transcriptional targets of EBF transcriptional activity. The identification of candidate targets that are transcription factor genes, including nscl-1, emx1 and aml1, improves our understanding of how EBF proteins participate in the hierarchy of transcription control during neuronal development, and suggests novel mechanisms by which EBF activity promotes migration and differentiation. Other candidate targets, including pcdh8 and kcnk5, expand our knowledge of the types of terminal differentiated neuronal functions that EBF proteins regulate.

Nano-hydroxyapatite modulates osteoblast lineage commitment by stimulation of DNA methylation and regulation of gene expression

PubMed Central

Ha, Shin-Woo; Jang, Hae Lin; Nam, Ki Tae; Beck, George R.

2015-01-01

Hydroxyapatite (HA) is the primary structural component of the skeleton and dentition. Under biological conditions, HA does not occur spontaneously and therefore must be actively synthesized by mineralizing cells such as osteoblasts. The mechanism(s) by which HA is actively synthesized by cells and deposited to create a mineralized matrix are not fully understood and the consequences of mineralization on cell function are even less well understood. HA can be chemically synthesized (HAp) and is therefore currently being investigated as a promising therapeutic biomaterial for use as a functional scaffold and implant coating for skeletal repair and dental applications. Here we investigated the biological effects of nano-HAp (10×100 nm) on the lineage commitment and differentiation of bone forming osteoblasts. Exposure of early stage differentiating osteoblasts resulted in dramatic and sustained changes in gene expression, both increased and decreased, whereas later stage osteoblasts were much less responsive. Analysis of the promoter region one of the most responsive genes, alkaline phosphatase, identified the stimulation of DNA methylation following cell exposure to nano-HAp. Collectively, the results reveal the novel epigenetic regulation of cell function by nano-HAp which has significant implication on lineage determination as well as identifying a novel potential therapeutic use of nanomaterials. PMID:26141836

Nano-hydroxyapatite modulates osteoblast lineage commitment by stimulation of DNA methylation and regulation of gene expression.

PubMed

Ha, Shin-Woo; Jang, Hae Lin; Nam, Ki Tae; Beck, George R

2015-10-01

Hydroxyapatite (HA) is the primary structural component of the skeleton and dentition. Under biological conditions, HA does not occur spontaneously and therefore must be actively synthesized by mineralizing cells such as osteoblasts. The mechanism(s) by which HA is actively synthesized by cells and deposited to create a mineralized matrix are not fully understood and the consequences of mineralization on cell function are even less well understood. HA can be chemically synthesized (HAp) and is therefore currently being investigated as a promising therapeutic biomaterial for use as a functional scaffold and implant coating for skeletal repair and dental applications. Here we investigated the biological effects of nano-HAp (10 × 100 nm) on the lineage commitment and differentiation of bone forming osteoblasts. Exposure of early stage differentiating osteoblasts resulted in dramatic and sustained changes in gene expression, both increased and decreased, whereas later stage osteoblasts were much less responsive. Analysis of the promoter region one of the most responsive genes, alkaline phosphatase, identified the stimulation of DNA methylation following cell exposure to nano-HAp. Collectively, the results reveal the novel epigenetic regulation of cell function by nano-HAp which has significant implication on lineage determination as well as identifying a novel potential therapeutic use of nanomaterials. Published by Elsevier Ltd.

Essential and Unexpected Role of YY1 to Promote Mesodermal Cardiac Differentiation

PubMed Central

Gregoire, Serge; Karra, Ravi; Passer, Derek; Deutsch, Marcus-Andre; Krane, Markus; Feistritzer, Rebecca; Sturzu, Anthony; Domian, Ibrahim; Saga, Yumiko; Wu, Sean M.

2013-01-01

Rational Cardiogenesis is regulated by a complex interplay between transcription factors. However, little is known about how these interactions regulate the transition from mesodermal precursors to cardiac progenitor cells (CPCs). Objective To identify novel regulators of mesodermal cardiac lineage commitment. Methods and Results We performed a bioinformatic-based transcription factor binding site analysis on upstream promoter regions of genes that are enriched in embryonic stem cell (ESC)-derived CPCs. From 32 candidate transcription factors screened, we found that YY1, a repressor of sarcomeric gene expression, is present in CPCs in vivo. Interestingly, we uncovered the ability of YY1 to transcriptionally activate Nkx2.5, a key marker of early cardiogenic commitment. YY1 regulates Nkx2.5 expression via a 2.1 kb cardiac-specific enhancer as demonstrated by in vitro luciferase-based assays and in vivo chromatin immunoprecipitation (ChIP) and genome-wide sequencing analysis. Furthermore, the ability of YY1 to activate Nkx2.5 expression depends on its cooperative interaction with Gata4 at a nearby chromatin. Cardiac mesoderm-specific loss-of-function of YY1 resulted in early embryonic lethality. This was corroborated in vitro by ESC-based assays where we show that the overexpression of YY1 enhanced the cardiogenic differentiation of ESCs into CPCs. Conclusion These results demonstrate an essential and unexpected role for YY1 to promote cardiogenesis as a transcriptional activator of Nkx2.5 and other CPC-enriched genes. PMID:23307821

The GMD1 and GMD2 genes of Arabidopsis encode isoforms of GDP-D-mannose 4,6-dehydratase with cell type-specific expression patterns.

PubMed

Bonin, Christopher P; Freshour, Glenn; Hahn, Michael G; Vanzin, Gary F; Reiter, Wolf-Dieter

2003-06-01

l-Fucose (l-Fuc) is a monosaccharide constituent of plant cell wall polysaccharides and glycoproteins. The committing step in the de novo synthesis of l-Fuc is catalyzed by GDP-d-mannose 4,6-dehydratase, which, in Arabidopsis, is encoded by the GMD1 and GMD2 (MUR1) genes. To determine the functional significance of this genetic redundancy, the expression patterns of both genes were investigated via promoter-beta-glucuronidase fusions and immunolocalization of a Fuc-containing epitope. GMD2 is expressed in most cell types of the root, with the notable exception of the root tip where strong expression of GMD1 is observed. Within shoot organs, GMD1::GUS expression is confined to stipules and pollen grains leading to fucosylation of the walls of these cell types in the mur1 mutant. These results suggest that GMD2 represents the major housekeeping gene for the de novo synthesis of GDP-l-Fuc, whereas GMD1 expression is limited to a number of specialized cell types. We conclude that the synthesis of GDP-l-Fuc is controlled in a cell-autonomous manner by differential expression of two isoforms of the same enzyme.

Mitotic inheritance of mRNA facilitates translational activation of the osteogenic-lineage commitment factor Runx2 in progeny of osteoblastic cells

PubMed Central

Varela, Nelson; Aranguiz, Alejandra; Lizama, Carlos; Sepulveda, Hugo; Antonelli, Marcelo; Thaler, Roman; Moreno, Ricardo D.; Montecino, Martin; Stein, Gary S.; van Wijnen, Andre J.; Galindo, Mario

2017-01-01

Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that ‘bookmark’ the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. PMID:26381402

Reverse engineering a mouse embryonic stem cell-specific transcriptional network reveals a new modulator of neuronal differentiation.

PubMed

De Cegli, Rossella; Iacobacci, Simona; Flore, Gemma; Gambardella, Gennaro; Mao, Lei; Cutillo, Luisa; Lauria, Mario; Klose, Joachim; Illingworth, Elizabeth; Banfi, Sandro; di Bernardo, Diego

2013-01-01

Gene expression profiles can be used to infer previously unknown transcriptional regulatory interaction among thousands of genes, via systems biology 'reverse engineering' approaches. We 'reverse engineered' an embryonic stem (ES)-specific transcriptional network from 171 gene expression profiles, measured in ES cells, to identify master regulators of gene expression ('hubs'). We discovered that E130012A19Rik (E13), highly expressed in mouse ES cells as compared with differentiated cells, was a central 'hub' of the network. We demonstrated that E13 is a protein-coding gene implicated in regulating the commitment towards the different neuronal subtypes and glia cells. The overexpression and knock-down of E13 in ES cell lines, undergoing differentiation into neurons and glia cells, caused a strong up-regulation of the glutamatergic neurons marker Vglut2 and a strong down-regulation of the GABAergic neurons marker GAD65 and of the radial glia marker Blbp. We confirmed E13 expression in the cerebral cortex of adult mice and during development. By immuno-based affinity purification, we characterized protein partners of E13, involved in the Polycomb complex. Our results suggest a role of E13 in regulating the division between glutamatergic projection neurons and GABAergic interneurons and glia cells possibly by epigenetic-mediated transcriptional regulation.

Ab initio reconstruction of transcriptomes of pluripotent and lineage committed cells reveals gene structures of thousands of lincRNAs

PubMed Central

Guttman, Mitchell; Garber, Manuel; Levin, Joshua Z.; Donaghey, Julie; Robinson, James; Adiconis, Xian; Fan, Lin; Koziol, Magdalena J.; Gnirke, Andreas; Nusbaum, Chad; Rinn, John L.; Lander, Eric S.; Regev, Aviv

2010-01-01

RNA-Seq provides an unbiased way to study a transcriptome, including both coding and non-coding genes. To date, most RNA-Seq studies have critically depended on existing annotations, and thus focused on expression levels and variation in known transcripts. Here, we present Scripture, a method to reconstruct the transcriptome of a mammalian cell using only RNA-Seq reads and the genome sequence. We apply it to mouse embryonic stem cells, neuronal precursor cells, and lung fibroblasts to accurately reconstruct the full-length gene structures for the vast majority of known expressed genes. We identify substantial variation in protein-coding genes, including thousands of novel 5′-start sites, 3′-ends, and internal coding exons. We then determine the gene structures of over a thousand lincRNA and antisense loci. Our results open the way to direct experimental manipulation of thousands of non-coding RNAs, and demonstrate the power of ab initio reconstruction to render a comprehensive picture of mammalian transcriptomes. PMID:20436462

A unique gene expression signature associated with serotonin 2C receptor RNA editing in the prefrontal cortex and altered in suicide

PubMed Central

Di Narzo, Antonio Fabio; Kozlenkov, Alexey; Roussos, Panos; Hao, Ke; Hurd, Yasmin; Lewis, David A.; Sibille, Etienne; Siever, Larry J.; Koonin, Eugene; Dracheva, Stella

2014-01-01

Editing of the pre-mRNA for the serotonin receptor 2C (5-HT2CR) by site-specific adenosine deamination (A-to-I pre-mRNA editing) substantially increases the functional plasticity of this key neurotransmitter receptor and is thought to contribute to homeostatic mechanisms in neurons. 5-HT2CR mRNA editing generates up to 24 different receptor isoforms. The extent of editing correlates with 5-HT2CR functional activity: more highly edited isoforms exhibit the least function. Altered 5-HT2CR editing has been reported in postmortem brains of suicide victims. We report a comparative analysis of the connections among 5-HT2CR editing, genome-wide gene expression and DNA methylation in suicide victims, individuals with major depressive disorder and non-psychiatric controls. The results confirm previous findings of an overrepresentation of highly edited mRNA variants (which encode hypoactive 5-HT2CR receptors) in the brains of suicide victims. A large set of genes for which the expression level is associated with editing was detected. This signature set of editing-associated genes is significantly enriched for genes that are involved in synaptic transmission, genes that are preferentially expressed in neurons, and genes whose expression is correlated with the level of DNA methylation. Notably, we report that the link between 5-HT2CR editing and gene expression is disrupted in suicide victims. The results suggest that the postulated homeostatic function of 5-HT2CR editing is dysregulated in individuals who committed suicide. PMID:24781207

A HaemAtlas: characterizing gene expression in differentiated human blood cells.

PubMed

Watkins, Nicholas A; Gusnanto, Arief; de Bono, Bernard; De, Subhajyoti; Miranda-Saavedra, Diego; Hardie, Debbie L; Angenent, Will G J; Attwood, Antony P; Ellis, Peter D; Erber, Wendy; Foad, Nicola S; Garner, Stephen F; Isacke, Clare M; Jolley, Jennifer; Koch, Kerstin; Macaulay, Iain C; Morley, Sarah L; Rendon, Augusto; Rice, Kate M; Taylor, Niall; Thijssen-Timmer, Daphne C; Tijssen, Marloes R; van der Schoot, C Ellen; Wernisch, Lorenz; Winzer, Thilo; Dudbridge, Frank; Buckley, Christopher D; Langford, Cordelia F; Teichmann, Sarah; Göttgens, Berthold; Ouwehand, Willem H

2009-05-07

Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are, in part, controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis, we have compared gene expression profiles of human erythroblasts, megakaryocytes, B cells, cytotoxic and helper T cells, natural killer cells, granulocytes, and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors, immunoglobulin superfamily members, and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude, ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition, we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg, GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data, which are freely accessible, will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies.

A HaemAtlas: characterizing gene expression in differentiated human blood cells

PubMed Central

Gusnanto, Arief; de Bono, Bernard; De, Subhajyoti; Miranda-Saavedra, Diego; Hardie, Debbie L.; Angenent, Will G. J.; Attwood, Antony P.; Ellis, Peter D.; Erber, Wendy; Foad, Nicola S.; Garner, Stephen F.; Isacke, Clare M.; Jolley, Jennifer; Koch, Kerstin; Macaulay, Iain C.; Morley, Sarah L.; Rendon, Augusto; Rice, Kate M.; Taylor, Niall; Thijssen-Timmer, Daphne C.; Tijssen, Marloes R.; van der Schoot, C. Ellen; Wernisch, Lorenz; Winzer, Thilo; Dudbridge, Frank; Buckley, Christopher D.; Langford, Cordelia F.; Teichmann, Sarah; Göttgens, Berthold; Ouwehand, Willem H.

2009-01-01

Hematopoiesis is a carefully controlled process that is regulated by complex networks of transcription factors that are, in part, controlled by signals resulting from ligand binding to cell-surface receptors. To further understand hematopoiesis, we have compared gene expression profiles of human erythroblasts, megakaryocytes, B cells, cytotoxic and helper T cells, natural killer cells, granulocytes, and monocytes using whole genome microarrays. A bioinformatics analysis of these data was performed focusing on transcription factors, immunoglobulin superfamily members, and lineage-specific transcripts. We observed that the numbers of lineage-specific genes varies by 2 orders of magnitude, ranging from 5 for cytotoxic T cells to 878 for granulocytes. In addition, we have identified novel coexpression patterns for key transcription factors involved in hematopoiesis (eg, GATA3-GFI1 and GATA2-KLF1). This study represents the most comprehensive analysis of gene expression in hematopoietic cells to date and has identified genes that play key roles in lineage commitment and cell function. The data, which are freely accessible, will be invaluable for future studies on hematopoiesis and the role of specific genes and will also aid the understanding of the recent genome-wide association studies. PMID:19228925

DNA replication fading as proliferating cells advance in their commitment to terminal differentiation.

PubMed

Estefanía, Monturus Ma; Ganier, Olivier; Hernández, Pablo; Schvartzman, Jorge B; Mechali, Marcel; Krimer, Dora B

2012-01-01

Terminal differentiation is the process by which cycling cells stop proliferating to start new specific functions. It involves dramatic changes in chromatin organization as well as gene expression. In the present report we used cell flow cytometry and genome wide DNA combing to investigate DNA replication during murine erythroleukemia-induced terminal cell differentiation. The results obtained indicated that the rate of replication fork movement slows down and the inter-origin distance becomes shorter during the precommitment and commitment periods before cells stop proliferating and accumulate in G1. We propose this is a general feature caused by the progressive heterochromatinization that characterizes terminal cell differentiation.

Canonical FGFs Prevent Osteogenic Lineage Commitment and Differentiation of Human Bone Marrow Stromal Cells Via ERK1/2 Signaling.

PubMed

Simann, Meike; Le Blanc, Solange; Schneider, Verena; Zehe, Viola; Lüdemann, Martin; Schütze, Norbert; Jakob, Franz; Schilling, Tatjana

2017-02-01

Controlling the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) in favor of osteogenesis represents a promising approach for osteoporosis therapy and prevention. Previously, Fibroblast Growth Factor 1 (FGF1) and its subfamily member FGF2 were scored as leading candidates to exercise control over skeletal precursor commitment and lineage decision albeit literature results are highly inconsistent. We show here that FGF1 and 2 strongly prevent the osteogenic commitment and differentiation of hBMSCs. Mineralization of extracellular matrix (ECM) and mRNA expression of osteogenic marker genes Alkaline Phosphatase (ALP), Collagen 1A1 (COL1A1), and Integrin-Binding Sialoprotein (IBSP) were significantly reduced. Furthermore, master regulators of osteogenic commitment like Runt-Related Transcription Factor 2 (RUNX2) and Bone Morphogenetic Protein 4 (BMP4) were downregulated. When administered under adipogenic culture conditions, canonical FGFs did not support osteogenic marker expression. Moreover despite the presence of osteogenic differentiation factors, FGFs even disabled the pro-osteogenic lineage decision of pre-differentiated adipocytic cells. In contrast to FGF Receptor 2 (FGFR2), FGFR1 was stably expressed throughout osteogenic and adipogenic differentiation and FGF addition. Moreover, FGFR1 and Extracellular Signal-Regulated Kinases 1 and 2 (ERK1/2) were found to be responsible for underlying signal transduction using respective inhibitors. Taken together, we present new findings indicating that canonical FGFR-ERK1/2 signaling entrapped hBMSCs in a pre-committed state and arrested further maturation of committed precursors. Our results might aid in unraveling and controlling check points relevant for ageing-associated aberrant adipogenesis with consequences for the treatment of degenerative diseases such as osteoporosis and for skeletal tissue engineering strategies. J. Cell. Biochem. 118: 263-275, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system

PubMed Central

Kutleša, Snježana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B.; Jurecic, Roland

2011-01-01

Objective Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. Materials and Methods EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. Results The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Tα, RAG-1, and T-cell receptor – Vβ genes; and 3) produced interferon-γ in response to T-cell receptor stimulation. Conclusions These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation. PMID:19447159

T-cell differentiation of multipotent hematopoietic cell line EML in the OP9-DL1 coculture system.

PubMed

Kutlesa, Snjezana; Zayas, Jennifer; Valle, Alexandra; Levy, Robert B; Jurecic, Roland

2009-08-01

Multipotent hematopoietic cell line EML can differentiate into myeloid, erythroid, megakaryocytic, and B-lymphoid lineages, but it remained unknown whether EML cells have T-cell developmental potential as well. The goal of this study was to determine whether the coculture with OP9 stromal cells expressing Notch ligand Delta-like 1 (OP9-DL1) could induce differentiation of EML cells into T-cell lineage. EML cells were cocultured with control OP9 or OP9-DL1 stromal cells in the presence of cytokines (stem cell factor, interleukin-7, and Fms-like tyrosine kinase 3 ligand). Their T-cell lineage differentiation was assessed through flow cytometry and reverse transcription polymerase chain reaction expression analysis of cell surface markers and genes characterizing and associated with specific stages of T-cell development. The phenotypic, molecular, and functional analysis has revealed that in EML/OP9-DL1 cocultures with cytokines, but not in control EML/OP9 cocultures, EML cell line undergoes T-cell lineage commitment and differentiation. In OP9-DL1 cocultures, EML cell line has differentiated into cells that 1) resembled double-negative, double-positive, and single-positive stages of T-cell development; 2) initiated expression of GATA-3, Pre-Talpha, RAG-1, and T-cell receptor-Vbeta genes; and 3) produced interferon-gamma in response to T-cell receptor stimulation. These results support the notion that EML cell line has the capacity for T-cell differentiation. Remarkably, induction of T-lineage gene expression and differentiation of EML cells into distinct stages of T-cell development were very similar to previously described T-cell differentiation of adult hematopoietic stem cells and progenitors in OP9-DL1 cocultures. Thus, EML/OP9-DL1 coculture could be a useful experimental system to study the role of particular genes in T-cell lineage specification, commitment, and differentiation.

Revealing complex function, process and pathway interactions with high-throughput expression and biological annotation data.

PubMed

Singh, Nitesh Kumar; Ernst, Mathias; Liebscher, Volkmar; Fuellen, Georg; Taher, Leila

2016-10-20

The biological relationships both between and within the functions, processes and pathways that operate within complex biological systems are only poorly characterized, making the interpretation of large scale gene expression datasets extremely challenging. Here, we present an approach that integrates gene expression and biological annotation data to identify and describe the interactions between biological functions, processes and pathways that govern a phenotype of interest. The product is a global, interconnected network, not of genes but of functions, processes and pathways, that represents the biological relationships within the system. We validated our approach on two high-throughput expression datasets describing organismal and organ development. Our findings are well supported by the available literature, confirming that developmental processes and apoptosis play key roles in cell differentiation. Furthermore, our results suggest that processes related to pluripotency and lineage commitment, which are known to be critical for development, interact mainly indirectly, through genes implicated in more general biological processes. Moreover, we provide evidence that supports the relevance of cell spatial organization in the developing liver for proper liver function. Our strategy can be viewed as an abstraction that is useful to interpret high-throughput data and devise further experiments.

Lineage determination of CD7+ CD5- CD2- and CD7+ CD5+ CD2- lymphoblasts: studies on phenotype, genotype, and gene expression of myeloperoxidase, CD3 epsilon, and CD3 delta.

PubMed

Yoneda, N; Tatsumi, E; Teshigawara, K; Nagata, S; Nagano, T; Kishimoto, Y; Kimura, T; Yasunaga, K; Yamaguchi, N

1994-04-01

The gene expression of myeloperoxidase (MPO), CD3 epsilon, and CD3 delta molecules, the gene rearrangement of T-cell receptor (TCR) delta, gamma, and beta and immunoglobulin heavy (IgH) chain, and the expression of cell-surface antigens were investigated in seven cases of CD7+ CD5- CD2- and four cases of CD7+ CD5+ CD2- acute lymphoblastic leukemia or lymphoblastic lymphoma (ALL/LBL) blasts, which were negative for cytochemical myeloperoxidase (cyMPO). More mature T-lineage blasts were also investigated in a comparative manner. In conclusion, the CD7+ CD5- CD2- blasts included four categories: undifferentiated blasts without lineage commitment, T-lineage blasts, T-/myeloid lineage blasts, and cyMPO-negative myeloblasts. The CD7+ CD5+ CD2- blasts included two categories; T-lineage and T-/myeloid lineage blasts. The 11 cases were of the germ-line gene (G) for TCR beta and IgH. Four cases were G for TCR delta and TCR gamma. The others were of the monoclonally rearranged gene (R) for TCR delta and G for TCR gamma or R for both TCR delta and TCR gamma. The expression or in vitro induction of CD13 and/or CD33 antigens correlated with the immaturity of these neoplastic T cells, since it was observed in all 11 CD7+ CD5- CD2- and CD7+ CD5+ CD2-, and some CD7+ CD5+ CD2+ (CD3- CD4- CD8-) cases, but not in CD3 +/- CD4+ CD8+ or CD3+ CD4+ CD8- cases. CD3 epsilon mRNA, but not CD3 delta mRNA, was detected in two CD7+ CD5- CD2- cases, while mRNA of neither of the two CD3 molecules was detected in the other tested CD7+ CD5- CD2- cases. In contrast, mRNA of both CD3 epsilon and CD3 delta were detected in all CD7+ CD5+ CD2- cases, indicating that CD7+ CD5- CD2- blasts at least belong to T-lineage. The blasts of two CD7+ CD5- CD2- cases with entire germ-line genes and without mRNA of the three molecules (MPO, CD3 epsilon, and CD3 delta) were regarded as being at an undifferentiated stage prior to their commitment to either T- or myeloid-lineage. The co-expression of the genes of MPO and CD3 epsilon in a CD7+ CD5- CD2- case MPO, CD3 epsilon, and CD3 delta in a CD7+ CD5+ CD2- case suggested the presence of some overlapping phase for T- and myeloid-lineage commitment during immature stages of differentiation. This helps understand the conversion of some T-ALL/LBL cases to acute myeloblastic leukemia (AML).(ABSTRACT TRUNCATED AT 400 WORDS)

Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berries.

PubMed

Castellarin, Simone D; Matthews, Mark A; Di Gaspero, Gabriele; Gambetta, Gregory A

2007-12-01

Water deficits consistently promote higher concentrations of anthocyanins in red winegrapes and their wines. However, controversy remains as to whether there is any direct effect on berry metabolism other than inhibition of growth. Early (ED) and late (LD) season water deficits, applied before or after the onset of ripening (veraison), were imposed on field grown Vitis vinifera "Cabernet Sauvignon", and the responses of gene expression in the flavonoid pathway and their corresponding metabolites were determined. ED accelerated sugar accumulation and the onset of anthocyanin synthesis. Both ED and LD increased anthocyanin accumulation after veraison. Expression profiling revealed that the increased anthocyanin accumulation resulted from earlier and greater expression of the genes controlling flux through the anthocyanin biosynthetic pathway, including F3H, DFR, UFGT and GST. Increases in total anthocyanins resulted predominantly from an increase of 3'4'5'-hydroxylated forms through the differential regulation of F3'H and F3'5'H. There were limited effects on proanthocyanidin, other flavonols, and on expression of genes committed to their synthesis. These results demonstrate that manipulation of abiotic stress through applied water deficits not only modulates compositional changes during berry ripening, but also alters the timing of particular aspects of the ripening process.

Reverse engineering a mouse embryonic stem cell-specific transcriptional network reveals a new modulator of neuronal differentiation

PubMed Central

De Cegli, Rossella; Iacobacci, Simona; Flore, Gemma; Gambardella, Gennaro; Mao, Lei; Cutillo, Luisa; Lauria, Mario; Klose, Joachim; Illingworth, Elizabeth; Banfi, Sandro; di Bernardo, Diego

2013-01-01

Gene expression profiles can be used to infer previously unknown transcriptional regulatory interaction among thousands of genes, via systems biology ‘reverse engineering’ approaches. We ‘reverse engineered’ an embryonic stem (ES)-specific transcriptional network from 171 gene expression profiles, measured in ES cells, to identify master regulators of gene expression (‘hubs’). We discovered that E130012A19Rik (E13), highly expressed in mouse ES cells as compared with differentiated cells, was a central ‘hub’ of the network. We demonstrated that E13 is a protein-coding gene implicated in regulating the commitment towards the different neuronal subtypes and glia cells. The overexpression and knock-down of E13 in ES cell lines, undergoing differentiation into neurons and glia cells, caused a strong up-regulation of the glutamatergic neurons marker Vglut2 and a strong down-regulation of the GABAergic neurons marker GAD65 and of the radial glia marker Blbp. We confirmed E13 expression in the cerebral cortex of adult mice and during development. By immuno-based affinity purification, we characterized protein partners of E13, involved in the Polycomb complex. Our results suggest a role of E13 in regulating the division between glutamatergic projection neurons and GABAergic interneurons and glia cells possibly by epigenetic-mediated transcriptional regulation. PMID:23180766

A Resource for Discovering Specific and Universal Biomarkers for Distributed Stem Cells

PubMed Central

Noh, Minsoo; Smith, Janet L.; Huh, Yang Hoon; Sherley, James L.

2011-01-01

Specific and universal biomarkers for distributed stem cells (DSCs) have been elusive. A major barrier to discovery of such ideal DSC biomarkers is difficulty in obtaining DSCs in sufficient quantity and purity. To solve this problem, we used cell lines genetically engineered for conditional asymmetric self-renewal, the defining DSC property. In gene microarray analyses, we identified 85 genes whose expression is tightly asymmetric self-renewal associated (ASRA). The ASRA gene signature prescribed DSCs to undergo asymmetric self-renewal to a greater extent than committed progenitor cells, embryonic stem cells, or induced pluripotent stem cells. This delineation has several significant implications. These include: 1) providing experimental evidence that DSCs in vivo undergo asymmetric self-renewal as individual cells; 2) providing an explanation why earlier attempts to define a common gene expression signature for DSCs were unsuccessful; and 3) predicting that some ASRA proteins may be ideal biomarkers for DSCs. Indeed, two ASRA proteins, CXCR6 and BTG2, and two other related self-renewal pattern associated (SRPA) proteins identified in this gene resource, LGR5 and H2A.Z, display unique asymmetric patterns of expression that have a high potential for universal and specific DSC identification. PMID:21818293

RNA-seq analysis of the gonadal transcriptome during Alligator mississippiensis temperature-dependent sex determination and differentiation.

PubMed

Yatsu, Ryohei; Miyagawa, Shinichi; Kohno, Satomi; Parrott, Benjamin B; Yamaguchi, Katsushi; Ogino, Yukiko; Miyakawa, Hitoshi; Lowers, Russell H; Shigenobu, Shuji; Guillette, Louis J; Iguchi, Taisen

2016-01-25

The American alligator (Alligator mississippiensis) displays temperature-dependent sex determination (TSD), in which incubation temperature during embryonic development determines the sexual fate of the individual. However, the molecular mechanisms governing this process remain a mystery, including the influence of initial environmental temperature on the comprehensive gonadal gene expression patterns occurring during TSD. Our characterization of transcriptomes during alligator TSD allowed us to identify novel candidate genes involved in TSD initiation. High-throughput RNA sequencing (RNA-seq) was performed on gonads collected from A. mississippiensis embryos incubated at both a male and a female producing temperature (33.5 °C and 30 °C, respectively) in a time series during sexual development. RNA-seq yielded 375.2 million paired-end reads, which were mapped and assembled, and used to characterize differential gene expression. Changes in the transcriptome occurring as a function of both development and sexual differentiation were extensively profiled. Forty-one differentially expressed genes were detected in response to incubation at male producing temperature, and included genes such as Wnt signaling factor WNT11, histone demethylase KDM6B, and transcription factor C/EBPA. Furthermore, comparative analysis of development- and sex-dependent differential gene expression revealed 230 candidate genes involved in alligator sex determination and differentiation, and early details of the suspected male-fate commitment were profiled. We also discovered sexually dimorphic expression of uncharacterized ncRNAs and other novel elements, such as unique expression patterns of HEMGN and ARX. Twenty-five of the differentially expressed genes identified in our analysis were putative transcriptional regulators, among which were MYBL2, MYCL, and HOXC10, in addition to conventional sex differentiation genes such as SOX9, and FOXL2. Inferred gene regulatory network was constructed, and the gene-gene and temperature-gene interactions were predicted. Gonadal global gene expression kinetics during sex determination has been extensively profiled for the first time in a TSD species. These findings provide insights into the genetic framework underlying TSD, and expand our current understanding of the developmental fate pathways during vertebrate sex determination.

A unique gene expression signature associated with serotonin 2C receptor RNA editing in the prefrontal cortex and altered in suicide.

PubMed

Di Narzo, Antonio Fabio; Kozlenkov, Alexey; Roussos, Panos; Hao, Ke; Hurd, Yasmin; Lewis, David A; Sibille, Etienne; Siever, Larry J; Koonin, Eugene; Dracheva, Stella

2014-09-15

Editing of the pre-mRNA for the serotonin receptor 2C (5-HT2CR) by site-specific adenosine deamination (A-to-I pre-mRNA editing) substantially increases the functional plasticity of this key neurotransmitter receptor and is thought to contribute to homeostatic mechanisms in neurons. 5-HT2CR mRNA editing generates up to 24 different receptor isoforms. The extent of editing correlates with 5-HT2CR functional activity: more highly edited isoforms exhibit the least function. Altered 5-HT2CR editing has been reported in postmortem brains of suicide victims. We report a comparative analysis of the connections among 5-HT2CR editing, genome-wide gene expression and DNA methylation in suicide victims, individuals with major depressive disorder and non-psychiatric controls. The results confirm previous findings of an overrepresentation of highly edited mRNA variants (which encode hypoactive 5-HT2CR receptors) in the brains of suicide victims. A large set of genes for which the expression level is associated with editing was detected. This signature set of editing-associated genes is significantly enriched for genes that are involved in synaptic transmission, genes that are preferentially expressed in neurons, and genes whose expression is correlated with the level of DNA methylation. Notably, we report that the link between 5-HT2CR editing and gene expression is disrupted in suicide victims. The results suggest that the postulated homeostatic function of 5-HT2CR editing is dysregulated in individuals who committed suicide. Published by Oxford University Press 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Role of Ox-PAPCs in the Differentiation of Mesenchymal Stem Cells (MSCs) and Runx2 and PPARγ2 Expression in MSCs-Like of Osteoporotic Patients

PubMed Central

Valenti, Maria Teresa; Garbin, Ulisse; Pasini, Andrea; Zanatta, Mirko; Stranieri, Chiara; Manfro, Stefania; Zucal, Chiara; Dalle Carbonare, Luca

2011-01-01

Background Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and adipocytes and conditions causing bone loss may induce a switch from the osteoblast to adipocyte lineage. In addition, the expression of Runx2 and the PPARγ2 transcription factor genes is essential for cellular commitment to an osteogenic and adipogenic differentiation, respectively. Modified lipoproteins derived from the oxidation of arachidonate-containing phospholipids (ox-PAPCs: POVPC, PGPC and PEIPC) are considered important factors in atherogenesis. Methodology We investigated the effect of ox-PAPCs on osteogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). In particular, we analyzed the transcription factor Runx2 and the PPARγ2 gene expression during osteogenic and adipogenic differentiation in absence and in presence of ox-PAPCs. We also analyzed gene expression level in a panel of osteoblastic and adipogenic differentiation markers. In addition, as circulating blood cells can be used as a “sentinel” that responds to changes in the macro- or micro-environment, we analyzed the Runx2 and the PPARγ2 gene expression in MSCs-like and ox-PAPC levels in serum of osteoporotic patients (OPs). Finally, we examined the effects of sera obtained from OPs in hMSCs comparing the results with age-matched normal donors (NDs). Principal findings Quantitative RT-PCR demonstrated that ox-PAPCs enhanced PPARγ2 and adipogenic gene expression and reduced Runx2 and osteoblast differentiation marker gene expression in differentiating hMSCs. In OPs, ox-PAPC levels and PPARγ2 expression were higher than in NDs, whereas Runx2 was lower than in ND circulant MSCs-like. Conclusions Ox-PAPCs affect the osteogenic differentiation by promoting adipogenic differentiation and this effect may appear involved in bone loss in OPs. PMID:21674037

The B-cell identity factor Pax5 regulates distinct transcriptional programmes in early and late B lymphopoiesis

PubMed Central

Revilla-i-Domingo, Roger; Bilic, Ivan; Vilagos, Bojan; Tagoh, Hiromi; Ebert, Anja; Tamir, Ido M; Smeenk, Leonie; Trupke, Johanna; Sommer, Andreas; Jaritz, Markus; Busslinger, Meinrad

2012-01-01

Pax5 controls the identity and development of B cells by repressing lineage-inappropriate genes and activating B-cell-specific genes. Here, we used genome-wide approaches to identify Pax5 target genes in pro-B and mature B cells. In these cell types, Pax5 bound to 40% of the cis-regulatory elements defined by mapping DNase I hypersensitive (DHS) sites, transcription start sites and histone modifications. Although Pax5 bound to 8000 target genes, it regulated only 4% of them in pro-B and mature B cells by inducing enhancers at activated genes and eliminating DHS sites at repressed genes. Pax5-regulated genes in pro-B cells account for 23% of all expression changes occurring between common lymphoid progenitors and committed pro-B cells, which identifies Pax5 as an important regulator of this developmental transition. Regulated Pax5 target genes minimally overlap in pro-B and mature B cells, which reflects massive expression changes between these cell types. Hence, Pax5 controls B-cell identity and function by regulating distinct target genes in early and late B lymphopoiesis. PMID:22669466

Pharmacologic Calcitriol Inhibits Osteoclast Lineage Commitment via the BMP-Smad1 and IκB-NF-κB Pathways.

PubMed

Li, Anna; Cong, Qian; Xia, Xuechun; Leong, Wai Fook; Yeh, James; Miao, Dengshun; Mishina, Yuji; Liu, Huijuan; Li, Baojie

2017-07-01

Vitamin D is involved in a range of physiological processes and its active form and analogs have been used to treat diseases such as osteoporosis. Yet how vitamin D executes its function remains unsolved. Here we show that the active form of vitamin D calcitriol increases the peak bone mass in mice by inhibiting osteoclastogenesis and bone resorption. Although calcitriol modestly promoted osteoclast maturation, it strongly inhibited osteoclast lineage commitment from its progenitor monocyte by increasing Smad1 transcription via the vitamin D receptor and enhancing BMP-Smad1 activation, which in turn led to increased IκBα expression and decreased NF-κB activation and NFATc1 expression, with IκBα being a Smad1 target gene. Inhibition of BMP type I receptor or ablation of Bmpr1a in monocytes alleviated the inhibitory effects of calcitriol on osteoclast commitment, bone resorption, and bone mass augmentation. These findings uncover crosstalk between the BMP-Smad1 and RANKL-NF-κB pathways during osteoclastogenesis that underlies the action of active vitamin D on bone health. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

The Nuclear Receptor DAF-12 Regulates Nutrient Metabolism and Reproductive Growth in Nematodes

PubMed Central

Wang, Zhu; Stoltzfus, Jonathan; You, Young-jai; Ranjit, Najju; Tang, Hao; Xie, Yang; Lok, James B.; Mangelsdorf, David J.; Kliewer, Steven A.

2015-01-01

Appropriate nutrient response is essential for growth and reproduction. Under favorable nutrient conditions, the C. elegans nuclear receptor DAF-12 is activated by dafachronic acids, hormones that commit larvae to reproductive growth. Here, we report that in addition to its well-studied role in controlling developmental gene expression, the DAF-12 endocrine system governs expression of a gene network that stimulates the aerobic catabolism of fatty acids. Thus, activation of the DAF-12 transcriptome coordinately mobilizes energy stores to permit reproductive growth. DAF-12 regulation of this metabolic gene network is conserved in the human parasite, Strongyloides stercoralis, and inhibition of specific steps in this network blocks reproductive growth in both of the nematodes. Our study provides a molecular understanding for metabolic adaptation of nematodes to their environment, and suggests a new therapeutic strategy for treating parasitic diseases. PMID:25774872

Epigenetic and Epitranscriptomic Factors Make a Mark on Hematopoietic Stem Cell Development.

PubMed

Kasper, Dionna M; Nicoli, Stefania

2018-03-01

Blood specification is a highly dynamic process, whereby committed hemogenic endothelial cells (ECs) progressively transdifferentiate into multipotent, self-renewing hematopoietic stem cells (HSCs). Massive changes in gene expression must occur to switch cell identity, however the factors that mediate such an effect were a mystery until recently. This review summarizes the higher-order mechanisms involved in endothelial to hematopoietic reprogramming identified thus far. Accumulating evidence from mouse and zebrafish studies reveal that numerous chromatin-modifying (epigenetic) and RNA-modifying (epitranscriptomic) factors are required for the formation of HSCs from hemogenic endothelium. These genes function throughout the endothelial-hematopoietic transition, suggesting a dynamic interplay between 'epi'-machineries. Epigenetic and epitranscriptomic regulation are key mechanisms for reshaping global EC gene expression patterns to those that support HSC production. Future studies that capture modification dynamics should bring us closer to a complete understanding of how HSCs transition from hemogenic endothelium at the molecular level.

Investigating Conservation of the Cell-Cycle-Regulated Transcriptional Program in the Fungal Pathogen, Cryptococcus neoformans

PubMed Central

Sierra, Crystal S.; Haase, Steven B.

2016-01-01

The pathogenic yeast Cryptococcus neoformans causes fungal meningitis in immune-compromised patients. Cell proliferation in the budding yeast form is required for C. neoformans to infect human hosts, and virulence factors such as capsule formation and melanin production are affected by cell-cycle perturbation. Thus, understanding cell-cycle regulation is critical for a full understanding of virulence factors for disease. Our group and others have demonstrated that a large fraction of genes in Saccharomyces cerevisiae is expressed periodically during the cell cycle, and that proper regulation of this transcriptional program is important for proper cell division. Despite the evolutionary divergence of the two budding yeasts, we found that a similar percentage of all genes (~20%) is periodically expressed during the cell cycle in both yeasts. However, the temporal ordering of periodic expression has diverged for some orthologous cell-cycle genes, especially those related to bud emergence and bud growth. Genes regulating DNA replication and mitosis exhibited a conserved ordering in both yeasts, suggesting that essential cell-cycle processes are conserved in periodicity and in timing of expression (i.e. duplication before division). In S. cerevisiae cells, we have proposed that an interconnected network of periodic transcription factors (TFs) controls the bulk of the cell-cycle transcriptional program. We found that temporal ordering of orthologous network TFs was not always maintained; however, the TF network topology at cell-cycle commitment appears to be conserved in C. neoformans. During the C. neoformans cell cycle, DNA replication genes, mitosis genes, and 40 genes involved in virulence are periodically expressed. Future work toward understanding the gene regulatory network that controls cell-cycle genes is critical for developing novel antifungals to inhibit pathogen proliferation. PMID:27918582

Circumvention of the multidrug-resistance protein (MRP-1) by an antitumor drug through specific inhibition of gene transcription in breast tumor cells.

PubMed

Mansilla, Sylvia; Rojas, Marta; Bataller, Marc; Priebe, Waldemar; Portugal, José

2007-04-01

Multidrug-resistance protein 1 (MRP-1) confers resistance to a number of clinically important chemotherapeutic agents. The promoter of the mrp-1 gene contains an Sp1-binding site, which we targeted using the antitumor bis-anthracycline WP631. When MCF-7/VP breast cancer cells, which overexpress MRP-1 protein, were incubated with WP631 the expression of the multidrug-resistance protein gene decreased. Conversely, doxorubicin did not alter mrp-1 gene expression. The inhibition of gene expression was followed by a decrease in the activity of the MRP-1 protein. The IC(75) for WP631 (drug concentration required to inhibit cell growth by 75%) circumvented the drug-efflux pump, without addition of resistant modifiers. After treatment with WP631, MCF-7/VP cells were committed to die after entering mitosis (mitotic catastrophe), while treatment with doxorubicin did not affect cell growth. This is the first report on an antitumor drug molecule inhibiting the mrp-1 gene directly, rather than being simply a poor substrate for the transporter-mediated efflux. However, both situations appeared to coexist, thereby a superior cytotoxic effect was attained. Ours results suggest that WP631 offers great potential for the clinical treatment of tumors displaying a multidrug-resistance phenotype.

Effect of human cytomegalovirus infection on the expression of Hoxb2 and Hoxb4 genes in the developmental process of cord blood erythroid progenitors.

PubMed

Liu, Wen-Jun; Huang, Mei-Xian; Guo, Qu-Lian; Chen, Jun-Hong; Shi, Han

2011-01-01

The aim of the present study was to investigate the role of Hoxb2 and Hoxb4 gene expression induced by human cytomegalovirus (HCMV) and/or all-trans retinoic acid (ATRA) on the proliferation and committed differentiation process of human cord blood hematopoietic stem cells (HSCs) to colony-forming erythroid progenitor cells (CFU-Es) in vitro. Cord blood was collected from the fetal placenta umbilical vein in 12 cases and cultured using hematopoietic stem cell culture technique in vitro. The proliferation and differentiation of cord blood HSCs to CFU-Es were continuously disrupted with HCMV-AD169 and/or 6 x 10⁻⁸ mol/l of ATRA. Expression levels of the Hoxb2 and Hoxb4 genes in the blank, ATRA, HCMV-AD169 and ATRA + HCMV treatment groups of CFU-Es were detected on day 3, 7 and 10 of culture by fluorescent quantitative reverse transcriptase-polymerase chain reaction method. Hoxb2 and Hoxb4 gene expression in each group began on day 3, obviously increased on day 7 and reached a peak on day 10. The expression levels of the Hoxb2 and Hoxb4 genes in the HCMV group were obviously down-regulated compared with the level in the blank group. However, expression levels of the Hoxb2 and Hoxb4 genes were significantly up-regulated in the HCMV + ATRA group compared with the HCMV group (P<0.05). Abnormal expression of the Hoxb2 and Hoxb4 genes induced by HCMV may play important roles in abnormal hematopoietic damage. They were also correlated with the process of erythroid hematopoiesis. ATRA (6 x 10⁻⁸ mol/l) significantly up-regulated expression of the Hoxb2 and Hoxb4 genes in the normal erythroid progenitor cells and in those cells infected with HCMV as well.

Unconventional function of an Achaete-Scute homolog as a terminal selector of nociceptive neuron identity

PubMed Central

Masoudi, Neda; Tavazoie, Saeed; Glenwinkel, Lori; Ryu, Leesun; Kim, Kyuhyung

2018-01-01

Proneural genes are among the most early-acting genes in nervous system development, instructing blast cells to commit to a neuronal fate. Drosophila Atonal and Achaete-Scute complex (AS-C) genes, as well as their vertebrate orthologs, are basic helix-loop-helix (bHLH) transcription factors with such proneural activity. We show here that a C. elegans AS-C homolog, hlh-4, functions in a fundamentally different manner. In the embryonic, larval, and adult nervous systems, hlh-4 is expressed exclusively in a single nociceptive neuron class, ADL, and its expression in ADL is maintained via transcriptional autoregulation throughout the life of the animal. However, in hlh-4 null mutants, the ADL neuron is generated and still appears neuronal in overall morphology and expression of panneuronal and pansensory features. Rather than acting as a proneural gene, we find that hlh-4 is required for the ADL neuron to function properly, to adopt its correct morphology, to express its unusually large repertoire of olfactory receptor–encoding genes, and to express other known features of terminal ADL identity, including neurotransmitter phenotype, neuropeptides, ion channels, and electrical synapse proteins. hlh-4 is sufficient to induce ADL identity features upon ectopic expression in other neuron types. The expression of ADL terminal identity features is directly controlled by HLH-4 via a phylogenetically conserved E-box motif, which, through bioinformatic analysis, we find to constitute a predictive feature of ADL-expressed terminal identity markers. The lineage that produces the ADL neuron was previously shown to require the conventional, transient proneural activity of another AS-C homolog, hlh-14, demonstrating sequential activities of distinct AS-C-type bHLH genes in neuronal specification. Taken together, we have defined here an unconventional function of an AS-C-type bHLH gene as a terminal selector of neuronal identity and we speculate that such function could be reflective of an ancestral function of an “ur-” bHLH gene. PMID:29672507

Directed Neural Differentiation of Mouse Embryonic Stem Cells Is a Sensitive System for the Identification of Novel Hox Gene Effectors

PubMed Central

Bami, Myrto; Episkopou, Vasso; Gavalas, Anthony; Gouti, Mina

2011-01-01

The evolutionarily conserved Hox family of homeodomain transcription factors plays fundamental roles in regulating cell specification along the anterior posterior axis during development of all bilaterian animals by controlling cell fate choices in a highly localized, extracellular signal and cell context dependent manner. Some studies have established downstream target genes in specific systems but their identification is insufficient to explain either the ability of Hox genes to direct homeotic transformations or the breadth of their patterning potential. To begin delineating Hox gene function in neural development we used a mouse ES cell based system that combines efficient neural differentiation with inducible Hoxb1 expression. Gene expression profiling suggested that Hoxb1 acted as both activator and repressor in the short term but predominantly as a repressor in the long run. Activated and repressed genes segregated in distinct processes suggesting that, in the context examined, Hoxb1 blocked differentiation while activating genes related to early developmental processes, wnt and cell surface receptor linked signal transduction and cell-to-cell communication. To further elucidate aspects of Hoxb1 function we used loss and gain of function approaches in the mouse and chick embryos. We show that Hoxb1 acts as an activator to establish the full expression domain of CRABPI and II in rhombomere 4 and as a repressor to restrict expression of Lhx5 and Lhx9. Thus the Hoxb1 patterning activity includes the regulation of the cellular response to retinoic acid and the delay of the expression of genes that commit cells to neural differentiation. The results of this study show that ES neural differentiation and inducible Hox gene expression can be used as a sensitive model system to systematically identify Hox novel target genes, delineate their interactions with signaling pathways in dictating cell fate and define the extent of functional overlap among different Hox genes. PMID:21637844

The Tribolium homeotic gene Abdominal is homologous to abdominal-A of the Drosophila bithorax complex

NASA Technical Reports Server (NTRS)

Stuart, J. J.; Brown, S. J.; Beeman, R. W.; Denell, R. E.; Spooner, B. S. (Principal Investigator)

1993-01-01

The Abdominal gene is a member of the single homeotic complex of the beetle, Tribolium castaneum. An integrated developmental genetic and molecular analysis shows that Abdominal is homologous to the abdominal-A gene of the bithorax complex of Drosophila. abdominal-A mutant embryos display strong homeotic transformations of the anterior abdomen (parasegments 7-9) to PS6, whereas developmental commitments in the posterior abdomen depend primarily on Abdominal-B. In beetle embryos lacking Abdominal function, parasegments throughout the abdomen are transformed to PS6. This observation demonstrates the general functional significance of parasegmental expression among insects and shows that the control of determinative decisions in the posterior abdomen by homeotic selector genes has undergone considerable evolutionary modification.

The Tribolium homeotic gene Abdominal is homologous to abdominal-A of the Drosophila bithorax complex.

PubMed

Stuart, J J; Brown, S J; Beeman, R W; Denell, R E

1993-01-01

The Abdominal gene is a member of the single homeotic complex of the beetle, Tribolium castaneum. An integrated developmental genetic and molecular analysis shows that Abdominal is homologous to the abdominal-A gene of the bithorax complex of Drosophila. abdominal-A mutant embryos display strong homeotic transformations of the anterior abdomen (parasegments 7-9) to PS6, whereas developmental commitments in the posterior abdomen depend primarily on Abdominal-B. In beetle embryos lacking Abdominal function, parasegments throughout the abdomen are transformed to PS6. This observation demonstrates the general functional significance of parasegmental expression among insects and shows that the control of determinative decisions in the posterior abdomen by homeotic selector genes has undergone considerable evolutionary modification.

Effects of active acromegaly on bone mRNA and microRNA expression patterns.

PubMed

Belaya, Zhanna; Grebennikova, Tatiana; Melnichenko, Galina; Nikitin, Alexey; Solodovnikov, Alexander; Brovkina, Olga; Grigoriev, Andrey; Rozhinskaya, Liudmila; Lutsenko, Alexander; Dedov, Ivan

2018-04-01

To evaluate the response of bone to chronic long-term growth hormone (GH) and insulin-like growth factor-1 (IGF1) excess by measuring the expression of selected mRNA and microRNA (miR) in bone tissue samples of patients with active acromegaly. Case-control study. Bone tissue samples were obtained during transsphenoidal adenomectomy from the sphenoid bone (sella turcica) from 14 patients with clinically and biochemically confirmed acromegaly and 10 patients with clinically non-functioning pituitary adenoma (NFPA) matched by sex and age. Expression of genes involved in the regulation of bone remodeling was studied using quantitative polymerase chain reaction (qPCR). Of the genes involved in osteoblast and osteoclast activity, only alkaline phosphatase (ALP) mRNA was 50% downregulated in patients with acromegaly. GH excess caused increased expression of the Wnt signaling antagonists ( DKK1) and agonists ( WNT10B) and changes in the levels of miR involved in mesenchymal stem cell commitment to chondrocytes (miR-199a-5p) or adipocytes (miR-27-5p, miR-125b-5p, miR-34a-5p, miR-188-3p) P  < 0.05; q  < 0.1. Relevant compensatory mechanisms were found through the changes in miR involved in osteoblastogenesis (miR-210-5p, miR-135a-5p, miR-211, miR-23a-3p, miR-204-5p), but the expression of TWIST1 was 50% downregulated and RUNX2 was unchanged. Acromegaly had minimal effects on tested mRNAs specific to osteoblast or osteoclast function except for downregulated ALP expression. The expressions of miR known to be involved in mesenchymal stem cell commitment and downregulated TWIST1 expression suggest acromegaly has a negative effect on osteoblastogenesis. © 2018 European Society of Endocrinology.

A defined Oct4 level governs cell state transitions of pluripotency entry and differentiation into all embryonic lineages.

PubMed

Radzisheuskaya, Aliaksandra; Chia, Gloryn Le Bin; dos Santos, Rodrigo L; Theunissen, Thorold W; Castro, L Filipe C; Nichols, Jennifer; Silva, José C R

2013-06-01

Oct4 is considered a master transcription factor for pluripotent cell self-renewal, but its biology remains poorly understood. Here, we investigated the role of Oct4 using the process of induced pluripotency. We found that a defined embryonic stem cell (ESC) level of Oct4 is required for pluripotency entry. However, once pluripotency is established, the Oct4 level can be decreased up to sevenfold without loss of self-renewal. Unexpectedly, cells constitutively expressing Oct4 at an ESC level robustly differentiated into all embryonic lineages and germline. In contrast, cells with low Oct4 levels were deficient in differentiation, exhibiting expression of naive pluripotency genes in the absence of pluripotency culture requisites. The restoration of Oct4 expression to an ESC level rescued the ability of these to restrict naive pluripotent gene expression and to differentiate. In conclusion, a defined Oct4 level controls the establishment of naive pluripotency as well as commitment to all embryonic lineages.

A role for RNA post-transcriptional regulation in satellite cell activation

PubMed Central

2012-01-01

Background Satellite cells are resident skeletal muscle stem cells responsible for muscle maintenance and repair. In resting muscle, satellite cells are maintained in a quiescent state. Satellite cell activation induces the myogenic commitment factor, MyoD, and cell cycle entry to facilitate transition to a population of proliferating myoblasts that eventually exit the cycle and regenerate muscle tissue. The molecular mechanism involved in the transition of a quiescent satellite cell to a transit-amplifying myoblast is poorly understood. Methods Satellite cells isolated by FACS from uninjured skeletal muscle and 12 h post-muscle injury from wild type and Syndecan-4 null mice were probed using Affymetrix 430v2 gene chips and analyzed by Spotfiretm and Ingenuity Pathway analysis to identify gene expression changes and networks associated with satellite cell activation, respectively. Additional analyses of target genes identify miRNAs exhibiting dynamic changes in expression during satellite cell activation. The function of the miRNAs was assessed using miRIDIAN hairpin inhibitors. Results An unbiased gene expression screen identified over 4,000 genes differentially expressed in satellite cells in vivo within 12 h following muscle damage and more than 50% of these decrease dramatically. RNA binding proteins and genes involved in post-transcriptional regulation were significantly over-represented whereas splicing factors were preferentially downregulated and mRNA stability genes preferentially upregulated. Furthermore, six computationally identified miRNAs demonstrated novel expression through muscle regeneration and in satellite cells. Three of the six miRNAs were found to regulate satellite cell fate. Conclusions The quiescent satellite cell is actively maintained in a state poised to activate in response to external signals. Satellite cell activation appears to be regulated by post-transcriptional gene regulation. PMID:23046558

Wnt/β-catenin signaling cascade down-regulation following massive small bowel resection in a rat.

PubMed

Sukhotnik, Igor; Roitburt, Alex; Pollak, Yulia; Dorfman, Tatiana; Matter, Ibrahim; Mogilner, Jorge G; Bejar, Jacob; Coran, Arnold G

2014-02-01

Growing evidence suggests that the Wnt/β-catenin signaling cascade is implicated in the control of stem cell activity, cell proliferation, lineage commitment, and cell survival during normal development and tissue regeneration of the gastrointestinal epithelium. The roles of this signaling cascade in stimulation of cell proliferation after massive small bowel resection are unknown. The purpose of this study was to evaluate the role of Wnt/β-catenin signaling during late stages of intestinal adaptation in a rat model of short bowel syndrome (SBS). Male rats were divided into two groups: sham rats underwent bowel transection and SBS rats underwent a 75 % bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined 2 weeks after operation. Illumina's digital gene expression analysis was used to determine Wnt/β-catenin signaling gene expression profiling. Twelve Wnt/β-catenin-related genes and β-catenin protein expression were determined using real-time PCR, western blotting and immunohistochemistry. From the total number of 20,000 probes, 20 genes related to Wnt/β-catenin signaling were investigated. From these genes, seven genes were found to be up-regulated and eight genes to be down-regulated in SBS vs. sham animals with a relative change in gene expression level of 20 % or more. From 12 genes determined by real-time PCR, nine genes were down-regulated in SBS rats compared to control animals including target gene c-Myc. SBS rats also showed a significant decrease in β-catenin protein compared to control animals. Two weeks following massive bowel resection in rats, Wnt/β-catenin signaling pathway is inhibited. In addition, it appears that cell differentiation rather than proliferation is most important in the late stages of intestinal adaptation.

The role of alternative splicing coupled to nonsense-mediated mRNA decay in human disease.

PubMed

da Costa, Paulo J; Menezes, Juliane; Romão, Luísa

2017-10-01

Alternative pre-mRNA splicing (AS) affects gene expression as it generates proteome diversity. Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that recognizes and selectively degrades mRNAs carrying premature translation-termination codons (PTCs), preventing the production of truncated proteins that could result in disease. Several studies have also implicated NMD in the regulation of steady-state levels of physiological mRNAs. In addition, it is known that several regulated AS events do not lead to generation of protein products, as they lead to transcripts that carry PTCs and thus, they are committed to NMD. Indeed, an estimated one-third of naturally occurring, alternatively spliced mRNAs is targeted for NMD, being AS coupled to NMD (AS-NMD) an efficient strategy to regulate gene expression. In this review, we will focus on how AS mechanism operates and how can be coupled to NMD to fine-tune gene expression levels. Furthermore, we will demonstrate the physiological significance of the interplay among AS and NMD in human disease, such as cancer and neurological disorders. The understanding of how AS-NMD orchestrates expression of vital genes is of utmost importance for the advance in diagnosis, prognosis and treatment of many human disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

Set regulation in asexual and sexual Plasmodium parasites reveals a novel mechanism of stage-specific expression.

PubMed

Pace, Tomasino; Olivieri, Anna; Sanchez, Massimo; Albanesi, Veronica; Picci, Leonardo; Siden Kiamos, Inga; Janse, Chris J; Waters, Andrew P; Pizzi, Elisabetta; Ponzi, Marta

2006-05-01

Transmission of the malaria parasite depends on specialized gamete precursors (gametocytes) that develop in the bloodstream of a vertebrate host. Gametocyte/gamete differentiation requires controlled patterns of gene expression and regulation not only of stage and gender-specific genes but also of genes associated with DNA replication and mitosis. Once taken up by mosquito, male gametocytes undergo three mitotic cycles within few minutes to produce eight motile gametes. Here we analysed, in two Plasmodium species, the expression of SET, a conserved nuclear protein involved in chromatin dynamics. SET is expressed in both asexual and sexual blood stages but strongly accumulates in male gametocytes. We demonstrated functionally the presence of two distinct promoters upstream of the set open reading frame, the one active in all blood stage parasites while the other active only in gametocytes and in a fraction of schizonts possibly committed to sexual differentiation. In ookinetes both promoters exhibit a basal activity, while in the oocysts the gametocyte-specific promoter is silent and the reporter gene is only transcribed from the constitutive promoter. This transcriptional control, described for the first time in Plasmodium, provides a mechanism by which single-copy genes can be differently modulated during parasite development. In male gametocytes an overexpression of SET might contribute to a prompt entry and execution of S/M phases within mosquito vector.

Notch Signaling Is Involved in Neurogenic Commitment of Human Periodontal Ligament-Derived Mesenchymal Stem Cells

PubMed Central

Osathanon, Thanaphum; Manokawinchoke, Jeeranan; Nowwarote, Nunthawan; Aguilar, Panuroot; Palaga, Tanapat

2013-01-01

Notch signaling plays critical roles in stem cells by regulating cell fate determination and differentiation. The aim of this study was to evaluate the participation of Notch signaling in neurogenic commitment of human periodontal ligament-derived mesenchymal stem cells (hPDLSCs) and to examine the ability to control differentiation of these cells using modified surfaces containing affinity immobilized Notch ligands. Neurogenic induction of hPDLSCs was performed via neurosphere formation. Cells were aggregated and form spheres as early 1 day in culture. In addition, the induced cells exhibited increased mRNA and protein expression of neuronal markers that is, β3-tubulin and neurofilament. During neuronal differentiation, a significant increase of Hes1 and Hey1 mRNA expression was noted. Using pharmacological inhibition (γ-secretase inhibitor) or genetic manipulation (overexpression of dominant negative mastermind-like transcription co-activators), neurosphere formation was attenuated and a marked decrease in neurogenic mRNA expression was observed. To confirm the role of Notch signaling in neuronal differentiation of hPDLSCs, the Notch ligand, Jagged-1, is bound to the surface using an affinity immobilization technique. The hPDLSC cultured on a Jagged-1-modified surface had increased expression of Notch signaling target genes, Hes-1 and Hey-1, confirming the activity and potency of surface-bound Jagged-1. Further, hPDLSC on surface-bound Jagged-1 under serum-free conditions showed multiple long and thin neurite-like extensions, and an increase in the expression of neurogenic mRNA markers was observed. Pretreatment of the cells with γ-secretase inhibitor, DAPT, before seeding on the Jagged-1-modified surface blocked development of the neurite-like morphology. Together, the results in this study suggest the involvement of Notch signaling in neurogenic commitment of hPDLSCs. PMID:23379739

Cellular Dichotomy Between Anchorage-Independent Growth Responses to bFGF and TA Reflects Molecular Switch in Commitment to Carcinogenesis

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

Waters, Katrina M.; Tan, Ruimin; Opresko, Lee K.

2009-11-01

We have investigated gene expression patterns underlying reversible and irreversible anchorage-independent growth (AIG) phenotypes to identify more sensitive markers of cell transformation for studies directed at interrogating carcinogenesis responses. In JB6 mouse epidermal cells, basic fibroblast growth factor (bFGF) induces an unusually efficient and reversible AIG response, relative to 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced AIG which is irreversible. The reversible and irreversible AIG phenotypes are characterized by largely non-overlapping global gene expression profiles. However, a subset of differentially expressed genes were identified as common to reversible and irreversible AIG phenotypes, including genes regulated in a reciprocal fashion. Hepatic leukemia factor (HLF) andmore » D-site albumin promoter-binding protein (DBP) were increased in both bFGF and TPA soft agar colonies and selected for functional validation. Ectopic expression of human HLF and DBP in JB6 cells resulted in a marked increase in TPA- and bFGF-regulated AIG responses. HLF and DBP expression were increased in soft agar colonies arising from JB6 cells exposed to gamma radiation and in a human basal cell carcinoma tumor tissue, relative to paired non-tumor tissue. Subsequent biological network analysis suggests that many of the differentially expressed genes that are common to bFGF- and TPA-dependent AIG are regulated by c-Myc, SP-1 and HNF-4 transcription factors. Collectively, we have identified a potential molecular switch that mediates the transition from reversible to irreversible AIG.« less

A comprehensive gene expression analysis at sequential stages of in vitro cardiac differentiation from isolated MESP1-expressing-mesoderm progenitors

PubMed Central

den Hartogh, Sabine C.; Wolstencroft, Katherine; Mummery, Christine L.; Passier, Robert

2016-01-01

In vitro cardiac differentiation of human pluripotent stem cells (hPSCs) closely recapitulates in vivo embryonic heart development, and therefore, provides an excellent model to study human cardiac development. We recently generated the dual cardiac fluorescent reporter MESP1mCherry/wNKX2-5eGFP/w line in human embryonic stem cells (hESCs), allowing the visualization of pre-cardiac MESP1+ mesoderm and their further commitment towards the cardiac lineage, marked by activation of the cardiac transcription factor NKX2-5. Here, we performed a comprehensive whole genome based transcriptome analysis of MESP1-mCherry derived cardiac-committed cells. In addition to previously described cardiac-inducing signalling pathways, we identified novel transcriptional and signalling networks indicated by transient activation and interactive network analysis. Furthermore, we found a highly dynamic regulation of extracellular matrix components, suggesting the importance to create a versatile niche, adjusting to various stages of cardiac differentiation. Finally, we identified cell surface markers for cardiac progenitors, such as the Leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4), belonging to the same subfamily of LGR5, and LGR6, established tissue/cancer stem cells markers. We provide a comprehensive gene expression analysis of cardiac derivatives from pre-cardiac MESP1-progenitors that will contribute to a better understanding of the key regulators, pathways and markers involved in human cardiac differentiation and development. PMID:26783251

Identification and characterisation of DfCHS, a chalcone synthase gene regulated by temperature and ultraviolet in Dryopteris fragrans.

PubMed

Sun, L L; Li, Y; Li, S S; Wu, X J; Hu, B Z; Chang, Y

2014-12-30

Chalcone synthase (CHS) is an enzyme that catalyzes the first committed step in flavonoid biosynthesis, and its transcription level is regulated by light conditions. By using homology cloning and rapid amplification of cDNA ends, we cloned a chalcone synthase gene (DfCHS) from Dryopteris fragrans (L.) Schott. The full-length cDNA of DfCHS is 1,737 bp, with an open reading frame (ORF) of 1,122 bp (deposited in GenBank under Accession Number KF530802) encoding a predicted protein of 373 amino acids. The calculated molecular mass of DfCHS is 41.3 kDa. We studied the expression of DfCHS and total flavonoid contents in tissue culture seedlings cultured under the low temperature at 4ºC, high temperature at 35ºC and UV conditions, respectively. The results show that the expression of DfCHS are not the same, but all present rising trends, then flavonoid contents were increased. Overall, our results imply that the expression of DfCHS gene provide a certain theory basis in the status of evolution among ferns.

IRF8 Governs Expression of Genes Involved in Innate and Adaptive Immunity in Human and Mouse Germinal Center B Cells

PubMed Central

Morse, Herbert C.

2011-01-01

IRF8 (Interferon Regulatory Factor 8) is a transcription factor expressed throughout B cell differentiation except for mature plasma cells. Previous studies showed it is part of the transcriptional network governing B cell specification and commitment in the bone marrow, regulates the distribution of mature B cells into the splenic follicular and marginal zone compartments, and is expressed at highest levels in germinal center (GC) B cells. Here, we investigated the transcriptional programs and signaling pathways affected by IRF8 in human and mouse GC B cells as defined by ChIP-chip analyses and transcriptional profiling. We show that IRF8 binds a large number of genes by targeting two distinct motifs, half of which are also targeted by PU.1. Over 70% of the binding sites localized to proximal and distal promoter regions with ∼25% being intragenic. There was significant enrichment among targeted genes for those involved in innate and adaptive immunity with over 30% previously defined as interferon stimulated genes. We also showed that IRF8 target genes contributes to multiple aspects of the biology of mature B cells including critical components of the molecular crosstalk among GC B cells, T follicular helper cells, and follicular dendritic cells. PMID:22096565

Isoform-level gene expression patterns in single-cell RNA-sequencing data.

PubMed

Vu, Trung Nghia; Wills, Quin F; Kalari, Krishna R; Niu, Nifang; Wang, Liewei; Pawitan, Yudi; Rantalainen, Mattias

2018-02-27

RNA sequencing of single cells enables characterization of transcriptional heterogeneity in seemingly homogeneous cell populations. Single-cell sequencing has been applied in a wide range of researches fields. However, few studies have focus on characterization of isoform-level expression patterns at the single-cell level. In this study we propose and apply a novel method, ISOform-Patterns (ISOP), based on mixture modeling, to characterize the expression patterns of isoform pairs from the same gene in single-cell isoform-level expression data. We define six principal patterns of isoform expression relationships and describe a method for differential-pattern analysis. We demonstrate ISOP through analysis of single-cell RNA-sequencing data from a breast cancer cell line, with replication in three independent datasets. We assigned the pattern types to each of 16,562 isoform-pairs from 4,929 genes. Among those, 26% of the discovered patterns were significant (p<0.05), while remaining patterns are possibly effects of transcriptional bursting, drop-out and stochastic biological heterogeneity. Furthermore, 32% of genes discovered through differential-pattern analysis were not detected by differential-expression analysis. The effect of drop-out events, mean expression level, and properties of the expression distribution on the performances of ISOP were also investigated through simulated datasets. To conclude, ISOP provides a novel approach for characterization of isoformlevel preference, commitment and heterogeneity in single-cell RNA-sequencing data. The ISOP method has been implemented as a R package and is available at https://github.com/nghiavtr/ISOP under a GPL-3 license. mattias.rantalainen@ki.se. Supplementary data are available at Bioinformatics online.

The ATP-sensitive potassium (KATP) channel-encoded dSUR gene is required for Drosophila heart function and is regulated by tinman

PubMed Central

Akasaka, Takeshi; Klinedinst, Susan; Ocorr, Karen; Bustamante, Erika L.; Kim, Seung K.; Bodmer, Rolf

2006-01-01

The homeobox transcription factor Tinman plays an important role in the initiation of heart development. Later functions of Tinman, including the target genes involved in cardiac physiology, are less well studied. We focused on the dSUR gene, which encodes an ATP-binding cassette transmembrane protein that is expressed in the heart. Mammalian SUR genes are associated with KATP (ATP-sensitive potassium) channels, which are involved in metabolic homeostasis. We provide experimental evidence that Tinman directly regulates dSUR expression in the developing heart. We identified a cis-regulatory element in the first intron of dSUR, which contains Tinman consensus binding sites and is sufficient for faithful dSUR expression in the fly’s myocardium. Site-directed mutagenesis of this element shows that these Tinman sites are critical to dSUR expression, and further genetic manipulations suggest that the GATA transcription factor Pannier is synergistically involved in cardiac-restricted dSUR expression in vivo. Physiological analysis of dSUR knock-down flies supports the idea that dSUR plays a protective role against hypoxic stress and pacing-induced heart failure. Because dSUR expression dramatically decreases with age, it is likely to be a factor involved in the cardiac aging phenotype of Drosophila. dSUR provides a model for addressing how embryonic regulators of myocardial cell commitment can contribute to the establishment and maintenance of cardiac performance. PMID:16882722

MicroRNA profiling of the murine hematopoietic system

PubMed Central

Monticelli, Silvia; Ansel, K Mark; Xiao, Changchun; Socci, Nicholas D; Krichevsky, Anna M; Thai, To-Ha; Rajewsky, Nikolaus; Marks, Debora S; Sander, Chris; Rajewsky, Klaus; Rao, Anjana; Kosik, Kenneth S

2005-01-01

Background MicroRNAs (miRNAs) are a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression. It is becoming clear that miRNAs play an important role in the regulation of gene expression during development. However, in mammals, expression data are principally based on whole tissue analysis and are still very incomplete. Results We used oligonucleotide arrays to analyze miRNA expression in the murine hematopoietic system. Complementary oligonucleotides capable of hybridizing to 181 miRNAs were immobilized on a membrane and probed with radiolabeled RNA derived from low molecular weight fractions of total RNA from several different hematopoietic and neuronal cells. This method allowed us to analyze cell type-specific patterns of miRNA expression and to identify miRNAs that might be important for cell lineage specification and/or cell effector functions. Conclusion This is the first report of systematic miRNA gene profiling in cells of the hematopoietic system. As expected, miRNA expression patterns were very different between hematopoietic and non-hematopoietic cells, with further subtle differences observed within the hematopoietic group. Interestingly, the most pronounced similarities were observed among fully differentiated effector cells (Th1 and Th2 lymphocytes and mast cells) and precursors at comparable stages of differentiation (double negative thymocytes and pro-B cells), suggesting that in addition to regulating the process of commitment to particular cellular lineages, miRNAs might have an important general role in the mechanism of cell differentiation and maintenance of cell identity. PMID:16086853

Clonal type I interferon-producing and dendritic cell precursors are contained in both human lymphoid and myeloid progenitor populations.

PubMed

Chicha, Laurie; Jarrossay, David; Manz, Markus G

2004-12-06

Because of different cytokine responsiveness, surface receptor, and transcription factor expression, human CD11c(-) natural type I interferon-producing cells (IPCs) and CD11c(+) dendritic cells were thought to derive through lymphoid and myeloid hematopoietic developmental pathways, respectively. To directly test this hypothesis, we used an in vitro assay allowing simultaneous IPC, dendritic cell, and B cell development and we tested lymphoid and myeloid committed hematopoietic progenitor cells for their developmental capacity. Lymphoid and common myeloid and granulocyte/macrophage progenitors were capable of developing into both functional IPCs, expressing gene transcripts thought to be associated with lymphoid lineage development, and into dendritic cells. However, clonal progenitors for both populations were about fivefold more frequent within myeloid committed progenitor cells. Thus, in humans as in mice, natural IPC and dendritic cell development robustly segregates with myeloid differentiation. This would fit with natural interferon type I-producing cell and dendritic cell activity in innate immunity, the evolutionary older arm of the cellular immune system.

A gene regulatory network armature for T-lymphocyte specification

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

Fung, Elizabeth-sharon

Choice of a T-lymphoid fate by hematopoietic progenitor cells depends on sustained Notch-Delta signaling combined with tightly-regulated activities of multiple transcription factors. To dissect the regulatory network connections that mediate this process, we have used high-resolution analysis of regulatory gene expression trajectories from the beginning to the end of specification; tests of the short-term Notchdependence of these gene expression changes; and perturbation analyses of the effects of overexpression of two essential transcription factors, namely PU.l and GATA-3. Quantitative expression measurements of >50 transcription factor and marker genes have been used to derive the principal components of regulatory change through whichmore » T-cell precursors progress from primitive multipotency to T-lineage commitment. Distinct parts of the path reveal separate contributions of Notch signaling, GATA-3 activity, and downregulation of PU.l. Using BioTapestry, the results have been assembled into a draft gene regulatory network for the specification of T-cell precursors and the choice of T as opposed to myeloid dendritic or mast-cell fates. This network also accommodates effects of E proteins and mutual repression circuits of Gfil against Egr-2 and of TCF-l against PU.l as proposed elsewhere, but requires additional functions that remain unidentified. Distinctive features of this network structure include the intense dose-dependence of GATA-3 effects; the gene-specific modulation of PU.l activity based on Notch activity; the lack of direct opposition between PU.l and GATA-3; and the need for a distinct, late-acting repressive function or functions to extinguish stem and progenitor-derived regulatory gene expression.« less

H2A.Z Acidic Patch Couples Chromatin Dynamics to Regulation of Gene Expression Programs during ESC Differentiation

PubMed Central

Subramanian, Vidya; Mazumder, Aprotim; Surface, Lauren E.; Butty, Vincent L.; Fields, Paul A.; Alwan, Allison; Torrey, Lillian; Thai, Kevin K.; Levine, Stuart S.; Bathe, Mark; Boyer, Laurie A.

2013-01-01

The histone H2A variant H2A.Z is essential for embryonic development and for proper control of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions of amino acid sequence of H2A.Z likely determine its functional specialization compared to core histone H2A. For example, H2A.Z contains three divergent residues in the essential C-terminal acidic patch that reside on the surface of the histone octamer as an uninterrupted acidic patch domain; however, we know little about how these residues contribute to chromatin structure and function. Here, we show that the divergent amino acids Gly92, Asp97, and Ser98 in the H2A.Z C-terminal acidic patch (H2A.ZAP3) are critical for lineage commitment during ESC differentiation. H2A.Z is enriched at most H3K4me3 promoters in ESCs including poised, bivalent promoters that harbor both activating and repressive marks, H3K4me3 and H3K27me3 respectively. We found that while H2A.ZAP3 interacted with its deposition complex and displayed a highly similar distribution pattern compared to wild-type H2A.Z, its enrichment levels were reduced at target promoters. Further analysis revealed that H2A.ZAP3 was less tightly associated with chromatin, suggesting that the mutant is more dynamic. Notably, bivalent genes in H2A.ZAP3 ESCs displayed significant changes in expression compared to active genes. Moreover, bivalent genes in H2A.ZAP3 ESCs gained H3.3, a variant associated with higher nucleosome turnover, compared to wild-type H2A.Z. We next performed single cell imaging to measure H2A.Z dynamics. We found that H2A.ZAP3 displayed higher mobility in chromatin compared to wild-type H2A.Z by fluorescent recovery after photobleaching (FRAP). Moreover, ESCs treated with the transcriptional inhibitor flavopiridol resulted in a decrease in the H2A.ZAP3 mobile fraction and an increase in its occupancy at target genes indicating that the mutant can be properly incorporated into chromatin. Collectively, our work suggests that the divergent residues in the H2A.Z acidic patch comprise a unique domain that couples control of chromatin dynamics to the regulation of developmental gene expression patterns during lineage commitment. PMID:23990805

Transcriptome Analysis of Nine Tissues to Discover Genes Involved in the Biosynthesis of Active Ingredients in Sophora flavescens.

PubMed

Han, Rongchun; Takahashi, Hiroki; Nakamura, Michimi; Bunsupa, Somnuk; Yoshimoto, Naoko; Yamamoto, Hirobumi; Suzuki, Hideyuki; Shibata, Daisuke; Yamazaki, Mami; Saito, Kazuki

2015-01-01

Sophora flavescens AITON (kurara) has long been used to treat various diseases. Although several research findings revealed the biosynthetic pathways of its characteristic chemical components as represented by matrine, insufficient analysis of transcriptome data hampered in-depth analysis of the underlying putative genes responsible for the biosynthesis of pharmaceutical chemical components. In this study, more than 200 million fastq format reads were generated by Illumina's next-generation sequencing approach using nine types of tissue from S. flavescens, followed by CLC de novo assembly, ultimately yielding 83,325 contigs in total. By mapping the reads back to the contigs, reads per kilobase of the transcript per million mapped reads values were calculated to demonstrate gene expression levels, and overrepresented gene ontology terms were evaluated using Fisher's exact test. In search of the putative genes relevant to essential metabolic pathways, all 1350 unique enzyme commission numbers were used to map pathways against the Kyoto Encyclopedia of Genes and Genomes. By analyzing expression patterns, we proposed some candidate genes involved in the biosynthesis of isoflavonoids and quinolizidine alkaloids. Adopting RNA-Seq analysis, we obtained substantially credible contigs for downstream work. The preferential expression of the gene for putative lysine/ornithine decarboxylase committed in the initial step of matrine biosynthesis in leaves and stems was confirmed in semi-quantitative polymerase chain reaction (PCR) analysis. The findings in this report may serve as a stepping-stone for further research into this promising medicinal plant.

Concise Review: Epigenetic Regulation of Myogenesis in Health and Disease

PubMed Central

Sincennes, Marie-Claude; Brun, Caroline E.

2016-01-01

Skeletal muscle regeneration is initiated by satellite cells, a population of adult stem cells that reside in the muscle tissue. The ability of satellite cells to self-renew and to differentiate into the muscle lineage is under transcriptional and epigenetic control. Satellite cells are characterized by an open and permissive chromatin state. The transcription factor Pax7 is necessary for satellite cell function. Pax7 is a nodal factor regulating the expression of genes associated with satellite cell growth and proliferation, while preventing differentiation. Pax7 recruits chromatin modifiers to DNA to induce expression of specific target genes involved in myogenic commitment following asymmetric division of muscle stem cells. Emerging evidence suggests that replacement of canonical histones with histone variants is an important regulatory mechanism controlling the ability of satellite cells and myoblasts to differentiate. Differentiation into the muscle lineage is associated with a global gene repression characterized by a decrease in histone acetylation with an increase in repressive histone marks. However, genes important for differentiation are upregulated by the specific action of histone acetyltransferases and other chromatin modifiers, in combination with several transcription factors, including MyoD and Mef2. Treatment with histone deacetylase (HDAC) inhibitors enhances muscle regeneration and is considered as a therapeutic approach in the treatment of muscular dystrophy. This review describes the recent findings on epigenetic regulation in satellite stem cells and committed myoblasts. The potential of epigenetic drugs, such as HDAC inhibitors, as well as their molecular mechanism of action in muscle cells, will be addressed. Significance This review summarizes recent findings concerning the epigenetic regulation of satellite cells in skeletal muscle. PMID:26798058

Pulsed magnetic therapy increases osteogenic differentiation of mesenchymal stem cells only if they are pre-committed.

PubMed

Ferroni, Letizia; Tocco, Ilaria; De Pieri, Andrea; Menarin, Martina; Fermi, Enrico; Piattelli, Adriano; Gardin, Chiara; Zavan, Barbara

2016-05-01

Pulsed electromagnetic field (PEMF) therapy has been documented to be an effective, non-invasive, safe treatment method for a variety of clinical conditions, especially in settings of recalcitrant healing. The underlying mechanisms on the different biological components of tissue regeneration are still to be elucidated. The aim of the present study was to characterize the effects of extremely low frequency (ELF)-PEMFs on commitment of mesenchymal stem cell (MSCs) culture system, through the determination of gene expression pattern and cellular morphology. Human MSCs derived from adipose tissue (ADSCs) were cultured in presence of adipogenic, osteogenic, neural, or glial differentiative medium and basal medium, then exposed to ELF-PEMFs daily stimulation for 21days. Control cultures were performed without ELF-PEMFs stimulation for all cell populations. Effects on commitment were evaluated after 21days of cultures. The results suggested ELF-PEMFs does not influence ADSCs commitment and does not promote adipogenic, osteogenic, neural or glial differentiation. However, ELF-PEMFs treatment on ADSCs cultured in osteogenic differentiative medium markedly increased osteogenesis. We concluded that PEMFs affect the osteogenic differentiation of ADSCs only if they are pre-commitment and that this therapy can be an appropriate candidate for treatment of conditions requiring an acceleration of repairing process. Copyright © 2016 Elsevier Inc. All rights reserved.

Braveheart, a long non-coding RNA required for cardiovascular lineage commitment

PubMed Central

Klattenhoff, Carla; Scheuermann, Johanna C.; Surface, Lauren E.; Bradley, Robert K.; Fields, Paul A.; Steinhauser, Matthew L.; Ding, Huiming; Butty, Vincent L.; Torrey, Lillian; Haas, Simon; Abo, Ryan; Tabebordbar, Mohammadsharif; Lee, Richard T.; Burge, Christopher B.; Boyer, Laurie A.

2013-01-01

Summary Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm towards a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of MesP1 (mesoderm posterior 1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of Polycomb Repressive Complex 2 (PRC2), during cardiomyocyte differentiation suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development. PMID:23352431

Expression of Essential B Cell Development Genes in Horses with Common Variable Immunodeficiency

PubMed Central

Tallmadge, R.L.; Such, K.A.; Miller, K.C.; Matychak, M.B.; Felippe, M.J.B.

2012-01-01

Common variable immunodeficiency (CVID) is a heterogeneous disorder of B cell differentiation or function with inadequate antibody production. Our laboratory studies a natural form of CVID in horses characterized by late-onset B cell lymphopenia due to impaired B cell production in the bone marrow. This study was undertaken to assess the status of B cell differentiation in the bone marrow of CVID-affected horses by measuring the expression of genes essential for early B cell commitment and development. Standard RT-PCR revealed that most of the transcription factors and key signaling molecules that directly regulate B cell differentiation in the bone marrow and precede PAX5 are expressed in the affected horses. Yet, the expression of PAX5 and relevant target genes was variable. Quantitative RT-PCR analysis confirmed that the mRNA expression of E2A, PAX5, CD19, and IGHD was significantly reduced in equine CVID patients when compared to healthy horses (p < 0.05). In addition, the PAX5/EBF1 and PAX5/B220 ratios were significantly reduced in CVID patients (p < 0.01). Immunohistochemical analysis confirmed the absence of PAX5-BSAP expression in the bone marrow of affected horses. Our data suggest that B cell development seems to be impaired at the transition between pre-pro-B cells and pro-B cells in equine CVID patients. PMID:22464097

The differential effects of 2% oxygen preconditioning on the subsequent differentiation of mouse and human pluripotent stem cells.

PubMed

Fynes, Kate; Tostoes, Rui; Ruban, Ludmila; Weil, Ben; Mason, Christopher; Veraitch, Farlan S

2014-08-15

A major challenge facing the development of effective cell therapies is the efficient differentiation of pluripotent stem cells (PSCs) into pure populations. Lowering oxygen tension to physiological levels can affect both the expansion and differentiation stages. However, to date, there are no studies investigating the knock-on effect of culturing PSCs under low oxygen conditions on subsequent lineage commitment at ambient oxygen levels. PSCs were passaged three times at 2% O2 before allowing cells to spontaneously differentiate as embryoid bodies (EBs) in high oxygen (20% O2) conditions. Maintenance of mouse PSCs in low oxygen was associated with a significant increase in the expression of early differentiation markers FGF5 and Eomes, while conversely we observed decreased expression of these genes in human PSCs. Low oxygen preconditioning primed mouse PSCs for their subsequent differentiation into mesodermal and endodermal lineages, as confirmed by increased gene expression of Eomes, Goosecoid, Brachyury, AFP, Sox17, FoxA2, and protein expression of Brachyury, Eomes, Sox17, FoxA2, relative to high oxygen cultures. The effects extended to the subsequent formation of more mature mesodermal lineages. We observed significant upregulation of cardiomyocyte marker Nkx2.5, and critically a decrease in the number of contaminant pluripotent cells after 12 days using a directed cardiomyocyte protocol. However, the impact of low oxygen preconditioning was to prime human cells for ectodermal lineage commitment during subsequent EB differentiation, with significant upregulation of Nestin and β3-tubulin. Our research demonstrates the importance of oxygen tension control during cell maintenance on the subsequent differentiation of both mouse and human PSCs, and highlights the differential effects.

Identification of candidate genes affecting Δ9-tetrahydrocannabinol biosynthesis in Cannabis sativa

PubMed Central

Marks, M. David; Tian, Li; Wenger, Jonathan P.; Omburo, Stephanie N.; Soto-Fuentes, Wilfredo; He, Ji; Gang, David R.; Weiblen, George D.; Dixon, Richard A.

2009-01-01

RNA isolated from the glands of a Δ9-tetrahydrocannabinolic acid (THCA)-producing strain of Cannabis sativa was used to generate a cDNA library containing over 100 000 expressed sequence tags (ESTs). Sequencing of over 2000 clones from the library resulted in the identification of over 1000 unigenes. Candidate genes for almost every step in the biochemical pathways leading from primary metabolites to THCA were identified. Quantitative PCR analysis suggested that many of the pathway genes are preferentially expressed in the glands. Hexanoyl-CoA, one of the metabolites required for THCA synthesis, could be made via either de novo fatty acids synthesis or via the breakdown of existing lipids. qPCR analysis supported the de novo pathway. Many of the ESTs encode transcription factors and two putative MYB genes were identified that were preferentially expressed in glands. Given the similarity of the Cannabis MYB genes to those in other species with known functions, these Cannabis MYBs may play roles in regulating gland development and THCA synthesis. Three candidates for the polyketide synthase (PKS) gene responsible for the first committed step in the pathway to THCA were characterized in more detail. One of these was identical to a previously reported chalcone synthase (CHS) and was found to have CHS activity. All three could use malonyl-CoA and hexanoyl-CoA as substrates, including the CHS, but reaction conditions were not identified that allowed for the production of olivetolic acid (the proposed product of the PKS activity needed for THCA synthesis). One of the PKS candidates was highly and specifically expressed in glands (relative to whole leaves) and, on the basis of these expression data, it is proposed to be the most likely PKS responsible for olivetolic acid synthesis in Cannabis glands. PMID:19581347

Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation and is also required for efficient chemotaxis.

PubMed

Mohanty, S; Jermyn, K A; Early, A; Kawata, T; Aubry, L; Ceccarelli, A; Schaap, P; Williams, J G; Firtel, R A

1999-08-01

Dd-STATa is a structural and functional homologue of the metazoan STAT (Signal Transducer and Activator of Transcription) proteins. We show that Dd-STATa null cells exhibit several distinct developmental phenotypes. The aggregation of Dd-STATa null cells is delayed and they chemotax slowly to a cyclic AMP source, suggesting a role for Dd-STATa in these early processes. In Dd-STATa null strains, slug-like structures are formed but they have an aberrant pattern of gene expression. In such slugs, ecmB/lacZ, a marker that is normally specific for cells on the stalk cell differentiation pathway, is expressed throughout the prestalk region. Stalk cell differentiation in Dictyostelium has been proposed to be under negative control, mediated by repressor elements present in the promoters of stalk cell-specific genes. Dd-STATa binds these repressor elements in vitro and the ectopic expression of ecmB/lacZ in the null strain provides in vivo evidence that Dd-STATa is the repressor protein that regulates commitment to stalk cell differentiation. Dd-STATa null cells display aberrant behavior in a monolayer assay wherein stalk cell differentiation is induced using the stalk cell morphogen DIF. The ecmB gene, a general marker for stalk cell differentiation, is greatly overinduced by DIF in Dd-STATa null cells. Also, Dd-STATa null cells are hypersensitive to DIF for expression of ST/lacZ, a marker for the earliest stages in the differentiation of one of the stalk cell sub-types. We suggest that both these manifestations of DIF hypersensitivity in the null strain result from the balance between activation and repression of the promoter elements being tipped in favor of activation when the repressor is absent. Paradoxically, although Dd-STATa null cells are hypersensitive to the inducing effects of DIF and readily form stalk cells in monolayer assay, the Dd-STATa null cells show little or no terminal stalk cell differentiation within the slug. Dd-STATa null slugs remain developmentally arrested for several days before forming very small spore masses supported by a column of apparently undifferentiated cells. Thus, complete stalk cell differentiation appears to require at least two events: a commitment step, whereby the repression exerted by Dd-STATa is lifted, and a second step that is blocked in a Dd-STATa null organism. This latter step may involve extracellular cAMP, a known repressor of stalk cell differentiation, because Dd-STATa null cells are abnormally sensitive to the inhibitory effects of extracellular cyclic AMP.

Spaceflight effects on cultured embryonic chick bone cells

NASA Technical Reports Server (NTRS)

Landis, W. J.; Hodgens, K. J.; Block, D.; Toma, C. D.; Gerstenfeld, L. C.

2000-01-01

A model calcifying system of primary osteoblast cell cultures derived from normal embryonic chicken calvaria has been flown aboard the shuttle, Endeavour, during the National Aeronautics and Space Administration (NASA) mission STS-59 (April 9-20, 1994) to characterize unloading and other spaceflight effects on the bone cells. Aliquots of cells (approximately 7 x 10(6)) grown in Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) were mixed with microcarrier beads, inoculated into cartridge culture units of artificial hollow fiber capillaries, and carried on the shuttle. To promote cell differentiation, cartridge media were supplemented with 12.5 microg/ml ascorbate and 10 mM beta-glycerophosphate for varying time periods before and during flight. Four cartridges contained cells from 17-day-old embryos grown for 5 days in the presence of ascorbate prior to launch (defined as flight cells committed to the osteoblastic lineage) and four cartridges supported cells from 14-day-old embryos grown for 10 days with ascorbate before launch (uncommitted flight cells). Eight cartridges prepared in the same manner were maintained under normal gravity throughout the flight (control cells) and four additional identical cartridges under normal gravity were terminated on the day of launch (basal cells). From shuttle launch to landing, all cartridges were contained in closed hardware units maintaining 5% CO2, 37 degrees C, and media delivery at a rate of approximately 1.5 ml/6 h. During day 3 and day 5 of flight, duplicate aliquots of conditioned media and accumulated cell products were collected in both the flight and the control hardware units. At the mission end, comparisons among flight, basal, and control samples were made in cell metabolism, gene expression for type I collagen and osteocalcin, and ultrastructure. Both committed and uncommitted flight cells were metabolically active, as measured by glucose uptake and lactate production, at approximately the same statistical levels as control counterparts. Flight cells elaborated a less extensive extracellular matrix, evidenced by a reduced collagen gene expression and collagen protein appearance compared with controls. Osteocalcin was expressed by all cells, a result indicating progressive differentiation of both flight and control osteoblasts, but its message levels also were reduced in flight cells compared with ground samples. This finding suggested that osteoblasts subjected to flight followed a slower progression toward a differentiated function. The summary of data indicates that spaceflight, including microgravity exposure, demonstrably affects bone cells by down-regulating type I collagen and osteocalcin gene expression and thereby inhibiting expression of the osteogenic phenotype notably by committed osteoblasts. The information is important for insight into the response of bone cells to changes of gravity and of force in general.

The Staphylococcus aureus RNome and Its Commitment to Virulence

PubMed Central

Felden, Brice; Vandenesch, François; Bouloc, Philippe; Romby, Pascale

2011-01-01

Staphylococcus aureus is a major human pathogen causing a wide spectrum of nosocomial and community-associated infections with high morbidity and mortality. S. aureus generates a large number of virulence factors whose timing and expression levels are precisely tuned by regulatory proteins and RNAs. The aptitude of bacteria to use RNAs to rapidly modify gene expression, including virulence factors in response to stress or environmental changes, and to survive in a host is an evolving concept. Here, we focus on the recently inventoried S. aureus regulatory RNAs, with emphasis on those with identified functions, two of which are directly involved in pathogenicity. PMID:21423670

The Ca2+-induced methyltransferase xPRMT1b controls neural fate in amphibian embryo.

PubMed

Batut, Julie; Vandel, Laurence; Leclerc, Catherine; Daguzan, Christiane; Moreau, Marc; Néant, Isabelle

2005-10-18

We have previously shown that an increase in intracellular Ca2+ is both necessary and sufficient to commit ectoderm to a neural fate in Xenopus embryos. However, the relationship between this Ca2+ increase and the expression of early neural genes has yet to be defined. Using a subtractive cDNA library between untreated and caffeine-treated animal caps, i.e., control ectoderm and ectoderm induced toward a neural fate by a release of Ca2+, we have isolated the arginine N-methyltransferase, xPRMT1b, a Ca2+-induced target gene, which plays a pivotal role in this process. First, we show in embryo and in animal cap that xPRMT1b expression is Ca2+-regulated. Second, overexpression of xPRMT1b induces the expression of early neural genes such as Zic3. Finally, in the whole embryo, antisense approach with morpholino oligonucleotide against xPRMT1b impairs neural development and in animal caps blocks the expression of neural markers induced by a release of internal Ca2+. Our results implicate an instructive role of an enzyme, an arginine methyltransferase protein, in the embryonic choice of determination between epidermal and neural fate. The results presented provide insights by which a Ca2+ increase induces neural fate.

Research resource: Tissue-specific transcriptomics and cistromics of nuclear receptor signaling: a web research resource.

PubMed

Ochsner, Scott A; Watkins, Christopher M; LaGrone, Benjamin S; Steffen, David L; McKenna, Neil J

2010-10-01

Nuclear receptors (NRs) are ligand-regulated transcription factors that recruit coregulators and other transcription factors to gene promoters to effect regulation of tissue-specific transcriptomes. The prodigious rate at which the NR signaling field has generated high content gene expression and, more recently, genome-wide location analysis datasets has not been matched by a committed effort to archiving this information for routine access by bench and clinical scientists. As a first step towards this goal, we searched the MEDLINE database for studies, which referenced either expression microarray and/or genome-wide location analysis datasets in which a NR or NR ligand was an experimental variable. A total of 1122 studies encompassing 325 unique organs, tissues, primary cells, and cell lines, 35 NRs, and 91 NR ligands were retrieved and annotated. The data were incorporated into a new section of the Nuclear Receptor Signaling Atlas Molecule Pages, Transcriptomics and Cistromics, for which we designed an intuitive, freely accessible user interface to browse the studies. Each study links to an abstract, the MEDLINE record, and, where available, Gene Expression Omnibus and ArrayExpress records. The resource will be updated on a regular basis to provide a current and comprehensive entrez into the sum of transcriptomic and cistromic research in this field.

Haploinsufficiency for the Six2 gene increases nephron progenitor proliferation promoting branching and nephron number.

PubMed

Combes, Alexander N; Wilson, Sean; Phipson, Belinda; Binnie, Brandon B; Ju, Adler; Lawlor, Kynan T; Cebrian, Cristina; Walton, Sarah L; Smyth, Ian M; Moritz, Karen M; Kopan, Raphael; Oshlack, Alicia; Little, Melissa H

2018-03-01

The regulation of final nephron number in the kidney is poorly understood. Cessation of nephron formation occurs when the self-renewing nephron progenitor population commits to differentiation. Transcription factors within this progenitor population, such as SIX2, are assumed to control expression of genes promoting self-renewal such that homozygous Six2 deletion results in premature commitment and an early halt to kidney development. In contrast, Six2 heterozygotes were assumed to be unaffected. Using quantitative morphometry, we found a paradoxical 18% increase in ureteric branching and final nephron number in Six2 heterozygotes, despite evidence for reduced levels of SIX2 protein and transcript. This was accompanied by a clear shift in nephron progenitor identity with a distinct subset of downregulated progenitor genes such as Cited1 and Meox1 while other genes were unaffected. The net result was an increase in nephron progenitor proliferation, as assessed by elevated EdU (5-ethynyl-2'-deoxyuridine) labeling, an increase in MYC protein, and transcriptional upregulation of MYC target genes. Heterozygosity for Six2 on an Fgf20-/- background resulted in premature differentiation of the progenitor population, confirming that progenitor regulation is compromised in Six2 heterozygotes. Overall, our studies reveal a unique dose response of nephron progenitors to the level of SIX2 protein in which the role of SIX2 in progenitor proliferation versus self-renewal is separable. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Dimorphic DNA methylation during temperature-dependent sex determination in the sea turtle Lepidochelys olivacea.

PubMed

Venegas, Daniela; Marmolejo-Valencia, Alejandro; Valdes-Quezada, Christian; Govenzensky, Tzipe; Recillas-Targa, Félix; Merchant-Larios, Horacio

2016-09-15

Sex determination in vertebrates depends on the expression of a conserved network of genes. Sea turtles such as Lepidochelys olivacea have temperature-dependent sex determination. The present work analyses some of the epigenetic processes involved in this. We describe sexual dimorphism in global DNA methylation patterns between ovaries and testes of L. olivacea and show that the differences may arise from a combination of DNA methylation and demethylation events that occur during sex determination. Irrespective of incubation temperature, 5-hydroxymethylcytosine was abundant in the bipotential gonad; however, following sex determination, this modification was no longer found in pre-Sertoli cells in the testes. These changes correlate with the establishment of the sexually dimorphic DNA methylation patterns, down regulation of Sox9 gene expression in ovaries and irreversible gonadal commitment towards a male or female differentiation pathway. Thus, DNA methylation changes may be necessary for the stabilization of the gene expression networks that drive the differentiation of the bipotential gonad to form either an ovary or a testis in L. olivacea and probably among other species that manifest temperature-dependent sex determination. Copyright © 2016 Elsevier Inc. All rights reserved.

Msx1-modulated muscle satellite cells retain a primitive state and exhibit an enhanced capacity for osteogenic differentiation

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

Ding, Ke, E-mail: dingke@med.uestc.edu.cn; Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, Chengdu 610072; Department of Orthopaedics, Southwest Hospital, Third Military Medical University, Chongqing 400038

Multipotent muscle satellite cells (MuSCs) have been identified as potential seed cells for bone tissue engineering. However, MuSCs exhibit a rapid loss of stemness after in vitro culturing, thereby compromising their therapeutic efficiency. Muscle segment homeobox gene 1 (msx1) has been found to induce the dedifferentiation of committed progenitor cells, as well as terminally differentiated myotubes. In this study, a Tet-off retroviral gene delivery system was used to modulate msx1 expression. After ten passages, MuSCs that did not express msx-1 (e.g., the non-msx1 group) were compared with MuSCs with induced msx-1 expression (e.g., the msx1 group). The latter group exhibitedmore » a more juvenile morphology, it contained a significantly lower percentage of senescent cells characterized by positive β-galactosidase staining, and it exhibited increased proliferation and a higher proliferation index. Immunocytochemical stainings further detected a more primitive gene expression profile for the msx1 group, while osteogenic differentiation assays and ectopic bone formation assays demonstrated an improved capacity for the msx1 group to undergo osteogenic differentiation. These results suggest that transient expression of msx1 in MuSCs can retain a primitive state, thereby enhancing their capacity for osteogenic differentiation and restoring the potential for MuSCs to serve as seed cells for bone tissue engineering.« less

Msx1-modulated muscle satellite cells retain a primitive state and exhibit an enhanced capacity for osteogenic differentiation.

PubMed

Ding, Ke; Liu, Wen-Ying; Zeng, Qiang; Hou, Fang; Xu, Jian-Zhong; Yang, Zhong

2017-03-01

Multipotent muscle satellite cells (MuSCs) have been identified as potential seed cells for bone tissue engineering. However, MuSCs exhibit a rapid loss of stemness after in vitro culturing, thereby compromising their therapeutic efficiency. Muscle segment homeobox gene 1 (msx1) has been found to induce the dedifferentiation of committed progenitor cells, as well as terminally differentiated myotubes. In this study, a Tet-off retroviral gene delivery system was used to modulate msx1 expression. After ten passages, MuSCs that did not express msx-1 (e.g., the non-msx1 group) were compared with MuSCs with induced msx-1 expression (e.g., the msx1 group). The latter group exhibited a more juvenile morphology, it contained a significantly lower percentage of senescent cells characterized by positive β-galactosidase staining, and it exhibited increased proliferation and a higher proliferation index. Immunocytochemical stainings further detected a more primitive gene expression profile for the msx1 group, while osteogenic differentiation assays and ectopic bone formation assays demonstrated an improved capacity for the msx1 group to undergo osteogenic differentiation. These results suggest that transient expression of msx1 in MuSCs can retain a primitive state, thereby enhancing their capacity for osteogenic differentiation and restoring the potential for MuSCs to serve as seed cells for bone tissue engineering. Copyright © 2017 Elsevier Inc. All rights reserved.

Validation of osteogenic properties of Cytochalasin D by high-resolution RNA-sequencing in mesenchymal stem cells derived from bone marrow and adipose tissues.

PubMed

Samsonraj, Rebekah; Paradise, Christopher R; Dudakovic, Amel; Sen, Buer; Nair, Asha A; Dietz, Allan B; Deyle, David R; Cool, Simon M; Rubin, Janet; van Wijnen, Andre

2018-06-08

Differentiation of mesenchymal stromal/stem cells (MSCs) involves a series of molecular signals and gene transcription events required for attaining cell lineage commitment. Modulation of the actin cytoskeleton using cytochalasin D (CytoD) drives osteogenesis at early time points in bone marrow-derived MSCs, and also initiates a robust osteogenic differentiation program in adipose-derived MSCs. To understand the molecular basis for these pronounced effects on osteogenic differentiation, we investigated global changes in gene expression in CytoD-treated murine and human MSCs by high-resolution RNA-sequencing (RNA-seq) analysis. A three-way bioinformatic comparison between human adipose-derived, human bone marrow-derived and mouse bone marrow-derived MSCs revealed significant upregulation of genes linked to extracellular matrix organization, cell adhesion and bone metabolism. As anticipated, the activation of these differentiation related genes is accompanied by a downregulation of nuclear and cell cycle-related genes presumably reflecting cytostatic effects of CytoD. We also identified eight novel CytoD activated genes - VGLL4, ARHGAP24, KLHL24, RCBTB2, BDH2, SCARF2, ACAD10, HEPH - which are commonly upregulated across the two species and tissue sources of our MSC samples. We selected the Hippo-pathway related VGLL4 gene, which encodes the transcriptional co-factor Vestigial-like 4, for further study because this pathway is linked to osteogenesis. VGLL4 siRNA depletion reduces mineralization of adipose-derived MSCs during CytoD-induced osteogenic differentiation. Together, our RNA-seq analyses suggest that while the stimulatory effects of CytoD on osteogenesis are pleiotropic and depend on the biological state of the cell type, a small group of genes including VGLL4 may contribute to MSC commitment towards the bone lineage.

The Architectural Organization of Human Stem Cell Cycle Regulatory Machinery

PubMed Central

Stein, Gary S.; Stein, Janet L.; Wijnen, Andre van J; Lian, Jane B.; Montecino, Martin; Medina, Ricardo; Kapinas, Kristie; Ghule, Prachi; Grandy, Rodrigo; Zaidi, Sayyed K.; Becker, Klaus A.

2013-01-01

Two striking features of human embryonic stem cells that support biological activity are an abbreviated cell cycle and reduced complexity to nuclear organization. The potential implications for rapid proliferation of human embryonic stem cells within the context of sustaining pluripotency, suppressing phenotypic gene expression and linkage to simplicity in the architectural compartmentalization of regulatory machinery in nuclear microenvironments is explored. Characterization of the molecular and architectural commitment steps that license human embryonic stem cells to initiate histone gene expression is providing understanding of the principal regulatory mechanisms that control the G1/S phase transition in primitive pluripotent cells. From both fundamental regulatory and clinical perspectives, further understanding of the pluripotent cell cycle in relation to compartmentalization of regulatory machinery in nuclear microenvironments is relevant to applications of stem cells for regenerative medicine and new dimensions to therapy where traditional drug discovery strategies have been minimally effective. PMID:22394165

Coregulation of terpenoid pathway genes and prediction of isoprene production in Bacillus subtilis using transcriptomics

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

Hess, Becky M.; Xue, Junfeng; Markillie, Lye Meng

2013-06-19

The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding the system level regulation and control of the pathway. To address this limitation, we examined Bacillus subtilis grown under multiple conditions and then determined the relationship between altered isoprene production and the pattern of gene expression. Wemore » found that terpenoid genes appeared to fall into two distinct subsets with opposing correlations with respect to the amount of isoprene produced. The group whose expression levels positively correlated with isoprene production included dxs, the gene responsible for the commitment step in the pathway, as well as ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. This analysis showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model which accurately predicts production of this secondary metabolite across many simulated environmental conditions.« less

Acute leukemia coexpressing myeloid, B- and T-lineage associated markers: multiparameter analysis of criteria defining lineage commitment and maturational stage in a case of undifferentiated leukemia.

PubMed

Meckenstock, G; Heyll, A; Schneider, E M; Hildebrandt, B; Runde, V; Aul, C; Bartram, C R; Ludwig, W D; Schneider, W

1995-02-01

Coexpression of myeloid, B-, and T-lineage associated markers was found in a patient with morphologically and cytochemically undifferentiated acute leukemia. Surface marker analysis using two-color immunofluorescence staining characterized blast cells to express CD34, CD38, CD117, and class II antigens, coexpressing TdT, CD4, CD7, CD13, CD19, and CD33. Cytoplasmic expression of myeloperoxidase, CD3, and CD22 could not be demonstrated. Monosomy for chromosome 7 was found by cytogenetic analysis. The absence of clonal rearrangements of immunoglobulin or T-cell receptor genes was shown by Southern blot analysis. Using a 3H-thymidine incorporation assay, DNA synthesis of leukemic blasts could be stimulated by IL-3, IL-6 and G-CSF in vitro. The present case did not offer specific criteria of lineage commitment. Corresponding to an equivalent counterpart in normal hematopoiesis, the involved cell population may reflect an early, most immature developmental stage within a multipotent progenitor cell compartment.

Clonal Type I Interferon–producing and Dendritic Cell Precursors Are Contained in Both Human Lymphoid and Myeloid Progenitor Populations

PubMed Central

Chicha, Laurie; Jarrossay, David; Manz, Markus G.

2004-01-01

Because of different cytokine responsiveness, surface receptor, and transcription factor expression, human CD11c− natural type I interferon–producing cells (IPCs) and CD11c+ dendritic cells were thought to derive through lymphoid and myeloid hematopoietic developmental pathways, respectively. To directly test this hypothesis, we used an in vitro assay allowing simultaneous IPC, dendritic cell, and B cell development and we tested lymphoid and myeloid committed hematopoietic progenitor cells for their developmental capacity. Lymphoid and common myeloid and granulocyte/macrophage progenitors were capable of developing into both functional IPCs, expressing gene transcripts thought to be associated with lymphoid lineage development, and into dendritic cells. However, clonal progenitors for both populations were about fivefold more frequent within myeloid committed progenitor cells. Thus, in humans as in mice, natural IPC and dendritic cell development robustly segregates with myeloid differentiation. This would fit with natural interferon type I–producing cell and dendritic cell activity in innate immunity, the evolutionary older arm of the cellular immune system. PMID:15557348

EBF proteins participate in transcriptional regulation of Xenopus muscle development.

PubMed

Green, Yangsook Song; Vetter, Monica L

2011-10-01

EBF proteins have diverse functions in the development of multiple lineages, including neurons, B cells and adipocytes. During Drosophila muscle development EBF proteins are expressed in muscle progenitors and are required for muscle cell differentiation, but there is no known function of EBF proteins in vertebrate muscle development. In this study, we examine the expression of ebf genes in Xenopus muscle tissue and show that EBF activity is necessary for aspects of Xenopus skeletal muscle development, including somite organization, migration of hypaxial muscle anlagen toward the ventral abdomen, and development of jaw muscle. From a microarray screen, we have identified multiple candidate targets of EBF activity with known roles in muscle development. The candidate targets we have verified are MYOD, MYF5, M-Cadherin and SEB-4. In vivo overexpression of the ebf2 and ebf3 genes leads to ectopic expression of these candidate targets, and knockdown of EBF activity causes downregulation of the endogenous expression of the candidate targets. Furthermore, we found that MYOD and MYF5 are likely to be direct targets. Finally we show that MYOD can upregulate the expression of ebf genes, indicating the presence of a positive feedback loop between EBF and MYOD that we find to be important for maintenance of MYOD expression in Xenopus. These results suggest that EBF activity is important for both stabilizing commitment and driving aspects of differentiation in Xenopus muscle cells. Copyright © 2010 Elsevier Inc. All rights reserved.

Notch/Delta signaling constrains reengineering of pro-T cells by PU.1

PubMed Central

Franco, Christopher B.; Scripture-Adams, Deirdre D.; Proekt, Irina; Taghon, Tom; Weiss, Angela H.; Yui, Mary A.; Adams, Stephanie L.; Diamond, Rochelle A.; Rothenberg, Ellen V.

2006-01-01

PU.1 is essential for early stages of mouse T cell development but antagonizes it if expressed constitutively. Two separable mechanisms are involved: attenuation and diversion. Dysregulated PU.1 expression inhibits pro-T cell survival, proliferation, and passage through β-selection by blocking essential T cell transcription factors, signaling molecules, and Rag gene expression, which expression of a rearranged T cell antigen receptor transgene cannot rescue. However, Bcl2 transgenic cells are protected from this attenuation and may even undergo β-selection, as shown by PU.1 transduction of defined subsets of Bcl2 transgenic fetal thymocytes with differentiation in OP9-DL1 and OP9 control cultures. The outcome of PU.1 expression in these cells depends on Notch/Delta signaling. PU.1 can efficiently divert thymocytes toward a myeloid-like state with multigene regulatory changes, but Notch/Delta signaling vetoes diversion. Gene expression analysis distinguishes sets of critical T lineage regulatory genes with different combinatorial responses to PU.1 and Notch/Delta signals, suggesting particular importance for inhibition of E proteins, Myb, and/or Gfi1 (growth factor independence 1) in diversion. However, Notch signaling only protects against diversion of cells that have undergone T lineage specification after Thy-1 and CD25 up-regulation. The results imply that in T cell precursors, Notch/Delta signaling normally acts to modulate and channel PU.1 transcriptional activities during the stages from T lineage specification until commitment. PMID:16880393

Cytochrome P450-Dependent Metabolism of Oxylipins in Tomato. Cloning and Expression of Allene Oxide Synthase and Fatty Acid Hydroperoxide Lyase1

PubMed Central

Howe, Gregg A.; Lee, Gyu In; Itoh, Aya; Li, Lei; DeRocher, Amy E.

2000-01-01

Allene oxide synthase (AOS) and fatty acid hydroperoxide lyase (HPL) are plant-specific cytochrome P450s that commit fatty acid hydroperoxides to different branches of oxylipin metabolism. Here we report the cloning and characterization of AOS (LeAOS) and HPL (LeHPL) cDNAs from tomato (Lycopersicon esculentum). Functional expression of the cDNAs in Escherichia coli showed that LeAOS and LeHPL encode enzymes that metabolize 13- but not 9-hydroperoxide derivatives of C18 fatty acids. LeAOS was active against both 13S-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13-HPOT) and 13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid, whereas LeHPL showed a strong preference for 13-HPOT. These results suggest a role for LeAOS and LeHPL in the metabolism of 13-HPOT to jasmonic acid and hexenal/traumatin, respectively. LeAOS expression was detected in all organs of the plant. In contrast, LeHPL expression was predominant in leaves and flowers. Damage inflicted to leaves by chewing insect larvae led to an increase in the local and systemic expression of both genes, with LeAOS showing the strongest induction. Wound-induced expression of LeAOS also occurred in the def-1 mutant that is deficient in octadecanoid-based signaling of defensive proteinase inhibitor genes. These results demonstrate that tomato uses genetically distinct signaling pathways for the regulation of different classes of wound responsive genes. PMID:10859201

Expression, subcellular localization, and cis-regulatory structure of duplicated phytoene synthase genes in melon (Cucumis melo L.).

PubMed

Qin, Xiaoqiong; Coku, Ardian; Inoue, Kentaro; Tian, Li

2011-10-01

Carotenoids perform many critical functions in plants, animals, and humans. It is therefore important to understand carotenoid biosynthesis and its regulation in plants. Phytoene synthase (PSY) catalyzes the first committed and rate-limiting step in carotenoid biosynthesis. While PSY is present as a single copy gene in Arabidopsis, duplicated PSY genes have been identified in many economically important monocot and dicot crops. CmPSY1 was previously identified from melon (Cucumis melo L.), but was not functionally characterized. We isolated a second PSY gene, CmPSY2, from melon in this work. CmPSY2 possesses a unique intron/exon structure that has not been observed in other plant PSYs. Both CmPSY1 and CmPSY2 are functional in vitro, but exhibit distinct expression patterns in different melon tissues and during fruit development, suggesting differential regulation of the duplicated melon PSY genes. In vitro chloroplast import assays verified the plastidic localization of CmPSY1 and CmPSY2 despite the lack of an obvious plastid target peptide in CmPSY2. Promoter motif analysis of the duplicated melon and tomato PSY genes and the Arabidopsis PSY revealed distinctive cis-regulatory structures of melon PSYs and identified gibberellin-responsive motifs in all PSYs except for SlPSY1, which has not been reported previously. Overall, these data provide new insights into the evolutionary history of plant PSY genes and the regulation of PSY expression by developmental and environmental signals that may involve different regulatory networks.

Coregulation of Terpenoid Pathway Genes and Prediction of Isoprene Production in Bacillus subtilis Using Transcriptomics.

PubMed

Hess, Becky M; Xue, Junfeng; Markillie, Lye Meng; Taylor, Ronald C; Wiley, H Steven; Ahring, Birgitte K; Linggi, Bryan

2013-01-01

The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes, as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding system level regulation and control of the pathway. To address these limitations, we examined Bacillus subtilis grown under multiple conditions and determined the relationship between altered isoprene production and gene expression patterns. We found that with respect to the amount of isoprene produced, terpenoid genes fall into two distinct subsets with opposing correlations. The group whose expression levels positively correlated with isoprene production included dxs, which is responsible for the commitment step in the pathway, ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome-wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. These analyses showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model that accurately predicts production of this secondary metabolite across many simulated environmental conditions.

Coregulation of Terpenoid Pathway Genes and Prediction of Isoprene Production in Bacillus subtilis Using Transcriptomics

PubMed Central

Hess, Becky M.; Xue, Junfeng; Markillie, Lye Meng; Taylor, Ronald C.; Wiley, H. Steven; Ahring, Birgitte K.; Linggi, Bryan

2013-01-01

The isoprenoid pathway converts pyruvate to isoprene and related isoprenoid compounds in plants and some bacteria. Currently, this pathway is of great interest because of the critical role that isoprenoids play in basic cellular processes, as well as the industrial value of metabolites such as isoprene. Although the regulation of several pathway genes has been described, there is a paucity of information regarding system level regulation and control of the pathway. To address these limitations, we examined Bacillus subtilis grown under multiple conditions and determined the relationship between altered isoprene production and gene expression patterns. We found that with respect to the amount of isoprene produced, terpenoid genes fall into two distinct subsets with opposing correlations. The group whose expression levels positively correlated with isoprene production included dxs, which is responsible for the commitment step in the pathway, ispD, and two genes that participate in the mevalonate pathway, yhfS and pksG. The subset of terpenoid genes that inversely correlated with isoprene production included ispH, ispF, hepS, uppS, ispE, and dxr. A genome-wide partial least squares regression model was created to identify other genes or pathways that contribute to isoprene production. These analyses showed that a subset of 213 regulated genes was sufficient to create a predictive model of isoprene production under different conditions and showed correlations at the transcriptional level. We conclude that gene expression levels alone are sufficiently informative about the metabolic state of a cell that produces increased isoprene and can be used to build a model that accurately predicts production of this secondary metabolite across many simulated environmental conditions. PMID:23840410

An Embryonic and Induced Pluripotent Stem Cell Model for Ovarian Granulosa Cell Development and Steroidogenesis.

PubMed

Lipskind, Shane; Lindsey, Jennifer S; Gerami-Naini, Behzad; Eaton, Jennifer L; O'Connell, Daniel; Kiezun, Adam; Ho, Joshua W K; Ng, Nicholas; Parasar, Parveen; Ng, Michelle; Nickerson, Michael; Demirci, Utkan; Maas, Richard; Anchan, Raymond M

2018-05-01

Embryoid bodies (EBs) can serve as a system for evaluating pluripotency, cellular differentiation, and tissue morphogenesis. In this study, we use EBs derived from mouse embryonic stem cells (mESCs) and human amniocyte-derived induced pluripotent stem cells (hAdiPSCs) as a model for ovarian granulosa cell (GC) development and steroidogenic cell commitment. We demonstrated that spontaneously differentiated murine EBs (mEBs) and human EBs (hEBs) displayed ovarian GC markers, such as aromatase (CYP19A1), FOXL2, AMHR2, FSHR, and GJA1. Comparative microarray analysis identified both shared and unique gene expression between mEBs and the maturing mouse ovary. Gene sets related to gonadogenesis, lipid metabolism, and ovarian development were significantly overrepresented in EBs. Of the 29 genes, 15 that were differentially regulated in steroidogenic mEBs displayed temporal expression changes between embryonic, postnatal, and mature ovarian tissues by polymerase chain reaction. Importantly, both mEBs and hEBs were capable of gonadotropin-responsive estradiol (E2) synthesis in vitro (217-759 pg/mL). Live fluorescence-activated cell sorting-sorted AMHR2 + granulosa-like cells from mEBs continued to produce E2 after purification (15.3 pg/mL) and secreted significantly more E2 than AMHR2 - cells (8.6 pg/mL, P < .05). We conclude that spontaneously differentiated EBs of both mESC and hAdiPSC origin can serve as a biologically relevant model for ovarian GC differentiation and steroidogenic cell commitment. These cells should be further investigated for therapeutic uses, such as stem cell-based hormone replacement therapy and in vitro maturation of oocytes.

EVI1 Interferes with Myeloid Maturation via Transcriptional Repression of Cebpa, via Binding to Two Far Downstream Regulatory Elements*

PubMed Central

Wilson, Michael; Tsakraklides, Vasiliki; Tran, Minh; Xiao, Ying-Yi; Zhang, Yi; Perkins, Archibald S.

2016-01-01

One mechanism by which oncoproteins work is through perturbation of cellular maturation; understanding the mechanisms by which this occurs can lead to the development of targeted therapies. EVI1 is a zinc finger oncoprotein involved in the development of acute myeloid leukemia; previous work has shown it to interfere with the maturation of granulocytes from immature precursors. Here we investigate the mechanism by which that occurs, using an immortalized hematopoietic progenitor cell line, EML-C1, as a model system. We document that overexpression of EVI1 abrogates retinoic acid-induced maturation of EML cells into committed myeloid cells, a process that can be documented by the down-regulation of stem cell antigen-1 and acquisition of responsiveness to granulocyte-macrophage colony-stimulating factor. We show that this requires DNA binding capacity of EVI1, suggesting that downstream target genes are involved. We identify the myeloid regulator Cebpa as a target gene and identify two EVI1 binding regions within evolutionarily conserved enhancer elements at +35 and +37 kb relative to the gene. EVI1 can strongly suppress Cebpa transcription, and add-back of Cebpa into EVI1-expressing EML cells partially corrects the block in maturation. We identify the DNA sequences to which EVI1 binds at +35 and +37 kb and show that mutation of one of these releases Cebpa from EVI1-induced suppression. We observe a more complex picture in primary bone marrow cells, where EVI1 suppresses Cebpa in stem cells but not in more committed progenitors. Our data thus identify a regulatory node by which EVI1 contributes to leukemia, and this represents a possible therapeutic target for treatment of EVI1-expressing leukemia. PMID:27129260

HLS7, a hemopoietic lineage switch gene homologous to the leukemia-inducing gene MLF1.

PubMed Central

Williams, J H; Daly, L N; Ingley, E; Beaumont, J G; Tilbrook, P A; Lalonde, J P; Stillitano, J P; Klinken, S P

1999-01-01

Hemopoietic lineage switching occurs when leukemic cells, apparently committed to one lineage, change and display the phenotype of another pathway. cDNA representational difference analysis was used to identify myeloid-specific genes that may be associated with an erythroid to myeloid lineage switch involving the murine J2E erythroleukemic cell line. One of the genes isolated (HLS7) is homologous to the novel human oncogene myeloid leukemia factor 1 (MLF1) involved in the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia. Enforced expression of HLS7 in J2E cells induced a monoblastoid phenotype, thereby recapitulating the spontaneous erythroid to myeloid lineage switch. HLS7 also inhibited erythropoietin- or chemically-induced differentiation of erythroleukemic cell lines and suppressed development of erythropoietin-responsive colonies in semi-solid culture. However, intracellular signaling activated by erythropoietin was not impeded by ectopic expression of HLS7. In contrast, HLS7 promoted maturation of M1 monoblastoid cells and increased myeloid colony formation in vitro. These data show that HLS7 can influence erythroid/myeloid lineage switching and the development of normal hemopoietic cells. PMID:10523300

HLS7, a hemopoietic lineage switch gene homologous to the leukemia-inducing gene MLF1.

PubMed

Williams, J H; Daly, L N; Ingley, E; Beaumont, J G; Tilbrook, P A; Lalonde, J P; Stillitano, J P; Klinken, S P

1999-10-15

Hemopoietic lineage switching occurs when leukemic cells, apparently committed to one lineage, change and display the phenotype of another pathway. cDNA representational difference analysis was used to identify myeloid-specific genes that may be associated with an erythroid to myeloid lineage switch involving the murine J2E erythroleukemic cell line. One of the genes isolated (HLS7) is homologous to the novel human oncogene myeloid leukemia factor 1 (MLF1) involved in the t(3;5)(q25.1;q34) translocation associated with acute myeloid leukemia. Enforced expression of HLS7 in J2E cells induced a monoblastoid phenotype, thereby recapitulating the spontaneous erythroid to myeloid lineage switch. HLS7 also inhibited erythropoietin- or chemically-induced differentiation of erythroleukemic cell lines and suppressed development of erythropoietin-responsive colonies in semi-solid culture. However, intracellular signaling activated by erythropoietin was not impeded by ectopic expression of HLS7. In contrast, HLS7 promoted maturation of M1 monoblastoid cells and increased myeloid colony formation in vitro. These data show that HLS7 can influence erythroid/myeloid lineage switching and the development of normal hemopoietic cells.

Analysis of the promoter of the cudA gene reveals novel mechanisms of Dictyostelium cell type differentiation.

PubMed

Fukuzawa, M; Williams, J G

2000-06-01

The cudA gene encodes a nuclear protein that is essential for normal multicellular development. At the slug stage cudA is expressed in the prespore cells and in a sub-region of the prestalk zone. We show that cap site distal promoter sequences direct cudA expression in prespore cells, while proximal sequences direct expression in the prestalk sub-region. The promoter domain that directs prespore-specific transcription consists of a positively acting region, that has the potential to direct expression in all cells within the slug, and a negatively acting region that prevents expression in the prestalk cells. Dd-STATa is the STAT protein that regulates commitment to stalk cell gene expression, where it is known to function as a transcriptional repressor. We show that Dd-STATa binds in vitro to the positively acting part of the prespore domain of the cudA promoter. However, Dd-STATa cannot be utilised for this purpose in vivo, because analysis of a Dd-STATa null mutant strain shows that Dd-STATa is not necessary for cudA transcription in prespore cells. In contrast, the part of the cudA promoter that directs prestalk-specific expression contains a binding site for Dd-STATa that is essential for its biological activity. Dd-STATa appears therefore to serve as a direct activator of cudA transcription in prestalk cells, while a protein with a DNA binding specificity highly related to that of Dd-STATa is utilised to activate cudA transcription in prespore cells.

Corrected and Republished from: BCL11A Is a Critical Component of a Transcriptional Network That Activates Recombinase Activating Gene Expression and V(D)J Recombination

PubMed Central

Lee, Baeck-Seung; Lee, Bum-Kyu; Iyer, Vishwanath R.; Sleckman, Barry P.; Shaffer, Arthur L.; Ippolito, Gregory C.

2017-01-01

ABSTRACT Recombination activating gene 1 (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining [V(D)J] segment recombination, an essential process for antigen receptor expression and lymphocyte development. The BCL11A transcription factor is required for B cell and plasmacytoid dendritic cell (pDC) development, but its molecular function(s) in early B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds directly to the RAG1 promoter as well as directly to regulatory regions of transcription factors previously implicated in both B cell and pDC development to activate RAG1 and RAG2 gene transcription in pro- and pre-B cells. We employed BCL11A overexpression with recombination substrates to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination. PMID:29038163

Polycomb-like 2 Associates with PRC2 and Regulates Transcriptional Networks during Mouse Embryonic Stem Cell Self-Renewal and Differentiation

PubMed Central

Walker, Emily; Chang, Wing Y.; Hunkapiller, Julie; Cagney, Gerard; Garcha, Kamal; Torchia, Joseph; Krogan, Nevan J.; Reiter, Jeremy F.; Stanford, William L.

2010-01-01

Summary Polycomb group (PcG) proteins are conserved epigenetic transcriptional repressors that control numerous developmental gene expression programs and have recently been implicated in modulating embryonic stem cell (ESC) fate. We identified the PcG protein PCL2 (polycomb-like 2) in a genome-wide screen for regulators of self-renewal and pluripotency and predicted that it would play an important role in mouse ESC fate determination. Using multiple biochemical strategies, we provide evidence that PCL2 is a Polycomb Repressive Complex 2 (PRC2)-associated protein in mouse ESCs. Knockdown of Pcl2 in ESCs resulted in heightened self-renewal characteristics, defects in differentiation and altered patterns of histone methylation. Integration of global gene expression and promoter occupancy analyses allowed us to identify PCL2 and PRC2 transcriptional targets and draft regulatory networks. We describe the role of PCL2 in both modulating transcription of ESC self-renewal genes in undifferentiated ESCs as well as developmental regulators during early commitment and differentiation. PMID:20144788

Memory T and memory B cells share a transcriptional program of self-renewal with long-term hematopoietic stem cells

PubMed Central

Luckey, Chance John; Bhattacharya, Deepta; Goldrath, Ananda W.; Weissman, Irving L.; Benoist, Christophe; Mathis, Diane

2006-01-01

The only cells of the hematopoietic system that undergo self-renewal for the lifetime of the organism are long-term hematopoietic stem cells and memory T and B cells. To determine whether there is a shared transcriptional program among these self-renewing populations, we first compared the gene-expression profiles of naïve, effector and memory CD8+ T cells with those of long-term hematopoietic stem cells, short-term hematopoietic stem cells, and lineage-committed progenitors. Transcripts augmented in memory CD8+ T cells relative to naïve and effector T cells were selectively enriched in long-term hematopoietic stem cells and were progressively lost in their short-term and lineage-committed counterparts. Furthermore, transcripts selectively decreased in memory CD8+ T cells were selectively down-regulated in long-term hematopoietic stem cells and progressively increased with differentiation. To confirm that this pattern was a general property of immunologic memory, we turned to independently generated gene expression profiles of memory, naïve, germinal center, and plasma B cells. Once again, memory-enriched and -depleted transcripts were also appropriately augmented and diminished in long-term hematopoietic stem cells, and their expression correlated with progressive loss of self-renewal function. Thus, there appears to be a common signature of both up- and down-regulated transcripts shared between memory T cells, memory B cells, and long-term hematopoietic stem cells. This signature was not consistently enriched in neural or embryonic stem cell populations and, therefore, appears to be restricted to the hematopoeitic system. These observations provide evidence that the shared phenotype of self-renewal in the hematopoietic system is linked at the molecular level. PMID:16492737

Homogentisate phytyltransferase from the unicellular green alga Chlamydomonas reinhardtii.

PubMed

Gálvez-Valdivieso, Gregorio; Cardeñosa, Rosa; Pineda, Manuel; Aguilar, Miguel

2015-09-01

Homogentisate phytyltransferase (HPT) (EC 2.5.1.-) catalyzes the first committed step of tocopherol biosynthesis in all photosynthetic organisms. This paper presents the molecular characterization and expression analysis of HPT1 gene, and a study on the accumulation of tocopherols under different environmental conditions in the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HPT1 protein conserves all the prenylphosphate- and divalent cation-binding sites that are found in polyprenyltransferases and all the amino acids that are essential for its catalytic activity. Its hydrophobicity profile confirms that HPT is a membrane-bound protein. Chlamydomonas genomic DNA analysis suggests that HPT is encoded by a single gene, HPT1, whose promoter region contains multiple motifs related to regulation by jasmonate, abscisic acid, low temperature and light, and an ATCTA motif presents in genes involved in tocopherol biosynthesis and some photosynthesis-related genes. Expression analysis revealed that HPT1 is strongly regulated by dark and low-temperature. Under the same treatments, α-tocopherol increased in cultures exposed to darkness or heat, whereas γ-tocopherol did it in low temperature. The regulatory expression pattern of HPT1 and the changes of tocopherol abundance support the idea that different tocopherols play specific functions, and suggest a role for γ-tocopherol in the adaptation to growth under low-temperature. Copyright © 2015 Elsevier GmbH. All rights reserved.

Expression levels of brown/beige adipocyte-related genes in fat depots of vitamin A-restricted fattening cattle.

PubMed

Chen, Hsuan-Ju; Ihara, Tsubasa; Yoshioka, Hidetugu; Itoyama, Erina; Kitamura, Shoko; Nagase, Hiroshi; Murakami, Hiroaki; Hoshino, Yoichiro; Murakami, Masaru; Tomonaga, Shozo; Matsui, Tohru; Funaba, Masayuki

2018-06-15

Brown/beige adipocytes dissipate energy as heat. We previously showed that brown/beige adipocytes are present in white adipose tissue (WAT) of fattening cattle. The present study examined the effect of vitamin A restriction on mRNA expression of brown/beige adipocyte-related genes. In Japan, fattening cattle are conventionally fed a vitamin A-restricted diet to improve beef marbling. Twelve Japanese Black steers aged 10 months were fed control feed (n=6) or vitamin A-restricted feed (n=6) for 20 months. Subcutaneous WAT (scWAT) and mesenteric WAT (mesWAT) were collected, and mRNA expression levels of molecules related to function of brown/beige adipocytes (Ucp1, Cidea, Dio2, Cox7a and Cox8b) as well as transcriptional regulators related to brown/beige adipogenesis (Zfp516, Nfia, Prdm16, and Pgc-1α) were evaluated. The vitamin A restriction significantly increased or tended to increase expression levels of Cidea and Pgc-1α in scWAT, and Cidea, Dio2, and Nfia in mesWAT. Previous studies revealed that the bone morphogenetic protein (BMP) pathway was responsible for commitment of mesenchymal stem cells to brown/beige adipocyte-lineage cells. The vitamin A restriction increased expression of Bmp7 and some Bmp receptors in WAT. The interrelationship between gene expression levels indicated that expression levels of Nfia, Prdm16, and Pgc-1α were closely related to those of genes related to function of brown/beige adipocytes in scWAT. Also, expression levels of Nfia, Prdm16, and Pgc-1α were highly correlated with those of Alk3 in scWAT. In summary, the present results suggest that the vitamin A restriction increases the number or activity of brown/beige adipocytes through regulatory expression of transcriptional regulators to induce brown/beige adipogenesis especially in scWAT of fattening cattle, which may be governed by the Bmp pathway.

Antineoplastic activity of the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine in anaplastic large cell lymphoma

PubMed Central

Hassler, Melanie R.; Klisaroska, Aleksandra; Kollmann, Karoline; Steiner, Irene; Bilban, Martin; Schiefer, Ana-Iris; Sexl, Veronika; Egger, Gerda

2012-01-01

DNA methylation is an epigenetic mechanism establishing long-term gene silencing during development and cell commitment, which is maintained in subsequent cell generations. Aberrant DNA methylation is found at gene promoters in most cancers and can lead to silencing of tumor suppressor genes. The DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-CdR) is able to reactivate genes silenced by DNA methylation and has been shown to be a very potent epigenetic drug in several hematological malignancies. In this report, we demonstrate that 5-aza-CdR exhibits high antineoplastic activity against anaplastic large cell lymphoma (ALCL), a rare CD30 positive non-Hodgkin lymphoma of T-cell origin. Low dose treatment of ALCL cell lines and xenografted tumors causes apoptosis and cell cycle arrest in vitro and in vivo. This is also reflected in genome-wide expression analyses, where genes related to apoptosis and cell death are amongst the most affected targets of 5-aza-CdR. Furthermore, we observed demethylation and re-expression of p16INK4A after drug administration and senescence associated β-galactosidase activity. Thus, our data provide evidence that 5-aza-CdR is highly efficient against ALCL and warrants further clinical evaluation for future therapeutic use. PMID:22687603

Wt-1 Expression Linked to Nitric Oxide Availability during Neonatal Obstructive Nephropathy

PubMed Central

Mazzei, Luciana; Manucha, Walter

2013-01-01

The wt-1 gene encodes a zinc finger DNA-binding protein that acts as a transcriptional activator or repressor depending on the cellular or chromosomal context. The wt-1 regulates the expression of a large number of genes that have a critical role in kidney development. Congenital obstructive nephropathy disrupts normal renal development and causes chronic progressive interstitial fibrosis, which contributes to renal growth arrest, ultimately leading to chronic renal failure. Wt-1 is downregulated during congenital obstructive nephropathy, leading to apoptosis. Of great interest, nitric oxide bioavailability associated with heat shock protein 70 (Hsp70) interaction may modulate wt-1 mRNA expression, preventing obstruction-induced cell death during neonatal unilateral ureteral obstruction. Moreover, recent genetic researches have allowed characterization of many of the complex interactions among the individual components cited, but the realization of new biochemical, molecular, and functional experiments as proposed in our and other research labs allows us to establish a deeper level of commitment among proteins involved and the potential pathogenic consequences of their imbalance. PMID:24288526

Efficient Direct Reprogramming of Mature Amniotic Cells into Endothelial Cells by ETS Factors and TGFβ Suppression

PubMed Central

Ginsberg, Michael; James, Daylon; Ding, Bi-Sen; Nolan, Daniel; Geng, Fuqiang; Butler, Jason M; Schachterle, William; Pulijaal, Venkat R; Mathew, Susan; Chasen, Stephen T; Xiang, Jenny; Rosenwaks, Zev; Shido, Koji; Elemento, Olivier; Rabbany, Sina Y; Rafii, Shahin

2012-01-01

ETS transcription factors ETV2, FLI1 and ERG1 specify pluripotent stem cells into endothelial cells (ECs). However, these ECs are unstable and drift towards non-vascular cell fates. We show that human mid-gestation c-Kit− lineage-committed amniotic cells (ACs) can be readily reprogrammed into induced vascular endothelial cells (iVECs). Transient ETV2 expression in ACs generated proliferative but immature iVECs, while co-expression with FLI1/ERG1 endowed iVECs with a vascular repertoire and morphology matching mature stable ECs. Brief TGFβ-inhibition functionalized VEGFR2 signaling, augmenting specification of ACs to iVECs. Genome-wide transcriptional analyses showed that iVECs are similar to adult ECs in which vascular-specific genes are turned on and non-vascular genes are silenced. Functionally, iVECs form long-lasting patent vasculature in Matrigel plugs and regenerating livers. Thus, short-term ETV2 expression and TGFβ-inhibition along with constitutive ERG1/FLI1 co-expression reprogram mature ACs into durable and functional iVECs with clinical-scale expansion potential. Public banking of HLA-typed iVECs would establish a vascular inventory for treatment of genetically diverse disorders. PMID:23084400

Early stages in the development of human T, natural killer and thymic dendritic cells.

PubMed

Spits, H; Blom, B; Jaleco, A C; Weijer, K; Verschuren, M C; van Dongen, J J; Heemskerk, M H; Res, P C

1998-10-01

T-cell development is initiated when CD34+ pluripotent stem cells or their immediate progeny leave the bone marrow to migrate to the thymus. Upon arrival in the thymus the stem cell progeny is not yet committed to the T-cell lineage as it has the capability to develop into T, natural killer (NK) and dendritic cells (DC). Primitive hematopoietic progenitor cells in the human thymus express CD34 and lack CD1a. When these progenitor cells develop into T cells they traverse a number of checkpoints. One early checkpoint is the induction of T-cell commitment, which correlates with appearance of CD1a and involves the loss of capacity to develop into NK cells and DC and the initiation of T-cell receptor (TCR) gene rearrangements. Basic helix-loop-helix transcription factors play a role in induction of T-cell commitment. CD1a+CD34+ cells develop into CD4+CD8 alpha+ beta+ cells by upregulating first CD4, followed by CD8 alpha and then CD8 beta. Selection for productive TCR beta gene rearrangements (beta selection) likely occurs in the CD4+CD8 alpha+ beta- and CD4+CD8 alpha+ beta+ populations. Although the T and NK-cell lineages are closely related to each other, NK cells can develop independently of the thymus. The fetal thymus is most likely one site of NK-cell development.

Green Supplier Network Manufacturer Commitment Form

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Online form expressing interest in committing to be a Green Supplier; this form expresses your intent to participate in a confidential Green Suppliers Network assessment, implement recommended environmental improvements and complete a NIST MEP follow-up.

Dopamine Signaling Leads to Loss of Polycomb Repression and Aberrant Gene Activation in Experimental Parkinsonism

PubMed Central

Lerdrup, Mads; Gomes, Ana-Luisa; Kryh, Hanna; Spigolon, Giada; Caboche, Jocelyne; Fisone, Gilberto; Hansen, Klaus

2014-01-01

Polycomb group (PcG) proteins bind to and repress genes in embryonic stem cells through lineage commitment to the terminal differentiated state. PcG repressed genes are commonly characterized by the presence of the epigenetic histone mark H3K27me3, catalyzed by the Polycomb repressive complex 2. Here, we present in vivo evidence for a previously unrecognized plasticity of PcG-repressed genes in terminally differentiated brain neurons of parkisonian mice. We show that acute administration of the dopamine precursor, L-DOPA, induces a remarkable increase in H3K27me3S28 phosphorylation. The induction of the H3K27me3S28p histone mark specifically occurs in medium spiny neurons expressing dopamine D1 receptors and is dependent on Msk1 kinase activity and DARPP-32-mediated inhibition of protein phosphatase-1. Chromatin immunoprecipitation (ChIP) experiments showed that increased H3K27me3S28p was accompanied by reduced PcG binding to regulatory regions of genes. An analysis of the genome wide distribution of L-DOPA-induced H3K27me3S28 phosphorylation by ChIP sequencing (ChIP-seq) in combination with expression analysis by RNA-sequencing (RNA-seq) showed that the induction of H3K27me3S28p correlated with increased expression of a subset of PcG repressed genes. We found that induction of H3K27me3S28p persisted during chronic L-DOPA administration to parkisonian mice and correlated with aberrant gene expression. We propose that dopaminergic transmission can activate PcG repressed genes in the adult brain and thereby contribute to long-term maladaptive responses including the motor complications, or dyskinesia, caused by prolonged administration of L-DOPA in Parkinson's disease. PMID:25254549

Insights into islet development and biology through characterization of a human iPSC-derived endocrine pancreas model

PubMed Central

van de Bunt, Martijn; Lako, Majlinda; Barrett, Amy; Gloyn, Anna L.; Hansson, Mattias; McCarthy, Mark I.; Honoré, Christian

2016-01-01

ABSTRACT Directed differentiation of stem cells offers a scalable solution to the need for human cell models recapitulating islet biology and T2D pathogenesis. We profiled mRNA expression at 6 stages of an induced pluripotent stem cell (iPSC) model of endocrine pancreas development from 2 donors, and characterized the distinct transcriptomic profiles associated with each stage. Established regulators of endodermal lineage commitment, such as SOX17 (log2 fold change [FC] compared to iPSCs = 14.2, p-value = 4.9 × 10−5) and the pancreatic agenesis gene GATA6 (log2 FC = 12.1, p-value = 8.6 × 10−5), showed transcriptional variation consistent with their known developmental roles. However, these analyses highlighted many other genes with stage-specific expression patterns, some of which may be novel drivers or markers of islet development. For example, the leptin receptor gene, LEPR, was most highly expressed in published data from in vivo-matured cells compared to our endocrine pancreas-like cells (log2 FC = 5.5, p-value = 2.0 × 10−12), suggesting a role for the leptin pathway in the maturation process. Endocrine pancreas-like cells showed significant stage-selective expression of adult islet genes, including INS, ABCC8, and GLP1R, and enrichment of relevant GO-terms (e.g. “insulin secretion”; odds ratio = 4.2, p-value = 1.9 × 10−3): however, principal component analysis indicated that in vitro-differentiated cells were more immature than adult islets. Integration of the stage-specific expression information with genetic data from T2D genome-wide association studies revealed that 46 of 82 T2D-associated loci harbor genes present in at least one developmental stage, facilitating refinement of potential effector transcripts. Together, these data show that expression profiling in an iPSC islet development model can further understanding of islet biology and T2D pathogenesis. PMID:27246810

Functional analysis of the Brassica napus L. phytoene synthase (PSY) gene family.

PubMed

López-Emparán, Ada; Quezada-Martinez, Daniela; Zúñiga-Bustos, Matías; Cifuentes, Víctor; Iñiguez-Luy, Federico; Federico, María Laura

2014-01-01

Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three "Arabidopsis-like" subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed.

Follow-up of an intelligent odd-mannered teenager with del(3)(p26). Remarks on authorship and ethical commitment.

PubMed

Rivera, H; Domínguez, M G; Matute, E

2006-01-01

A male teenager formerly found to have a 46,XY,del(3)(p26)de novo karyotype was restudied. At the age of 14(8/12) yr, he attends the last grade of middle school and was a cooperating teenager with slender habitus, severe myopia, prominent nose, sacral dimples, pubertal stage Tanner III, and multiple surgical scars. Neuropsychological studies revealed a full scale IQ of 95 with slow performance (WISC-IV Spanish test) as well as an internalizing behavioral profile, poor social skills, a mild attention deficit, somatic complaints, and a feminized gender role. FISH with the 3p subtelomeric probe revealed that the deleted chromosome actually lacked the specific signal (n=10 cells). The patient's average intelligence confirms that euchromatic imbalances do not necessarily cause mental retardation and suggests that his deletion actually included the CALL gene, the Contactin 4 gene and other 3p26 genes related to intellectual capabilities; yet, the resulting hemizygosity either did not lead to haploinsufficiency or was minimally expressed. Moreover, the patient's peculiar cognitive and behavioral profile suggests that the 3p26 deletion is associated with a distinctive neuropsychological phenotype. Incidentally we comment on authorship and publication ethics in order to urge our institutional ethics committee to arbitrate authorship conflicts and thereby be consistent with its ethical commitment.

Sall4 is essential for stabilization, but not for pluripotency, of embryonic stem cells by repressing aberrant trophectoderm gene expression.

PubMed

Yuri, Shunsuke; Fujimura, Sayoko; Nimura, Keisuke; Takeda, Naoki; Toyooka, Yayoi; Fujimura, Yu-Ichi; Aburatani, Hiroyuki; Ura, Kiyoe; Koseki, Haruhiko; Niwa, Hitoshi; Nishinakamura, Ryuichi

2009-04-01

Sall4 is a mouse homolog of a causative gene of the autosomal dominant disorder Okihiro syndrome. We previously showed that the absence of Sall4 leads to lethality during peri-implantation and that Sall4-null embryonic stem (ES) cells proliferate poorly with intact pluripotency when cultured on feeder cells. Here, we report that, in the absence of feeder cells, Sall4-null ES cells express the trophectoderm marker Cdx2, but are maintained for a long period in an undifferentiated state with minimally affected Oct3/4 expression. Feeder-free Sall4-null ES cells contribute solely to the inner cell mass and epiblast in vivo, indicating that these cells still retain pluripotency and do not fully commit to the trophectoderm. These phenotypes could arise from derepression of the Cdx2 promoter, which is normally suppressed by Sall4 and the Mi2/NuRD HDAC complex. However, proliferation was impaired and G1 phase prolonged in the absence of Sall4, suggesting another role for Sall4 in cell cycle control. Although Sall1, also a Sall family gene, is known to genetically interact with Sall4 in vivo, Sall1-null ES cells have no apparent defects and no exacerbation is observed in ES cells lacking both Sall1 and Sall4, compared with Sall4-null cells. This suggests a unique role for Sall4 in ES cells. Thus, though Sall4 does not contribute to the central machinery of the pluripotency, it stabilizes ES cells by repressing aberrant trophectoderm gene expression.

Sex, stem cells and tumors in the Drosophila ovary.

PubMed

Salz, Helen K

2013-01-01

The Drosophila Sex-lethal (Sxl) gene encodes a female-specific RNA binding protein that in somatic cells globally regulates all aspects of female-specific development and behavior. Sxl also has a critical, but less well understood, role in female germ cells. Germ cells without Sxl protein can adopt a stem cell fate when housed in a normal ovary, but fail to successfully execute the self-renewal differentiation fate switch. The failure to differentiate is accompanied by the inappropriate expression of a set of male specific markers, continued proliferation, and formation of a tumor. The findings in Chau et al., (2012) identify the germline stem cell maintenance factor nanos as one of its target genes, and suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional downregulation of nanos expression. These studies provide the basis for a new model in which Sxl directly couples sexual identity with the self-renewal differentiation decision and raises several interesting questions about the genesis of the tumor phenotype.

Myeloid leukemia factor 1 associates with a novel heterogeneous nuclear ribonucleoprotein U-like molecule.

PubMed

Winteringham, Louise N; Endersby, Raelene; Kobelke, Simon; McCulloch, Ross K; Williams, James H; Stillitano, Justin; Cornwall, Scott M; Ingley, Evan; Klinken, S Peter

2006-12-15

Myeloid leukemia factor 1 (MLF1) is an oncoprotein associated with hemopoietic lineage commitment and acute myeloid leukemia. Here we show that Mlf1 associated with a novel binding partner, Mlf1-associated nuclear protein (Manp), a new heterogeneous nuclear ribonucleoprotein (hnRNP) family member, related to hnRNP-U. Manp localized exclusively in the nucleus and could redirect Mlf1 from the cytoplasm into the nucleus. The nuclear content of Mlf1 was also regulated by 14-3-3 binding to a canonical 14-3-3 binding motif within the N terminus of Mlf1. Significantly Mlf1 contains a functional nuclear export signal and localized primarily to the nuclei of hemopoietic cells. Mlf1 was capable of binding DNA, and microarray analysis revealed that it affected the expression of several genes, including transcription factors. In summary, this study reveals that Mlf1 translocates between nucleus and cytoplasm, associates with a novel hnRNP, and influences gene expression.

The BCL11A Transcription Factor Directly Activates RAG Gene Expression and V(D)J Recombination

PubMed Central

Lee, Baeck-seung; Dekker, Joseph D.; Lee, Bum-kyu; Iyer, Vishwanath R.; Sleckman, Barry P.; Shaffer, Arthur L.; Ippolito, Gregory C.

2013-01-01

Recombination-activating gene 1 protein (RAG1) and RAG2 are critical enzymes for initiating variable-diversity-joining (VDJ) segment recombination, an essential process for antigen receptor expression and lymphocyte development. The transcription factor BCL11A is required for B cell development, but its molecular function(s) in B cell fate specification and commitment is unknown. We show here that the major B cell isoform, BCL11A-XL, binds the RAG1 promoter and Erag enhancer to activate RAG1 and RAG2 transcription in pre-B cells. We employed BCL11A overexpression with recombination substrates in a cultured pre-B cell line as well as Cre recombinase-mediated Bcl11alox/lox deletion in explanted murine pre-B cells to demonstrate direct consequences of BCL11A/RAG modulation on V(D)J recombination. We conclude that BCL11A is a critical component of a transcriptional network that regulates B cell fate by controlling V(D)J recombination. PMID:23438597

Gonadal morphogenesis and gene expression in reptiles with temperature-dependent sex determination.

PubMed

Merchant-Larios, H; Díaz-Hernández, V; Marmolejo-Valencia, A

2010-01-01

In reptiles with temperature-dependent sexual determination, the thermosensitive period (TSP) is the interval in which the sex is defined during gonadal morphogenesis. One-shift experiments in a group of eggs define the onset and the end of the TSP as all and none responses, respectively. Timing for sex-undetermined (UG) and -determined gonads (DG) differs at male- (MPT) or female-producing temperatures (FPT). During the TSP a decreasing number of embryos respond to temperature shifts indicating that in this period embryos with both UG and DG exist. Although most UG correspond to undifferentiated gonads, some embryos extend UG after the onset of histological differentiation. Thus, temperature affects gonadal cells during the process of morphogenesis, but timing of commitment depends on individual embryos. A correlation between gonadal morphogenesis, TSP, and gene expression suggests that determination of the molecular pathways modulated by temperature in epithelial cells (surface epithelium and medullary cords) holds the key for a unifying hypothesis on temperature-dependent sex determination. (c) 2010 S. Karger AG, Basel.

Ectopic expression of a fruit phytoene synthase from Citrus paradisi Macf. promotes abiotic stress tolerance in transgenic tobacco.

PubMed

Cidade, Luciana C; de Oliveira, Tahise M; Mendes, Amanda F S; Macedo, Amanda F; Floh, Eny I S; Gesteira, Abelmon S; Soares-Filho, Walter S; Costa, Marcio G C

2012-12-01

Abscisic acid (ABA) is an important regulator of plant responses to environmental stresses and an absolute requirement for stress tolerance. Recently, a third phytoene synthase (PSY3) gene paralog was identified in monocots and demonstrated to play a specialized role in stress-induced ABA formation, thus suggesting that the first committed step in carotenogenesis is a key limiting step in ABA biosynthesis. To examine whether the ectopic expression of PSY, other than PSY3, would similarly affect ABA level and stress tolerance, we have produced transgenic tobacco containing a fruit-specific PSY (CpPSY) of grapefruit (Citrus paradisi Macf.). The transgenic plants contained a single- or double-locus insertion and expressed CpPSY at varying transcript levels. In comparison with the wild-type plants, the CpPSY expressing transgenic plants showed a significant increase on root length and shoot biomass under PEG-, NaCl- and mannitol-induced osmotic stress. The enhanced stress tolerance of transgenic plants was correlated with the increased endogenous ABA level and expression of stress-responsive genes, which in turn was correlated with the CpPSY copy number and expression level in different transgenic lines. Collectively, these results provide further evidence that PSY is a key enzyme regulating ABA biosynthesis and that the altered expression of other PSYs in transgenic plants may provide a similar function to that of the monocot's PSY3 in ABA biosynthesis and stress tolerance. The results also pave the way for further use of CpPSY, as well as other PSYs, as potential candidate genes for engineering tolerance to drought and salt stress in crop plants.

Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing.

PubMed

Han, Qianqian; Yang, Pishan; Wu, Yuwei; Meng, Shu; Sui, Lei; Zhang, Lan; Yu, Liming; Tang, Yin; Jiang, Hua; Xuan, Dongying; Kaplan, David L; Kim, Sung Hoon; Tu, Qisheng; Chen, Jake

2015-08-01

Epigenetic regulation of gene expression is a central mechanism that governs cell stemness, determination, commitment, and differentiation. It has been recently found that PHF8, a major H4K20/H3K9 demethylase, plays a critical role in craniofacial and bone development. In this study, we hypothesize that PHF8 promotes osteoblastogenesis by epigenetically regulating the expression of a nuclear matrix protein, special AT-rich sequence-binding protein 2 (SATB2) that plays pivotal roles in skeletal patterning and osteoblast differentiation. Our results showed that expression levels of PHF8 and SATB2 in preosteoblasts and bone marrow stromal cells (BMSCs) increased simultaneously during osteogenic induction. Overexpressing PHF8 in these cells upregulated the expression of SATB2, Runx2, osterix, and bone matrix proteins. Conversely, knockdown of PHF8 reduced the expression of these genes. Furthermore, ChIP assays confirmed that PHF8 specifically bound to the transcription start site (TSS) of the SATB2 promoter, and the expression of H3K9me1 at the TSS region of SATB2 decreased in PHF8 overexpressed group. Implantation of the BMSCs overexpressing PHF8 with silk protein scaffolds promoted bone regeneration in critical-sized defects in mouse calvaria. Taken together, our results demonstrated that PHF8 epigenetically modulates SATB2 activity, triggering BMSCs osteogenic differentiation and facilitating bone formation and regeneration in biodegradable silk scaffolds.

CRISPR/Cas9-mediated heterozygous knockout of the autism gene CHD8 and characterization of its transcriptional networks in neurodevelopment.

PubMed

Wang, Ping; Lin, Mingyan; Pedrosa, Erika; Hrabovsky, Anastasia; Zhang, Zheng; Guo, Wenjun; Lachman, Herbert M; Zheng, Deyou

2015-01-01

Disruptive mutation in the CHD8 gene is one of the top genetic risk factors in autism spectrum disorders (ASDs). Previous analyses of genome-wide CHD8 occupancy and reduced expression of CHD8 by shRNA knockdown in committed neural cells showed that CHD8 regulates multiple cell processes critical for neural functions, and its targets are enriched with ASD-associated genes. To further understand the molecular links between CHD8 functions and ASD, we have applied the CRISPR/Cas9 technology to knockout one copy of CHD8 in induced pluripotent stem cells (iPSCs) to better mimic the loss-of-function status that would exist in the developing human embryo prior to neuronal differentiation. We then carried out transcriptomic and bioinformatic analyses of neural progenitors and neurons derived from the CHD8 mutant iPSCs. Transcriptome profiling revealed that CHD8 hemizygosity (CHD8 (+/-)) affected the expression of several thousands of genes in neural progenitors and early differentiating neurons. The differentially expressed genes were enriched for functions of neural development, β-catenin/Wnt signaling, extracellular matrix, and skeletal system development. They also exhibited significant overlap with genes previously associated with autism and schizophrenia, as well as the downstream transcriptional targets of multiple genes implicated in autism. Providing important insight into how CHD8 mutations might give rise to macrocephaly, we found that seven of the twelve genes associated with human brain volume or head size by genome-wide association studies (e.g., HGMA2) were dysregulated in CHD8 (+/-) neural progenitors or neurons. We have established a renewable source of CHD8 (+/-) iPSC lines that would be valuable for investigating the molecular and cellular functions of CHD8. Transcriptomic profiling showed that CHD8 regulates multiple genes implicated in ASD pathogenesis and genes associated with brain volume.

The Role of Cellular Proliferation in Adipogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

PubMed

Marquez, Maribel P; Alencastro, Frances; Madrigal, Alma; Jimenez, Jossue Loya; Blanco, Giselle; Gureghian, Alex; Keagy, Laura; Lee, Cecilia; Liu, Robert; Tan, Lun; Deignan, Kristen; Armstrong, Brian; Zhao, Yuanxiang

2017-11-01

Mitotic clonal expansion has been suggested as a prerequisite for adipogenesis in murine preadipocytes, but the precise role of cell proliferation during human adipogenesis is unclear. Using adipose tissue-derived human mesenchymal stem cells as an in vitro cell model for adipogenic study, a group of cell cycle regulators, including Cdk1 and CCND1, were found to be downregulated as early as 24 h after adipogenic initiation and consistently, cell proliferation activity was restricted to the first 48 h of adipogenic induction. Cell proliferation was either further inhibited using siRNAs targeting cell cycle genes or enhanced by supplementing exogenous growth factor, basic fibroblast growth factor (bFGF), at specific time intervals during adipogenesis. Expression knockdown of Cdk1 at the initiation of adipogenic induction resulted in significantly increased adipocytes, even though total number of cells was significantly reduced compared to siControl-treated cells. bFGF stimulated proliferation throughout adipogenic differentiation, but exerted differential effect on adipogenic outcome at different phases, promoting adipogenesis during mitotic phase (first 48 h), but significantly inhibiting adipogenesis during adipogenic commitment phase (days 3-6). Our results demonstrate that cellular proliferation is counteractive to adipogenic commitment in human adipogenesis. However, cellular proliferation stimulation can be beneficial for adipogenesis during the mitotic phase by increasing the population of cells capable of committing to adipocytes before adipogenic commitment.

Characterization and genetic manipulation of human umbilical cord vein mesenchymal stem cells: potential application in cell-based gene therapy.

PubMed

Kermani, Abbas Jafari; Fathi, Fardin; Mowla, Seyed Javad

2008-04-01

Stem cells are defined by two main characteristics: self-renewal capacity and commitment to multi-lineage differentiation. The cells have a great therapeutic potential in repopulating damaged tissues as well as being genetically manipulated and used in cell-based gene therapy. Umbilical cord vein is a readily available and inexpensive source of stem cells that are capable of generating various cell types. Despite the recent isolation of human umbilical cord vein mesenchymal stem cells (UVMSC), the self-renewal capacity and the potential clinical application of the cells are not well known. In the present study, we have successfully isolated and cultured human UVMSCs. Our data further revealed that the isolated cells express the self-renewal genes Oct-4, Nanog, ZFX, Bmi-1, and Nucleostemin; but not Zic-3, Hoxb-4, TCL-1, Tbx-3 and Esrrb. In addition, our immunocytochemistry results revealed the expression of SSEA-4, but not SSEA-3, TRA-1-60, and TRA-1-81 embryonic stem cell surface markers in the cells. Also, we were able to transfect the cells with a reporter, enhanced green fluorescent protein (EGFP), and a therapeutic human brain-derived neurotrophic factor (hBDNF) gene by means of electroporation and obtained a stable cell line, which could constantly express both transgenes. The latter data provide further evidence on the usefulness of umbilical cord vein mesenchymal stem cells as a readily available source of stem cells, which could be genetically manipulated and used in cell-based gene therapy applications.

Engineering metabolic pathways in plants by multigene transformation.

PubMed

Zorrilla-López, Uxue; Masip, Gemma; Arjó, Gemma; Bai, Chao; Banakar, Raviraj; Bassie, Ludovic; Berman, Judit; Farré, Gemma; Miralpeix, Bruna; Pérez-Massot, Eduard; Sabalza, Maite; Sanahuja, Georgina; Vamvaka, Evangelia; Twyman, Richard M; Christou, Paul; Zhu, Changfu; Capell, Teresa

2013-01-01

Metabolic engineering in plants can be used to increase the abundance of specific valuable metabolites, but single-point interventions generally do not improve the yields of target metabolites unless that product is immediately downstream of the intervention point and there is a plentiful supply of precursors. In many cases, an intervention is necessary at an early bottleneck, sometimes the first committed step in the pathway, but is often only successful in shifting the bottleneck downstream, sometimes also causing the accumulation of an undesirable metabolic intermediate. Occasionally it has been possible to induce multiple genes in a pathway by controlling the expression of a key regulator, such as a transcription factor, but this strategy is only possible if such master regulators exist and can be identified. A more robust approach is the simultaneous expression of multiple genes in the pathway, preferably representing every critical enzymatic step, therefore removing all bottlenecks and ensuring completely unrestricted metabolic flux. This approach requires the transfer of multiple enzyme-encoding genes to the recipient plant, which is achieved most efficiently if all genes are transferred at the same time. Here we review the state of the art in multigene transformation as applied to metabolic engineering in plants, highlighting some of the most significant recent advances in the field.

Nucleosome eviction along with H3K9ac deposition enhances Sox2 binding during human neuroectodermal commitment

PubMed Central

Du, Yanhua; Liu, Zhenping; Cao, Xinkai; Chen, Xiaolong; Chen, Zhenyu; Zhang, Xiaobai; Zhang, Xiaoqing; Jiang, Cizhong

2017-01-01

Neuroectoderm is an important neural precursor. However, chromatin remodeling and its epigenetic regulatory roles during the differentiation of human neuroectodermal cells (hNECs) from human embryonic stem cells (hESCs) remain largely unexplored. Here, we obtained hNECs through directed differentiation from hESCs, and determined chromatin states in the two cell types. Upon differentiation, H2A.Z-mediated nucleosome depletion leads to an open chromatin structure in promoters and upregulates expression of neuroectodermal genes. Increase in H3K9ac signals and decrease in H3K27me3 signals in promoters result in an active chromatin state and activate neuroectodermal genes. Conversely, decrease in H3K9ac signals and increase in H3K27me3 signals in promoters repress pluripotency genes. Moreover, H3K9ac signals facilitate the pluripotency factor Sox2 binding to target sites unique to hNECs. Knockdown of the acetyltransferase Kat2b erases H3K9ac signals, disrupts Sox2 binding, and fails the differentiation. Our results demonstrate a hierarchy of epigenetic regulation of gene expression during the differentiation of hNECs from hESCs through chromatin remodeling. PMID:28475175

Disruption of alpha beta but not of gamma delta T cell development by overexpression of the helix-loop-helix protein Id3 in committed T cell progenitors.

PubMed Central

Blom, B; Heemskerk, M H; Verschuren, M C; van Dongen, J J; Stegmann, A P; Bakker, A Q; Couwenberg, F; Res, P C; Spits, H

1999-01-01

Enforced expression of Id3, which has the capacity to inhibit many basic helix-loop-helix (bHLH) transcription factors, in human CD34(+) hematopoietic progenitor cells that have not undergone T cell receptor (TCR) gene rearrangements inhibits development of the transduced cells into TCRalpha beta and gamma delta cells in a fetal thymic organ culture (FTOC). Here we document that overexpression of Id3, in progenitors that have initiated TCR gene rearrangements (pre-T cells), inhibits development into TCRalpha beta but not into TCRgamma delta T cells. Furthermore, Id3 impedes expression of recombination activating genes and downregulates pre-Talpha mRNA. These observations suggest possible mechanisms by which Id3 overexpression can differentially affect development of pre-T cells into TCRalpha beta and gamma delta cells. We also observed that cell surface CD4(-)CD8(-)CD3(-) cells with rearranged TCR genes developed from Id3-transduced but not from control-transduced pre-T cells in an FTOC. These cells had properties of both natural killer (NK) and pre-T cells. These findings suggest that bHLH factors are required to control T cell development after the T/NK developmental checkpoint. PMID:10329625

A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

PubMed Central

Walko, Gernot; Viswanathan, Priyalakshmi; Tihy, Matthieu; Nijjher, Jagdeesh; Dunn, Sara-Jane; Lamond, Angus I

2017-01-01

Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states, termed commitment, is poorly understood. Here, we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension to induce differentiation. Cell detachment induces several protein phosphatases, five of which - DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Conversely, DUSP10 expression antagonises commitment. The phosphatases form a dynamic network of transient positive and negative interactions that change over time, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem and differentiated) via an unstable (committed) state. Phosphatase expression is also spatially regulated in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment. PMID:29043977

Milk: an exosomal microRNA transmitter promoting thymic regulatory T cell maturation preventing the development of atopy?

PubMed

Melnik, Bodo C; John, Swen Malte; Schmitz, Gerd

2014-02-12

Epidemiological evidence confirmed that raw cow's milk consumption in the first year of life protects against the development of atopic diseases and increases the number of regulatory T-cells (Tregs). However, milk's atopy-protective mode of action remains elusive.This review supported by translational research proposes that milk-derived microRNAs (miRs) may represent the missing candidates that promote long-term lineage commitment of Tregs downregulating IL-4/Th2-mediated atopic sensitization and effector immune responses. Milk transfers exosomal miRs including the ancient miR-155, which is important for the development of the immune system and controls pivotal target genes involved in the regulation of FoxP3 expression, IL-4 signaling, immunoglobulin class switching to IgE and FcϵRI expression. Boiling of milk abolishes milk's exosomal miR-mediated bioactivity. Infant formula in comparison to human breast- or cow's milk is deficient in bioactive exosomal miRs that may impair FoxP3 expression. The boost of milk-mediated miR may induce pivotal immunoregulatory and epigenetic modifications required for long-term thymic Treg lineage commitment explaining the atopy-protective effect of raw cow's milk consumption.The presented concept offers a new option for the prevention of atopic diseases by the addition of physiological amounts of miR-155-enriched exosomes to infant formula for mothers incapable of breastfeeding.

The Phytoene synthase gene family of apple (Malus x domestica) and its role in controlling fruit carotenoid content.

PubMed

Ampomah-Dwamena, Charles; Driedonks, Nicky; Lewis, David; Shumskaya, Maria; Chen, Xiuyin; Wurtzel, Eleanore T; Espley, Richard V; Allan, Andrew C

2015-07-28

Carotenoid compounds play essential roles in plants such as protecting the photosynthetic apparatus and in hormone signalling. Coloured carotenoids provide yellow, orange and red colour to plant tissues, as well as offering nutritional benefit to humans and animals. The enzyme phytoene synthase (PSY) catalyses the first committed step of the carotenoid biosynthetic pathway and has been associated with control of pathway flux. We characterised four PSY genes found in the apple genome to further understand their involvement in fruit carotenoid accumulation. The apple PSY gene family, containing six members, was predicted to have three functional members, PSY1, PSY2, and PSY4, based on translation of the predicted gene sequences and/or corresponding cDNAs. However, only PSY1 and PSY2 showed activity in a complementation assay. Protein localisation experiments revealed differential localization of the PSY proteins in chloroplasts; PSY1 and PSY2 localized to the thylakoid membranes, while PSY4 localized to plastoglobuli. Transcript levels in 'Granny Smith' and 'Royal Gala' apple cultivars showed PSY2 was most highly expressed in fruit and other vegetative tissues. We tested the transient activation of the apple PSY1 and PSY2 promoters and identified potential and differential regulation by AP2/ERF transcription factors, which suggested that the PSY genes are controlled by different transcriptional mechanisms. The first committed carotenoid pathway step in apple is controlled by MdPSY1 and MdPSY2, while MdPSY4 play little or no role in this respect. This has implications for apple breeding programmes where carotenoid enhancement is a target and would allow co-segregation with phenotypes to be tested during the development of new cultivars.

The Abbreviated Pluripotent Cell Cycle

PubMed Central

Kapinas, Kristina; Grandy, Rodrigo; Ghule, Prachi; Medina, Ricardo; Becker, Klaus; Pardee, Arthur; Zaidi, Sayyed K.; Lian, Jane; Stein, Janet; van Wijnen, Andre; Stein, Gary

2013-01-01

Human embryonic stem cells and induced pluripotent stem cells proliferate rapidly and divide symmetrically producing equivalent progeny cells. In contrast, lineage committed cells acquire an extended symmetrical cell cycle. Self-renewal of tissue-specific stem cells is sustained by asymmetric cell division where one progeny cell remains a progenitor while the partner progeny cell exits the cell cycle and differentiates. There are three principal contexts for considering the operation and regulation of the pluripotent cell cycle: temporal, regulatory andstructural. The primary temporal context that the pluripotent self-renewal cell cycle of human embryonic stem cells (hESCs) is a short G1 period without reducing periods of time allocated to S phase, G2, and mitosis. The rules that govern proliferation in hESCs remain to be comprehensively established. However, several lines of evidence suggest a key role for the naïve transcriptome of hESCs, which is competent to stringently regulate the ESC cell cycle. This supports the requirements of pluripotent cells to self propagate while suppressing expression of genes that confer lineage commitment and/or tissue specificity. However, for the first time, we consider unique dimensions to the architectural organization and assembly of regulatory machinery for gene expression in nuclear microenviornments that define parameters of pluripotency. From both fundamental biological and clinical perspectives, understanding control of the abbreviated embryonic stem cell cycle can provide options to coordinate control of proliferation versus differentiation. Wound healing, tissue engineering, and cell-based therapy to mitigate developmental aberrations illustrate applications that benefit from knowledge of the biology of the pluripotent cell cycle. PMID:22552993

Gene expression changes in the prefrontal cortex, anterior cingulate cortex and nucleus accumbens of mood disorders subjects that committed suicide.

PubMed

Sequeira, Adolfo; Morgan, Ling; Walsh, David M; Cartagena, Preston M; Choudary, Prabhakara; Li, Jun; Schatzberg, Alan F; Watson, Stanley J; Akil, Huda; Myers, Richard M; Jones, Edward G; Bunney, William E; Vawter, Marquis P

2012-01-01

Suicidal behaviors are frequent in mood disorders patients but only a subset of them ever complete suicide. Understanding predisposing factors for suicidal behaviors in high risk populations is of major importance for the prevention and treatment of suicidal behaviors. The objective of this project was to investigate gene expression changes associated with suicide in brains of mood disorder patients by microarrays (Affymetrix HG-U133 Plus2.0) in the dorsolateral prefrontal cortex (DLPFC: 6 Non-suicides, 15 suicides), the anterior cingulate cortex (ACC: 6NS, 9S) and the nucleus accumbens (NAcc: 8NS, 13S). ANCOVA was used to control for age, gender, pH and RNA degradation, with P ≤ 0.01 and fold change ± 1.25 as criteria for significance. Pathway analysis revealed serotonergic signaling alterations in the DLPFC and glucocorticoid signaling alterations in the ACC and NAcc. The gene with the lowest p-value in the DLPFC was the 5-HT2A gene, previously associated both with suicide and mood disorders. In the ACC 6 metallothionein genes were down-regulated in suicide (MT1E, MT1F, MT1G, MT1H, MT1X, MT2A) and three were down-regulated in the NAcc (MT1F, MT1G, MT1H). Differential expression of selected genes was confirmed by qPCR, we confirmed the 5-HT2A alterations and the global down-regulation of members of the metallothionein subfamilies MT 1 and 2 in suicide completers. MTs 1 and 2 are neuro-protective following stress and glucocorticoid stimulations, suggesting that in suicide victims neuroprotective response to stress and cortisol may be diminished. Our results thus suggest that suicide-specific expression changes in mood disorders involve both glucocorticoids regulated metallothioneins and serotonergic signaling in different regions of the brain.

Gene Expression Changes in the Prefrontal Cortex, Anterior Cingulate Cortex and Nucleus Accumbens of Mood Disorders Subjects That Committed Suicide

PubMed Central

Sequeira, Adolfo; Morgan, Ling; Walsh, David M.; Cartagena, Preston M.; Choudary, Prabhakara; Li, Jun; Schatzberg, Alan F.; Watson, Stanley J.; Akil, Huda; Myers, Richard M.; Jones, Edward G.; Bunney, William E.; Vawter, Marquis P.

2012-01-01

Suicidal behaviors are frequent in mood disorders patients but only a subset of them ever complete suicide. Understanding predisposing factors for suicidal behaviors in high risk populations is of major importance for the prevention and treatment of suicidal behaviors. The objective of this project was to investigate gene expression changes associated with suicide in brains of mood disorder patients by microarrays (Affymetrix HG-U133 Plus2.0) in the dorsolateral prefrontal cortex (DLPFC: 6 Non-suicides, 15 suicides), the anterior cingulate cortex (ACC: 6NS, 9S) and the nucleus accumbens (NAcc: 8NS, 13S). ANCOVA was used to control for age, gender, pH and RNA degradation, with P≤0.01 and fold change±1.25 as criteria for significance. Pathway analysis revealed serotonergic signaling alterations in the DLPFC and glucocorticoid signaling alterations in the ACC and NAcc. The gene with the lowest p-value in the DLPFC was the 5-HT2A gene, previously associated both with suicide and mood disorders. In the ACC 6 metallothionein genes were down-regulated in suicide (MT1E, MT1F, MT1G, MT1H, MT1X, MT2A) and three were down-regulated in the NAcc (MT1F, MT1G, MT1H). Differential expression of selected genes was confirmed by qPCR, we confirmed the 5-HT2A alterations and the global down-regulation of members of the metallothionein subfamilies MT 1 and 2 in suicide completers. MTs 1 and 2 are neuro-protective following stress and glucocorticoid stimulations, suggesting that in suicide victims neuroprotective response to stress and cortisol may be diminished. Our results thus suggest that suicide-specific expression changes in mood disorders involve both glucocorticoids regulated metallothioneins and serotonergic signaling in different regions of the brain. PMID:22558144

Transcriptome profiling of sleeping, waking, and sleep deprived adult heterozygous Aldh1L1 - eGFP-L10a mice.

PubMed

Bellesi, Michele; de Vivo, Luisa; Tononi, Giulio; Cirelli, Chiara

2015-12-01

Transcriptomic studies revealed that hundreds of mRNAs show differential expression in the brains of sleeping relative to awake rats, mice, flies, and sparrows. Although these results have offered clues regarding the molecular consequences of sleep and sleep loss, their functional significance thus far has been limited. This is probably because the previous studies pooled transcripts from all brain cells, including neurons and glia. In Bellesi et al. 2015 [1], we used the translating ribosome affinity purification technology (TRAP) and microarray analysis to obtain a genome-wide mRNA profiling of astrocytes as a function of sleep and wake. We used bacterial artificial chromosome (BAC) transgenic mice expressing eGFP tagged ribosomal protein L10a under the promoter of the Aldh1L1 gene, a highly expressed astrocytic gene. Using this approach, we could extract only the astrocytic mRNAs, and only those already committed to be translated into proteins (L10a is part of the translational machinery). Here, we report a detailed description of the protocol used in the study [1]. Array data have been submitted to NCBI GEO under accession number (GSE69079).

Genomic hardwiring and phenotypic plasticity of terpenoid-based defenses in conifers.

PubMed

Huber, Dezene P W; Ralph, Steven; Bohlmann, Jörg

2004-12-01

Over evolutionary history, conifers have faced a myriad of threats from phloem- and xylem-feeding insects, defoliating insects, and fungal pathogens. Among the trees' defenses, terpenoids appear to play a major role by harming, disabling, deterring, repelling, or otherwise reducing the fitness of potential invaders. Each of the three classes of terpenoids in conifers, monoterpenes, sesquiterpenes, and diterpenes, are composed of a large number of representative compounds. In most cases, the presence of a particular terpenoid compound in the oleoresin or volatile emissions from a specific conifer can be accounted for by the expression of one of many committed terpene synthase (TPS) genes. However, while each TPS may produce one or a few major products, many produce a variety of minor products with relatively constant component ratios in the product blends. TPS genes exist in conifers in large and functionally diverse, yet monophyletic, gene families. Within these gene families, new biochemical functions of TPS appear to have evolved by gene duplication and changes in the amino acid sequence of the enzyme's active site. In addition, TPS genes may be differentially expressed prior to, during, and following attack by insects or pathogens. Thus, while the production of any particular terpenoid is hardwired into a conifer's genome, these trees have the capacity to change the mixture of terpenoids in oleoresin secretions and volatile emissions. Anatomical changes may also accompany induced terpenoid production, supplementing the plasticity of the molecular and biochemical events.

Inducible transgenics. New lessons on events governing the induction and commitment in mammary tumorigenesis.

PubMed

Hulit, J; Di Vizio, D; Pestell, R G

2001-01-01

Breast cancer arises from multiple genetic events that together contribute to the established, irreversible malignant phenotype. The development of inducible tissue-specific transgenics has allowed a careful dissection of the events required for induction and subsequent maintenance of tumorigenesis. Mammary gland targeted expression of oncogenic Ras or c-Myc is sufficient for the induction of mammary gland tumorigenesis in the rodent, and when overexpressed together the rate of tumor onset is substantially enhanced. In an exciting recent finding, D'Cruz et al discovered tetracycline-regulated c-Myc overexpression in the mammary gland induced invasive mammary tumors that regressed upon withdrawal of c-Myc expression. Almost one-half of the c-Myc-induced tumors harbored K-ras or N-ras gene point mutations, correlating with tumor persistence on withdrawal of c-Myc transgene expression. These findings suggest maintenance of tumorigenesis may involve a second mutation within the Ras pathway.

Forced expression of the Ikaros 6 isoform in human placental blood CD34(+) cells impairs their ability to differentiate toward the B-lymphoid lineage.

PubMed

Tonnelle, C; Bardin, F; Maroc, C; Imbert, A M; Campa, F; Dalloul, A; Schmitt, C; Chabannon, C

2001-11-01

Studies in mice suggest that the Ikaros (Ik) gene encodes several isoforms and is a critical regulator of hematolymphoid differentiation. Little is known on the role of Ikaros in human stem cell differentiation. Herein, the biological consequences of the forced expression of Ikaros 6 (Ik6) in human placental blood CD34(+) progenitors are evaluated. Ik6 is one of the isoforms produced from the Ikaros premessenger RNA by alternative splicing and is thought to behave as a dominant negative isoform of the gene product because it lacks the DNA binding domain present in transcriptionally active isoforms. The results demonstrate that human cord blood CD34(+) cells that express high levels of Ik6 as a result of retrovirally mediated gene transfer have a reduced capacity to produce lymphoid B cells in 2 independent assays: (1) in vitro reinitiation of human hematopoiesis during coculture with the MS-5 murine stromal cell line and (2) xenotransplantation in nonobese diabetic-severe combined immunodeficient mice. These results suggest that Ikaros plays an important role in stem cell commitment in humans and that the balance between the different isoforms is a key element of this regulatory system; they support the hypothesis that posttranscriptional events can participate in the control of human hematopoietic differentiation.

Molecular stages of rapid and uniform neuralization of human embryonic stem cells.

PubMed

Bajpai, R; Coppola, G; Kaul, M; Talantova, M; Cimadamore, F; Nilbratt, M; Geschwind, D H; Lipton, S A; Terskikh, A V

2009-06-01

Insights into early human development are fundamental for our understanding of human biology. Efficient differentiation of human embryonic stem cells (hESCs) into neural precursor cells is critical for future cell-based therapies. Here, using defined conditions, we characterized a new method for rapid and uniform differentiation of hESCs into committed neural precursor cells (designated C-NPCs). Dynamic gene expression analysis identified several distinct stages of ESC neuralization and revealed functional modules of coregulated genes and pathways. The first wave of gene expression changes, likely corresponding to the transition through primitive ectoderm, started at day 3, preceding the formation of columnar neuroepithelial rosettes. The second wave started at day 5, coinciding with the formation of rosettes. The majority of C-NPCs were positive for both anterior and posterior markers of developing neuroepithelium. In culture, C-NPCs became electrophysiologically functional neurons; on transplantation into neonatal mouse brains, C-NPCs integrated into the cortex and olfactory bulb, acquiring appropriate neuronal morphologies and markers. Compared to rosette-NPCs,(1) C-NPCs exhibited limited in vitro expansion capacity and did not express potent oncogenes such as PLAG1 or RSPO3. Concordantly, we never detected tumors or excessive neural proliferation after transplantation of C-NPCs into mouse brains. In conclusion, our study provides a framework for future analysis of molecular signaling during ESC neuralization.

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

Zienkiewicz, Krzysztof; Zienkiewicz, Agnieszka; Poliner, Eric

Photosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail. We chose six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6),more » based on their expression profile, for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds. Taken together, these results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.« less

Nannochloropsis, a rich source of diacylglycerol acyltransferases for engineering of triacylglycerol content in different hosts

DOE PAGES

Zienkiewicz, Krzysztof; Zienkiewicz, Agnieszka; Poliner, Eric; ...

2017-01-03

Photosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail. We chose six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6),more » based on their expression profile, for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds. Taken together, these results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.« less

Transcriptomic profile adaptations following exposure of equine satellite cells to nutriactive phytochemical gamma-oryzanol.

PubMed

Szcześniak, K A; Ciecierska, A; Ostaszewski, P; Sadkowski, T

2016-01-01

Adult skeletal muscle myogenesis depends on the activation of satellite cells that have the potential to differentiate into new fibers. Gamma-oryzanol (GO), a commercially available nutriactive phytochemical, has gained global interest on account of its muscle-building and regenerating effects. Here, we investigated GO for its potential influence on myogenesis, using equine satellite cell culture model, since the horse is a unique animal, bred and exercised for competitive sport. To our knowledge, this is the first report where the global gene expression in cultured equine satellite cells has been described. Equine satellite cells were isolated from semitendinosus muscle and cultured until the second day of differentiation. Differentiating cells were incubated with GO for the next 24 h. Subsequently, total RNA from GO-treated and control cells was isolated, amplified, labeled, and hybridized to two-color Horse Gene Expression Microarray slides. Quantitative PCR was used for the validation of microarray data. Our results revealed 58 genes with changed expression in GO-treated vs. control cells. Analysis of expression changes suggests that various processes are reinforced by GO in differentiating equine satellite cells, including inhibition of myoblast differentiation, increased proliferation and differentiation, stress response, and increased myogenic lineage commitment. The present study may confirm putative muscle-enhancing abilities of GO; however, the collective role of GO in skeletal myogenesis remains equivocal. The diversity of these changes is likely due to heterogenous growth rate of cells in primary culture. Genes identified in our study, modulated by the presence of GO, may become potential targets of future research investigating impact of this supplement in skeletal muscle on proteomic and biochemical level.

Genetic and epigenetic mechanisms of gene regulation during lens development

PubMed Central

Cvekl, Ales; Duncan, Melinda K.

2007-01-01

Recent studies demonstrated a number of links between chromatin structure, gene expression, extracellular signaling and cellular differentiation during lens development. Lens progenitor cells originate from a pool of common progenitor cells, the pre-placodal region (PPR) which is formed due to a complex exchange of extracellular signals between the neural plate, naïve ectoderm and mesendoderm. A specific commitment to the lens program over alternate choices such as the formation of olfactory epithelium or the anterior pituitary is manifested by the formation of a thickened surface ectoderm, the lens placode. Mouse lens progenitor cells are characterized by the expression of a complement of lens lineage-specific transcription factors including Pax6, Six3 and Sox2, controlled by FGF and BMP signaling, followed later by c-Maf, Mab21like1, Prox1 and FoxE3. Proliferation of lens progenitors together with their morphogenetic movements results in the formation of the lens vesicle. This transient structure, comprised of lens precursor cells, is polarized with its anterior cells retaining their epithelial morphology and proliferative capacity, whereas the posterior lens precursor cells initiate terminal differentiation forming the primary lens fibers. Lens differentiation is marked by expression and accumulation of crystallins and other structural proteins. The transcriptional control of crystallin genes is characterized by the reiterative use of transcription factors required for the establishment of lens precursors in combination with more ubiquitously expressed factors (e.g. AP-1, AP-2α, CREB and USF) and recruitment of histone acetyltransferases (HATs) CBP and p300, and chromatin remodeling complexes SWI/SNF and ISWI. These studies have poised the study of lens development at the forefront of efforts to understand the connections between development, cell signaling, gene transcription and chromatin remodeling. PMID:17905638

Cardiac stem cell genetic engineering using the alphaMHC promoter.

PubMed

Bailey, Brandi; Izarra, Alberto; Alvarez, Roberto; Fischer, Kimberlee M; Cottage, Christopher T; Quijada, Pearl; Díez-Juan, Antonio; Sussman, Mark A

2009-11-01

Cardiac stem cells (CSCs) show potential as a cellular therapeutic approach to blunt tissue damage and facilitate reparative and regenerative processes after myocardial infarction. Despite multiple published reports of improvement, functional benefits remain modest using normal stem cells delivered by adoptive transfer into damaged myocardium. The goal of this study is to enhance survival and proliferation of CSCs that have undergone lineage commitment in early phases as evidenced by expression of proteins driven by the alpha-myosin heavy chain (alphaMHC) promoter. The early increased expression of survival kinases augments expansion of the cardiogenic CSC pool and subsequent daughter progeny. Normal CSCs engineered with fluorescent reporter protein constructs under control of the alphaMHC promoter show transgene protein expression, confirming activity of the promoter in CSCs. Cultured CSCs from both nontransgenic and cardiac-specific transgenic mice expressing survival kinases driven by the alphaMHC promoter were analyzed to characterize transgene expression following treatments to promote differentiation in culture. Therapeutic genes controlled by the alphaMHC promoter can be engineered into and expressed in CSCs and cardiomyocyte progeny with the goal of improving the efficacy of cardiac stem cell therapy.

Endothelial ERK signaling controls lymphatic fate specification

PubMed Central

Deng, Yong; Atri, Deepak; Eichmann, Anne; Simons, Michael

2013-01-01

Lymphatic vessels are thought to arise from PROX1-positive endothelial cells (ECs) in the cardinal vein in response to induction of SOX18 expression; however, the molecular event responsible for increased SOX18 expression has not been established. We generated mice with endothelial-specific, inducible expression of an RAF1 gene with a gain-of-function mutation (RAF1S259A) that is associated with Noonan syndrome. Expression of mutant RAF1S259A in ECs activated ERK and induced SOX18 and PROX1 expression, leading to increased commitment of venous ECs to the lymphatic fate. Excessive production of lymphatic ECs resulted in lymphangiectasia that was highly reminiscent of abnormal lymphatics seen in Noonan syndrome and similar “RASopathies.” Inhibition of ERK signaling during development abrogated the lymphatic differentiation program and rescued the lymphatic phenotypes induced by expression of RAF1S259A. These data suggest that ERK activation plays a key role in lymphatic EC fate specification and that excessive ERK activation is the basis of lymphatic abnormalities seen in Noonan syndrome and related diseases. PMID:23391722

Identification of Regulatory Elements That Control PPARγ Expression in Adipocyte Progenitors

PubMed Central

Chou, Wen-Ling; Galmozzi, Andrea; Partida, David; Kwan, Kevin; Yeung, Hui; Su, Andrew I.; Saez, Enrique

2013-01-01

Adipose tissue renewal and obesity-driven expansion of fat cell number are dependent on proliferation and differentiation of adipose progenitors that reside in the vasculature that develops in coordination with adipose depots. The transcriptional events that regulate commitment of progenitors to the adipose lineage are poorly understood. Because expression of the nuclear receptor PPARγ defines the adipose lineage, isolation of elements that control PPARγ expression in adipose precursors may lead to discovery of transcriptional regulators of early adipocyte determination. Here, we describe the identification and validation in transgenic mice of 5 highly conserved non-coding sequences from the PPARγ locus that can drive expression of a reporter gene in a manner that recapitulates the tissue-specific pattern of PPARγ expression. Surprisingly, these 5 elements appear to control PPARγ expression in adipocyte precursors that are associated with the vasculature of adipose depots, but not in mature adipocytes. Characterization of these five PPARγ regulatory sequences may enable isolation of the transcription factors that bind these cis elements and provide insight into the molecular regulation of adipose tissue expansion in normal and pathological states. PMID:24009687

Cellular and Molecular Characterization of Human Cardiac Stem Cells Reveals Key Features Essential for Their Function and Safety

PubMed Central

Vahdat, Sadaf; Mousavi, Seyed Ahmad; Omrani, Gholamreza; Gholampour, Maziar; Sotoodehnejadnematalahi, Fattah; Ghazizadeh, Zaniar; Gharechahi, Javad

2015-01-01

Cell therapy of heart diseases is emerging as one of the most promising known treatments in recent years. Transplantation of cardiac stem cells (CSCs) may be one of the best strategies to cure adult or pediatric heart diseases. As these patient-derived stem cells need to be isolated from small heart biopsies, it is important to select the best isolation method and CSC subpopulation with the best cardiogenic functionality. We employed three different protocols including c-KIT+ cell sorting, clonogenic expansion, and explants culture to isolate c-KIT+ cells, clonogenic expansion-derived cells (CEDCs), and cardiosphere-derived cells (CDCs), respectively. Evaluation of isolated CSC characteristics in vitro and after rat myocardial infarction (MI) model transplantation revealed that although c-KIT+ and CDCs had higher MI regenerative potential, CEDCs had more commitment into cardiomyocytes and needed lower passages that were essential to reach a definite cell count. Furthermore, genome-wide expression analysis showed that subsequent passages caused changes in characteristics of cells, downregulation of cell cycle-related genes, and upregulation of differentiation and carcinogenic genes, which might lead to senescence, commitment, and possible tumorigenicity of the cells. Because of different properties of CSC subpopulations, we suggest that appropriate CSCs subpopulation should be chosen based on their experimental or clinical use. PMID:25867933

Haemopoietic stem cells.

PubMed

Bellantuono, Ilaria

2004-04-01

Considerable effort has been made in recent years in understanding the mechanisms that govern stem cell generation, proliferation, self-renewal, commitment and lately plasticity. In the development of the haemopoietic system during embryonic and fetal life the notion of different pools of stem cells arising from the endothelium is gaining consensus. Gene expression profiling of populations of stem cells is bringing to light categories of genes important for self-renewal or commitment. Besides the role of transcription factors in lineage decision, the role of soluble factors and transmembrane proteins, very active at the time of embryo development, are taking central stage in the maintenance and in vitro expansion of haemopoietic stem cells (HSCs). The hierarchical model of haemopoietic development is being questioned with reports of lineage switching and plasticity of haemopoietic stem cells to non-haemopoietic cells. Yet the understanding of the overall process is still very fragmented and hypothetical. This is mainly due to the absence of appropriate markers to enable selection of homogeneous stem cell populations and the need to rely on retrospective functional assays, able only to determine the overall behaviour of a population of cells. This review is intended to be an overview of the haemopoietic system and a critical re-visitation of issues such as plasticity and self-renewal important for therapeutic applications of haemopoietic stem cells.

Information Commons for Rice (IC4R)

PubMed Central

2016-01-01

Rice is the most important staple food for a large part of the world's human population and also a key model organism for plant research. Here, we present Information Commons for Rice (IC4R; http://ic4r.org), a rice knowledgebase featuring adoption of an extensible and sustainable architecture that integrates multiple omics data through community-contributed modules. Each module is developed and maintained by different committed groups, deals with data collection, processing and visualization, and delivers data on-demand via web services. In the current version, IC4R incorporates a variety of rice data through multiple committed modules, including genome-wide expression profiles derived entirely from RNA-Seq data, resequencing-based genomic variations obtained from re-sequencing data of thousands of rice varieties, plant homologous genes covering multiple diverse plant species, post-translational modifications, rice-related literatures and gene annotations contributed by the rice research community. Unlike extant related databases, IC4R is designed for scalability and sustainability and thus also features collaborative integration of rice data and low costs for database update and maintenance. Future directions of IC4R include incorporation of other omics data and association of multiple omics data with agronomically important traits, dedicating to build IC4R into a valuable knowledgebase for both basic and translational researches in rice. PMID:26519466

Transcription factor COUP-TFII is indispensable for venous and lymphatic development in zebrafish and Xenopus laevis

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

Aranguren, Xabier L., E-mail: xabier.lopezaranguren@med.kuleuven.be; Beerens, Manu, E-mail: manu.beerens@med.kuleuven.be; Vandevelde, Wouter, E-mail: woutervandevelde@gmail.com

Highlights: {yields} COUP-TFII deficiency in zebrafish affects arterio-venous EC specification. {yields} COUP-TFII is indispensable for lymphatic development in zebrafish. {yields} COUP-TFII knockdown in Xenopus disrupts lymphatic EC differentiation and migration. {yields} COUP-TFII's role in EC fate decisions is evolutionary conserved. -- Abstract: Transcription factors play a central role in cell fate determination. Gene targeting in mice revealed that Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also known as Nuclear Receptor 2F2 or NR2F2) induces a venous phenotype in endothelial cells (ECs). More recently, NR2F2 was shown to be required for initiating the expression of Prox1, responsible for lymphatic commitment ofmore » venous ECs. Small animal models like zebrafish embryos and Xenopus laevis tadpoles have been very useful to elucidate mechanisms of (lymph) vascular development. Therefore, the role of NR2F2 in (lymph) vascular development was studied by eliminating its expression in these models. Like in mice, absence of NR2F2 in zebrafish resulted in distinct vascular defects including loss of venous marker expression, major trunk vessel fusion and vascular leakage. Both in zebrafish and Xenopus the development of the main lymphatic structures was severely hampered. NR2F2 knockdown significantly decreased prox1 expression in zebrafish ECs and the same manipulation affected lymphatic (L)EC commitment, migration and function in Xenopus tadpoles. Therefore, the role of NR2F2 in EC fate determination is evolutionary conserved.« less

Clonal precursor of bone, cartilage, and hematopoietic niche stromal cells

PubMed Central

Chan, Charles K. F.; Lindau, Paul; Jiang, Wen; Chen, James Y.; Zhang, Lillian F.; Chen, Ching-Cheng; Seita, Jun; Sahoo, Debashis; Kim, Jae-Beom; Lee, Andrew; Park, Sujin; Nag, Divya; Gong, Yongquan; Kulkarni, Subhash; Luppen, Cynthia A.; Theologis, Alexander A.; Wan, Derrick C.; DeBoer, Anthony; Seo, Eun Young; Vincent-Tompkins, Justin D.; Loh, Kyle; Walmsley, Graham G.; Kraft, Daniel L.; Wu, Joseph C.; Longaker, Michael T.; Weissman, Irving L.

2013-01-01

Organs are composites of tissue types with diverse developmental origins, and they rely on distinct stem and progenitor cells to meet physiological demands for cellular production and homeostasis. How diverse stem cell activity is coordinated within organs is not well understood. Here we describe a lineage-restricted, self-renewing common skeletal progenitor (bone, cartilage, stromal progenitor; BCSP) isolated from limb bones and bone marrow tissue of fetal, neonatal, and adult mice. The BCSP clonally produces chondrocytes (cartilage-forming) and osteogenic (bone-forming) cells and at least three subsets of stromal cells that exhibit differential expression of cell surface markers, including CD105 (or endoglin), Thy1 [or CD90 (cluster of differentiation 90)], and 6C3 [ENPEP glutamyl aminopeptidase (aminopeptidase A)]. These three stromal subsets exhibit differential capacities to support hematopoietic (blood-forming) stem and progenitor cells. Although the 6C3-expressing subset demonstrates functional stem cell niche activity by maintaining primitive hematopoietic stem cell (HSC) renewal in vitro, the other stromal populations promote HSC differentiation to more committed lines of hematopoiesis, such as the B-cell lineage. Gene expression analysis and microscopic studies further reveal a microenvironment in which CD105-, Thy1-, and 6C3-expressing marrow stroma collaborate to provide cytokine signaling to HSCs and more committed hematopoietic progenitors. As a result, within the context of bone as a blood-forming organ, the BCSP plays a critical role in supporting hematopoiesis through its generation of diverse osteogenic and hematopoietic-promoting stroma, including HSC supportive 6C3(+) niche cells. PMID:23858471

Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive G{sub s}–G protein signaling in osteoblasts

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

Wattanachanya, Lalita, E-mail: lalita_md@yahoo.com; Division of Endocrinology and Metabolism, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok; Wang, Liping, E-mail: lipingwang05@yahoo.com

G protein-coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active G{sub s}-coupled GPCR, under the control of the 2.3 kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced G{sub s} signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1(2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated G{sub s} G protein-coupled receptor signaling, at the cellularmore » level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3 kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of G{sub s}-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of G{sub s} signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to G{sub s} signaling in mature OBs. - Highlights: • OB expression of an engineered G{sub s}-coupled receptor dramatically increases bone mass. • We investigated the changes in gene expression in vivo in enhanced OB G{sub s} signaling. • Genes in cell cycle and transcription were increased in enhanced OB G{sub s} signaling. • GPCRs and paracrine mediators of the effect of G{sub s} signaling in OBs were determined.« less

Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing

PubMed Central

Han, Qianqian; Yang, Pishan; Wu, Yuwei; Meng, Shu; Sui, Lei; Zhang, Lan; Yu, Liming; Tang, Yin; Jiang, Hua; Xuan, Dongying; Kaplan, David L.; Kim, Sung Hoon

2015-01-01

Epigenetic regulation of gene expression is a central mechanism that governs cell stemness, determination, commitment, and differentiation. It has been recently found that PHF8, a major H4K20/H3K9 demethylase, plays a critical role in craniofacial and bone development. In this study, we hypothesize that PHF8 promotes osteoblastogenesis by epigenetically regulating the expression of a nuclear matrix protein, special AT-rich sequence-binding protein 2 (SATB2) that plays pivotal roles in skeletal patterning and osteoblast differentiation. Our results showed that expression levels of PHF8 and SATB2 in preosteoblasts and bone marrow stromal cells (BMSCs) increased simultaneously during osteogenic induction. Overexpressing PHF8 in these cells upregulated the expression of SATB2, Runx2, osterix, and bone matrix proteins. Conversely, knockdown of PHF8 reduced the expression of these genes. Furthermore, ChIP assays confirmed that PHF8 specifically bound to the transcription start site (TSS) of the SATB2 promoter, and the expression of H3K9me1 at the TSS region of SATB2 decreased in PHF8 overexpressed group. Implantation of the BMSCs overexpressing PHF8 with silk protein scaffolds promoted bone regeneration in critical-sized defects in mouse calvaria. Taken together, our results demonstrated that PHF8 epigenetically modulates SATB2 activity, triggering BMSCs osteogenic differentiation and facilitating bone formation and regeneration in biodegradable silk scaffolds. PMID:25923143

Haploinsufficiency of BAZ1B contributes to Williams syndrome through transcriptional dysregulation of neurodevelopmental pathways.

PubMed

Lalli, Matthew A; Jang, Jiwon; Park, Joo-Hye C; Wang, Yidi; Guzman, Elmer; Zhou, Hongjun; Audouard, Morgane; Bridges, Daniel; Tovar, Kenneth R; Papuc, Sorina M; Tutulan-Cunita, Andreea C; Huang, Yadong; Budisteanu, Magdalena; Arghir, Aurora; Kosik, Kenneth S

2016-04-01

Williams syndrome (WS) is a neurodevelopmental disorder caused by a genomic deletion of ∼28 genes that results in a cognitive and behavioral profile marked by overall intellectual impairment with relative strength in expressive language and hypersocial behavior. Advancements in protocols for neuron differentiation from induced pluripotent stem cells allowed us to elucidate the molecular circuitry underpinning the ontogeny of WS. In patient-derived stem cells and neurons, we determined the expression profile of the Williams-Beuren syndrome critical region-deleted genes and the genome-wide transcriptional consequences of the hemizygous genomic microdeletion at chromosome 7q11.23. Derived neurons displayed disease-relevant hallmarks and indicated novel aberrant pathways in WS neurons including over-activated Wnt signaling accompanying an incomplete neurogenic commitment. We show that haploinsufficiency of the ATP-dependent chromatin remodeler, BAZ1B, which is deleted in WS, significantly contributes to this differentiation defect. Chromatin-immunoprecipitation (ChIP-seq) revealed BAZ1B target gene functions are enriched for neurogenesis, neuron differentiation and disease-relevant phenotypes. BAZ1B haploinsufficiency caused widespread gene expression changes in neural progenitor cells, and together with BAZ1B ChIP-seq target genes, explained 42% of the transcriptional dysregulation in WS neurons. BAZ1B contributes to regulating the balance between neural precursor self-renewal and differentiation and the differentiation defect caused by BAZ1B haploinsufficiency can be rescued by mitigating over-active Wnt signaling in neural stem cells. Altogether, these results reveal a pivotal role for BAZ1B in neurodevelopment and implicate its haploinsufficiency as a likely contributor to the neurological phenotypes in WS. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Transcriptome of Atoh7 retinal progenitor cells identifies new Atoh7-dependent regulatory genes for retinal ganglion cell formation.

PubMed

Gao, Zhiguang; Mao, Chai-An; Pan, Ping; Mu, Xiuqian; Klein, William H

2014-11-01

The bHLH transcription factor ATOH7 (Math5) is essential for establishing retinal ganglion cell (RGC) fate. However, Atoh7-expressing retinal progenitor cells (RPCs) can give rise to all retinal cell types, suggesting that other factors are involved in specifying RGCs. The basis by which a subpopulation of Atoh7-expressing RPCs commits to an RGC fate remains uncertain but is of critical importance to retinal development since RGCs are the earliest cell type to differentiate. To better understand the regulatory mechanisms leading to cell-fate specification, a binary genetic system was generated to specifically label Atoh7-expressing cells with green fluorescent protein (GFP). Fluorescence-activated cell sorting (FACS)-purified GFP(+) and GFP(-) cells were profiled by RNA-seq. Here, we identify 1497 transcripts that were differentially expressed between the two RPC populations. Pathway analysis revealed diminished growth factor signaling in Atoh7-expressing RPCs, indicating that these cells had exited the cell cycle. In contrast, axon guidance signals were enriched, suggesting that axons of Atoh7-expressing RPCs were already making synaptic connections. Notably, many genes enriched in Atoh7-expressing RPCs encoded transcriptional regulators, and several were direct targets of ATOH7, including, and unexpectedly, Ebf3 and Eya2. We present evidence for a Pax6-Atoh7-Eya2 pathway that acts downstream of Atoh7 but upstream of differentiation factor Pou4f2. EYA2 is a protein phosphatase involved in protein-protein interactions and posttranslational regulation. These properties, along with Eya2 as an early target gene of ATOH7, suggest that EYA2 functions in RGC specification. Our results expand current knowledge of the regulatory networks operating in Atoh7-expressing RPCs and offer new directions for exploring the earliest aspects of retinogenesis. © 2014 Wiley Periodicals, Inc.

Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment

PubMed Central

Gauthier, Philippe; Yu, Zongdong; Tran, Quynh T.; Bhatti, Fazal-Ur-Rehman; Zhu, Xiaofei

2016-01-01

Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1+/CD146+, STRO-1−/CD146+ and STRO-1−/CD146− subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were down-regulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration. PMID:27757536

Cementogenic genes in human periodontal ligament stem cells are downregulated in response to osteogenic stimulation while upregulated by vitamin C treatment.

PubMed

Gauthier, Philippe; Yu, Zongdong; Tran, Quynh T; Bhatti, Fazal-Ur-Rehman; Zhu, Xiaofei; Huang, George T-J

2017-04-01

Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1 + /CD146 + , STRO-1 - /CD146 + and STRO-1 - /CD146 - subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the stimulation with osteogenic differentiation medium, CAP and CEMP1 were downregulated while osteogenic markers bone sialoprotein (BSP) and osteocalcin (OCN) were upregulated. Both CAP and CEMP1 were upregulated by VC treatment. Transplantation of VC-treated PDLSCs into immunocompromised mice resulted in forming significantly more ectopic cementum- and bone-like mineral tissues in vivo. Immunohistochemical analysis of the ectopic growth showed that CAP and CEMP1 were mainly expressed in the mineral tissue and in some cells of the fibrous tissues. We conclude that osteogenic stimulation is not inductive but appears to be inhibitory of cementogenic pathways, whereas VC induces cementogenic lineage commitment by PDLSCs and may be a useful stimulus for cementogenesis in periodontal regeneration.

Helper T Cell Identity and Evolution of Differential Transcriptomes and Epigenomes

PubMed Central

Vahedi, Golnaz; Poholek, Amanda; Hand, Timothy W.; Laurence, Arian; Kann, Yuka; O’Shea, John J.; Hirahara, Kiyoshi

2013-01-01

Summary CD4+ T cells are critical for the elimination of an immense array of microbial pathogens. Among the ways they accomplish this task is to generate progeny with specialized, characteristic patterns of gene expression. From this perspective, helper cells can be viewed as pluripotent precursors that adopt distinct cell fates. Although there are aspects of helper cell differentiation that can be modeled as a classic cell fate commitment, CD4+ T cells also maintain considerable flexibility in their transcriptional program. This makes sense in terms of host defense but raises the question of how these remarkable cells balance both these requirements, a high degree of specific gene expression and the capacity for plasticity. In this review, we discuss recent advances in our understanding of CD4+ T-cell specification, focusing on how genomic perspectives have influenced our views of these processes. The relative contributions of sensors of the cytokine milieu, especially the signal transducer and activator of transcription (STAT) family transcription factors, ‘master regulators’, and other transcription factors are considered as they relate to the helper cell transcriptome and epigenome. PMID:23405893

Constitutive expression of ftsZ overrides the whi developmental genes to initiate sporulation of Streptomyces coelicolor.

PubMed

Willemse, Joost; Mommaas, A Mieke; van Wezel, Gilles P

2012-03-01

The filamentous soil bacteria Streptomyces undergo a highly complex developmental programme. Before streptomycetes commit themselves to sporulation, distinct morphological checkpoints are passed in the aerial hyphae that are subject to multi-level control by the whi sporulation genes. Here we show that whi-independent expression of FtsZ restores sporulation to the early sporulation mutants whiA, whiB, whiG, whiH, whiI and whiJ. Viability, stress resistance and high-resolution electron microscopy underlined that viable spores were formed. However, spores from sporulation-restored whiA and whiG mutants showed defects in DNA segregation/condensation, while spores from the complemented whiB mutant had increased stress sensitivity, perhaps as a result of changes in the spore sheath. In contrast to the whi mutants, normal sporulation of ssgB null mutants-which fail to properly localise FtsZ-could not be restored by enhancing FtsZ protein levels, forming spore-like bodies that lack spore walls. Our data strongly suggest that the whi genes control a decisive event towards sporulation of streptomycetes, namely the correct timing of developmental ftsZ transcription. The biological significance may be to ensure that sporulation-specific cell division will only start once sufficient aerial mycelium biomass has been generated. Our data shed new light on the longstanding question as to how whi genes control sporulation, which has intrigued scientists for four decades.

Characterization of a cinnamoyl-CoA reductase 1 (CCR1) mutant in maize: effects on lignification, fibre development, and global gene expression

PubMed Central

Tamasloukht, Barek; Wong Quai Lam, Mary Sarah-Jane; Martinez, Yves; Tozo, Koffi; Barbier, Odile; Jourda, Cyril; Jauneau, Alain; Borderies, Gisèle; Balzergue, Sandrine; Renou, Jean-Pierre; Huguet, Stéphanie; Martinant, Jean Pierre; Tatout, Christophe; Lapierre, Catherine; Barrière, Yves; Goffner, Deborah; Pichon, Magalie

2011-01-01

Cinnamoyl-CoA reductase (CCR), which catalyses the first committed step of the lignin-specific branch of monolignol biosynthesis, has been extensively characterized in dicot species, but few data are available in monocots. By screening a Mu insertional mutant collection in maize, a mutant in the CCR1 gene was isolated named Zmccr1–. In this mutant, CCR1 gene expression is reduced to 31% of the residual wild-type level. Zmccr1– exhibited enhanced digestibility without compromising plant growth and development. Lignin analysis revealed a slight decrease in lignin content and significant changes in lignin structure. p-Hydroxyphenyl units were strongly decreased and the syringyl/guaiacyl ratio was slightly increased. At the cellular level, alterations in lignin deposition were mainly observed in the walls of the sclerenchymatic fibre cells surrounding the vascular bundles. These cell walls showed little to no staining with phloroglucinol. These histochemical changes were accompanied by an increase in sclerenchyma surface area and an alteration in cell shape. In keeping with this cell type-specific phenotype, transcriptomics performed at an early stage of plant development revealed the down-regulation of genes specifically associated with fibre wall formation. To the present authors’ knowledge, this is the first functional characterization of CCR1 in a grass species. PMID:21493812

Modulation of basal cell fate during productive and transforming HPV-16 infection is mediated by progressive E6-driven depletion of Notch.

PubMed

Kranjec, Christian; Holleywood, Christina; Libert, Diane; Griffin, Heather; Mahmood, Radma; Isaacson, Erin; Doorbar, John

2017-08-01

In stratified epithelia such as the epidermis, homeostasis is maintained by the proliferation of cells in the lower epithelial layers and the concomitant loss of differentiated cells from the epithelial surface. These differentiating keratinocytes progressively stratify and form a self-regenerating multi-layered barrier that protects the underlying dermis. In such tissue, the continual loss and replacement of differentiated cells also limits the accumulation of oncogenic mutations within the tissue. Inactivating mutations in key driver genes, such as TP53 and NOTCH1, reduce the proportion of differentiating cells allowing for the long-term persistence of expanding mutant clones in the tissue. Here we show that through the expression of E6, HPV-16 prevents the early fate commitment of human keratinocytes towards differentiation and confers a strong growth advantage to human keratinocytes. When E6 is expressed either alone or with E7, it promotes keratinocyte proliferation at high cell densities, through the combined inactivation of p53 and Notch1. In organotypic raft culture, the activity of E6 is restricted to the basal layer of the epithelium and is enhanced during the progression from productive to abortive or transforming HPV-16 infection. Consistent with this, the expression of p53 and cleaved Notch1 becomes progressively more disrupted, and is associated with increased basal cell density and reduced commitment to differentiation. The expression of cleaved Notch1 is similarly disrupted also in HPV-16-positive cervical lesions, depending on neoplastic grade. When taken together, these data depict an important role of high-risk E6 in promoting the persistence of infected keratinocytes in the basal and parabasal layers through the inactivation of gene products that are commonly mutated in non-HPV-associated neoplastic squamous epithelia. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Functional Analysis of the Brassica napus L. Phytoene Synthase (PSY) Gene Family

PubMed Central

López-Emparán, Ada; Quezada-Martinez, Daniela; Zúñiga-Bustos, Matías; Cifuentes, Víctor; Iñiguez-Luy, Federico; Federico, María Laura

2014-01-01

Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three “Arabidopsis-like” subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed. PMID:25506829

Oral administration of Lactobacillus plantarum 299v modulates gene expression in the ileum of pigs: prediction of crosstalk between intestinal immune cells and sub-mucosal adipocytes.

PubMed

Hulst, Marcel; Gross, Gabriele; Liu, Yaping; Hoekman, Arjan; Niewold, Theo; van der Meulen, Jan; Smits, Mari

2015-05-01

To study host-probiotic interactions in parts of the intestine only accessible in humans by surgery (jejunum, ileum and colon), pigs were used as model for humans. Groups of eight 6-week-old pigs were repeatedly orally administered with 5 × 10(12) CFU Lactobacillus plantarum 299v (L. plantarum 299v) or PBS, starting with a single dose followed by three consecutive daily dosings 10 days later. Gene expression was assessed with pooled RNA samples isolated from jejunum, ileum and colon scrapings of the eight pigs per group using Affymetrix porcine microarrays. Comparison of gene expression profiles recorded from L. plantarum 299v-treated pigs with PBS-treated pigs indicated that L. plantarum 299v affected metabolic and immunological processes, particularly in the ileum. A higher expression level of several B cell-specific transcription factors/regulators was observed, suggesting that an influx of B cells from the periphery to the ileum and/or the proliferation of progenitor B cells to IgA-committed plasma cells in the Peyer's patches of the ileum was stimulated. Genes coding for enzymes that metabolize leukotriene B4, 1,25-dihydroxyvitamin D3 and steroids were regulated in the ileum. Bioinformatics analysis predicted that these metabolites may play a role in the crosstalk between intestinal immune cells and sub-mucosal adipocytes. Together with regulation of genes that repress NFKB- and PPARG-mediated transcription, this crosstalk may contribute to tempering of inflammatory reactions. Furthermore, the enzyme adenosine deaminase, responsible for the breakdown of the anti-inflammatory mediator adenosine, was strongly down-regulated in response to L. plantarum 299v. This suggested that L. plantarum 299v-regulated production of adenosine by immune cells like regulatory T cells may also be a mechanism that tempers inflammation in the ileum, and perhaps also in other parts of the pig's body.

Epigenetic Marks Define the Lineage and Differentiation Potential of Two Distinct Neural Crest-Derived Intermediate Odontogenic Progenitor Populations

PubMed Central

Gopinathan, Gokul; Kolokythas, Antonia

2013-01-01

Epigenetic mechanisms, such as histone modifications, play an active role in the differentiation and lineage commitment of mesenchymal stem cells. In the present study, epigenetic states and differentiation profiles of two odontogenic neural crest-derived intermediate progenitor populations were compared: dental pulp (DP) and dental follicle (DF). ChIP on chip assays revealed substantial H3K27me3-mediated repression of odontoblast lineage genes DSPP and dentin matrix protein 1 (DMP1) in DF cells, but not in DP cells. Mineralization inductive conditions caused steep increases of mineralization and patterning gene expression levels in DP cells when compared to DF cells. In contrast, mineralization induction resulted in a highly dynamic histone modification response in DF cells, while there was only a subdued effect in DP cells. Both DF and DP progenitors featured H3K4me3-active marks on the promoters of early mineralization genes RUNX2, MSX2, and DLX5, while OSX, IBSP, and BGLAP promoters were enriched for H3K9me3 or H3K27me3. Compared to DF cells, DP cells expressed higher levels of three pluripotency-associated genes, OCT4, NANOG, and SOX2. Finally, gene ontology comparison of bivalent marks unique for DP and DF cells highlighted cell–cell attachment genes in DP cells and neurogenesis genes in DF cells. In conclusion, the present study indicates that the DF intermediate odontogenic neural crest lineage is distinguished from its DP counterpart by epigenetic repression of DSPP and DMP1 genes and through dynamic histone enrichment responses to mineralization induction. Findings presented here highlight the crucial role of epigenetic regulatory mechanisms in the terminal differentiation of odontogenic neural crest lineages. PMID:23379639

Transcription factors in melanocyte development: distinct roles for Pax-3 and Mitf.

PubMed

Hornyak, T J; Hayes, D J; Chiu, L Y; Ziff, E B

2001-03-01

A transgenic mouse model was used to examine the roles of the murine transcription factors Pax-3 and Mitf in melanocyte development. Transgenic mice expressing beta-galactosidase from the dopachrome tautomerase (Dct) promoter were generated and found to express the transgene in developing melanoblasts as early as embryonic day (E) 9.5. These mice express the transgene in a pattern characteristic of endogenous Dct expression. Transgenic mice were intercrossed with two murine coat color mutants, Splotch (Sp), containing a mutation in the murine Pax3 gene, and Mitf(mi), with a mutation in the basic-helix-loop-helix-leucine zipper gene Mitf. Transgenic heterozygous mutant animals were crossed to generate transgenic embryos for analysis. Examination of beta-galactosidase-expressing melanoblasts in mutant embryos reveals that Mitf is required in vivo for survival of melanoblasts up to the migration staging area in neural crest development. Examination of Mitf(mi)/+ embryos shows that there are diminished numbers of melanoblasts in the heterozygous state early in melanocyte development, consistent with a gene dosage-dependent effect upon cell survival. However, quantification and analysis of melanoblast growth during the migratory phase suggests that melanoblasts then increase in number more rapidly in the heterozygous embryo. In contrast to Mitf(mi)/Mitf(mi) embryos, Sp/Sp embryos exhibit melanoblasts that have migrated to characteristic locations along the melanoblast migratory pathway, but are greatly reduced in number compared to control littermates. Together, these results support a model for melanocyte development whereby Pax3 is required to expand a pool of committed melanoblasts or restricted progenitor cells early in development, whereas Mitf facilitates survival of the melanoblast in a gene dosage-dependent manner within and immediately after emigration from the dorsal neural tube, and may also directly or indirectly affect the rate at which melanoblast number increases during dorsolateral pathway migration.

runt Homology Domain Transcription Factors (Runx, Cbfa, and AML) Mediate Repression of the Bone Sialoprotein Promoter: Evidence for Promoter Context-Dependent Activity of Cbfa Proteins

PubMed Central

Javed, Amjad; Barnes, George L.; Jasanya, B. O.; Stein, Janet L.; Gerstenfeld, Louis; Lian, Jane B.; Stein, Gary S.

2001-01-01

Expression of the bone sialoprotein (BSP) gene, a marker of bone formation, is largely restricted to cells in mineralized tissues. Recent studies have shown that the Cbfa1 (also known as Runx2, AML-3, and PEBP2αA) transcription factor supports commitment and differentiation of progenitor cells to hypertrophic chondrocytes and osteoblasts. This study addresses the functional involvement of Cbfa sites in expression of the Gallus BSP gene. Gel mobility shift analyses with nuclear extracts from ROS 17/2.8 osteoblastic cells revealed that multiple Cbfa consensus sequences are functional Cbfa DNA binding sites. Responsiveness of the 1.2-kb Gallus BSP promoter to Cbfa factors Cbfa1, Cbfa2, and Cbfa3 was assayed in osseous and nonosseous cells. Each of the Cbfa factors mediated repression of the wild-type BSP promoter, in contrast to their well known activation of various hematopoietic and skeletal phenotypic genes. Suppression of BSP by Cbfa factors was not observed in BSP promoters in which Cbfa sites were deleted or mutated. Expression of the endogenous BSP gene in Gallus osteoblasts was similarly downregulated by forced expression of Cbfa factors. Our data indicate that Cbfa repression of the BSP promoter does not involve the transducin-like enhancer (TLE) proteins. Neither coexpression of TLE1 or TLE2 nor the absence of the TLE interaction motif of Cbfa1 (amino acids 501 to 513) influenced repressor activity. However, removal of the C terminus of Cbfa1 (amino acids 362 to 513) relieved suppression of the BSP promoter. Our results, together with the evolutionary conservation of the seven Cbfa sites in the Gallus and human BSP promoters, suggest that suppressor activity by Cbfa is of significant physiologic consequence and may contribute to spatiotemporal expression of BSP during bone development. PMID:11283267

Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation

PubMed Central

Ng, Ashley P.; Kauppi, Maria; Metcalf, Donald; Hyland, Craig D.; Josefsson, Emma C.; Lebois, Marion; Zhang, Jian-Guo; Baldwin, Tracey M.; Di Rago, Ladina; Hilton, Douglas J.; Alexander, Warren S.

2014-01-01

Thrombopoietin (TPO) acting via its receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO-dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (MplPF4cre/PF4cre). MplPF4cre/PF4cre mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO overstimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool. PMID:24711413

Mpl expression on megakaryocytes and platelets is dispensable for thrombopoiesis but essential to prevent myeloproliferation.

PubMed

Ng, Ashley P; Kauppi, Maria; Metcalf, Donald; Hyland, Craig D; Josefsson, Emma C; Lebois, Marion; Zhang, Jian-Guo; Baldwin, Tracey M; Di Rago, Ladina; Hilton, Douglas J; Alexander, Warren S

2014-04-22

Thrombopoietin (TPO) acting via its receptor, the cellular homologue of the myeloproliferative leukemia virus oncogene (Mpl), is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO-dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (Mpl(PF4cre/PF4cre)). Mpl(PF4cre/PF4cre) mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO overstimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool.

Isolation of Oct4-Expressing Extraembryonic Endoderm Precursor Cell Lines

PubMed Central

Debeb, Bisrat G.; Galat, Vasiliy; Epple-Farmer, Jessica; Iannaccone, Steve; Woodward, Wendy A.; Bader, Michael; Iannaccone, Philip; Binas, Bert

2009-01-01

Background The extraembryonic endoderm (ExEn) defines the yolk sac, a set of membranes that provide essential support for mammalian embryos. Recent findings suggest that the committed ExEn precursor is present already in the embryonic Inner Cell Mass (ICM) as a group of cells that intermingles with the closely related epiblast precursor. All ICM cells contain Oct4, a key transcription factor that is first expressed at the morula stage. In vitro, the epiblast precursor is most closely represented by the well-characterized embryonic stem (ES) cell lines that maintain the expression of Oct4, but analogous ExEn precursor cell lines are not known and it is unclear if they would express Oct4. Methodology/Principal Findings Here we report the isolation and characterization of permanently proliferating Oct4-expressing rat cell lines (“XEN-P cell lines”), which closely resemble the ExEn precursor. We isolated the XEN-P cell lines from blastocysts and characterized them by plating and gene expression assays as well as by injection into embryos. Like ES cells, the XEN-P cells express Oct4 and SSEA1 at high levels and their growth is stimulated by leukemia inhibitory factor, but instead of the epiblast determinant Nanog, they express the ExEn determinants Gata6 and Gata4. Further, they lack markers characteristic of the more differentiated primitive/visceral and parietal ExEn stages, but exclusively differentiate into these stages in vitro and contribute to them in vivo. Conclusions/Significance Our findings (i) suggest strongly that the ExEn precursor is a self-renewable entity, (ii) indicate that active Oct4 gene expression (transcription plus translation) is part of its molecular identity, and (iii) provide an in vitro model of early ExEn differentiation. PMID:19784378

Regulation of osteogenesis by long noncoding RNAs: An epigenetic mechanism contributing to bone formation.

PubMed

Tye, Coralee E; Boyd, Joseph R; Page, Natalie A; Falcone, Michelle M; Stein, Janet L; Stein, Gary S; Lian, Jane B

2018-12-01

Long noncoding RNAs (lncRNAs) have recently emerged as novel regulators of lineage commitment, differentiation, development, viability, and disease progression. Few studies have examined their role in osteogenesis; however, given their critical and wide-ranging roles in other tissues, lncRNAs are most likely vital regulators of osteogenesis. In this study, we extensively characterized lncRNA expression in mesenchymal cells during commitment and differentiation to the osteoblast lineage using a whole transcriptome sequencing approach (RNA-Seq). Using mouse primary mesenchymal stromal cells (mMSC), we identified 1438 annotated lncRNAs expressed during MSC differentiation, 462 of which are differentially expressed. We performed guilt-by-association analysis using lncRNA and mRNA expression profiles to identify lncRNAs influencing MSC commitment and differentiation. These findings open novel dimensions for exploring lncRNAs in regulating normal bone formation and in skeletal disorders.

PIOX, a new pathogen-induced oxygenase with homology to animal cyclooxygenase.

PubMed

Sanz, A; Moreno, J I; Castresana, C

1998-09-01

Changes in gene expression induced in tobacco leaves by the harpin HrpN protein elicitor were examined, and a new cDNA, piox (for pathogen-induced oxygenase), with homology to genes encoding cyclooxygenase or prostaglandin endoperoxide synthase (PGHS), was identified. In addition to the amino acid identity determined, the protein encoded by piox is predicted to have a structural core similar to that of ovine PGHS-1. Moreover, studies of protein functionality demonstrate that the PIOX recombinant protein possesses at least one of the two enzymatic activities of PGHSs, that of catalyzing the oxygenation of polyunsaturated fatty acids. piox transcripts accumulated after protein elicitor treatment or inoculation with bacteria. Expression of piox was induced in tissues responding to inoculation with both incompatible and compatible bacteria, but RNA and protein accumulation differed for both types of interactions. We show that expression of piox is rapidly induced in response to various cellular signals mediating plant responses to pathogen infection and that activation of piox expression is most likely related to the oxidative burst that takes place during the cell death processes examined. Cyclooxygenase catalyzes the first committed step in the formation of prostaglandins and thromboxanes, which are lipid-derived signal molecules that mediate many cellular processes, including the immune response in vertebrates. The finding of tobacco PIOX suggests that more similarities than hitherto expected will be found between the lipid-based responses for plant and animal systems.

Romidepsin Promotes Osteogenic and Adipocytic Differentiation of Human Mesenchymal Stem Cells through Inhibition of Histondeacetylase Activity

PubMed Central

Ali, Dalia; Manikandan, Muthurangan; Hamam, Rimi; Alfayez, Musaad; Aldahmash, Abdullah

2018-01-01

Bone marrow mesenchymal stem cells (BMSCs) are adult multipotent stem cells that can differentiate into mesodermal lineage cells, including adipocytes and osteoblasts. However, the epigenetic mechanisms governing the lineage-specific commitment of BMSCs into adipocytes or osteoblasts are under investigation. Herein, we investigated the epigenetic effect of romidepsin, a small molecule dual inhibitor targeting HDAC1 and HDAC2 identified through an epigenetic library functional screen. BMSCs exposed to romidepsin (5 nM) exhibited enhanced adipocytic and osteoblastic differentiation. Global gene expression and signaling pathway analyses of differentially expressed genes revealed a strong enrichment of genes involved in adipogenesis and osteogenesis in romidepsin-treated BMSCs during induction into adipocytes or osteoblasts, respectively. Pharmacological inhibition of FAK signaling during adipogenesis or inhibition of FAK or TGFβ signaling during osteogenesis diminished the biological effects of romidepsin on BMSCs. The results of chromatin immunoprecipitation combined with quantitative polymerase chain reaction indicated a significant increase in H3K9Ac epigenetic markers in the promoter regions of peroxisome proliferator-activated receptor gamma (PPARγ) and KLF15 (related to adipogenesis) or SP7 (Osterix) and alkaline phosphatase (ALP) (related to osteogenesis) in romidepsin-treated BMSCs. Our data indicated that romidepsin is a novel in vitro modulator of adipocytic and osteoblastic differentiation of BMSCs. PMID:29731773

Romidepsin Promotes Osteogenic and Adipocytic Differentiation of Human Mesenchymal Stem Cells through Inhibition of Histondeacetylase Activity.

PubMed

Ali, Dalia; Chalisserry, Elna P; Manikandan, Muthurangan; Hamam, Rimi; Alfayez, Musaad; Kassem, Moustapha; Aldahmash, Abdullah; Alajez, Nehad M

2018-01-01

Bone marrow mesenchymal stem cells (BMSCs) are adult multipotent stem cells that can differentiate into mesodermal lineage cells, including adipocytes and osteoblasts. However, the epigenetic mechanisms governing the lineage-specific commitment of BMSCs into adipocytes or osteoblasts are under investigation. Herein, we investigated the epigenetic effect of romidepsin, a small molecule dual inhibitor targeting HDAC1 and HDAC2 identified through an epigenetic library functional screen. BMSCs exposed to romidepsin (5 nM) exhibited enhanced adipocytic and osteoblastic differentiation. Global gene expression and signaling pathway analyses of differentially expressed genes revealed a strong enrichment of genes involved in adipogenesis and osteogenesis in romidepsin-treated BMSCs during induction into adipocytes or osteoblasts, respectively. Pharmacological inhibition of FAK signaling during adipogenesis or inhibition of FAK or TGF β signaling during osteogenesis diminished the biological effects of romidepsin on BMSCs. The results of chromatin immunoprecipitation combined with quantitative polymerase chain reaction indicated a significant increase in H3K9Ac epigenetic markers in the promoter regions of peroxisome proliferator-activated receptor gamma (PPAR γ ) and KLF15 (related to adipogenesis) or SP7 (Osterix) and alkaline phosphatase (ALP) (related to osteogenesis) in romidepsin-treated BMSCs. Our data indicated that romidepsin is a novel in vitro modulator of adipocytic and osteoblastic differentiation of BMSCs.

A new look at immune privilege of the eye: dual role for the vision-related molecule retinoic acid.

PubMed

Zhou, Ru; Horai, Reiko; Mattapallil, Mary J; Caspi, Rachel R

2011-10-15

The eye is an immunologically privileged and profoundly immunosuppressive environment. Early studies reported inhibition of T cell proliferation, IFN-γ production, and generation of regulatory T cells (Tregs) by aqueous humor (AH) and identified TGF-β as a critical factor. However, T cell subsets including Foxp3(+) Treg and Th17 were unknown at that time, as was the role of retinoic acid (RA) in Treg induction. Consequently, the effect of the ocular microenvironment on T cell lineage commitment and function, and the role of RA in this process, had not been explored. We now use gene-manipulated mice and highly purified T cell populations to demonstrate that AH suppresses lineage commitment and acquisition of Th1 and Th17 effector function of naive T cells, manifested as reduction of lineage-specific transcription factors and cytokines. Instead, AH promoted its massive conversion to Foxp3(+) Tregs that expressed CD25, GITR, CTLA-4, and CD103 and were functionally suppressive. TGF-β and RA were both needed and synergized for Treg conversion by AH, with TGF-β-enhancing T cell expression of RA receptor α. Newly converted Foxp3(+) Tregs were unstable, but were stabilized upon continued exposure to AH or by the DNA demethylating agent 5-aza-2'-deoxycytidine. In contrast, T cells already committed to effector function were resistant to the suppressive and Treg-inducing effects of AH. We conclude that RA in the eye plays a dual role: in vision and in immune privilege. Nevertheless, primed effector T cells are relatively insensitive to AH, helping to explain their ability to induce uveitis despite an inhibitory ocular microenvironment.

Dedifferentiation of Human Primary Thyrocytes into Multilineage Progenitor Cells without Gene Introduction

PubMed Central

Saenko, Vladimir; Suzuki, Masatoshi; Matsuse, Michiko; Ohtsuru, Akira; Kumagai, Atsushi; Uga, Tatsuya; Yano, Hiroshi; Nagayama, Yuji; Yamashita, Shunichi

2011-01-01

While identification and isolation of adult stem cells have potentially important implications, recent reports regarding dedifferentiation/reprogramming from differentiated cells have provided another clue to gain insight into source of tissue stem/progenitor cells. In this study, we developed a novel culture system to obtain dedifferentiated progenitor cells from normal human thyroid tissues. After enzymatic digestion, primary thyrocytes, expressing thyroglobulin, vimentin and cytokeratin-18, were cultured in a serum-free medium called SAGM. Although the vast majority of cells died, a small proportion (∼0.5%) survived and proliferated. During initial cell expansion, thyroglobulin/cytokeratin-18 expression was gradually declined in the proliferating cells. Moreover, sorted cells expressing thyroid peroxidase gave rise to proliferating clones in SAGM. These data suggest that those cells are derived from thyroid follicular cells or at least thyroid-committed cells. The SAGM-grown cells did not express any thyroid-specific genes. However, after four-week incubation with FBS and TSH, cytokeratin-18, thyroglobulin, TSH receptor, PAX8 and TTF1 expressions re-emerged. Moreover, surprisingly, the cells were capable of differentiating into neuronal or adipogenic lineage depending on differentiating conditions. In summary, we have developed a novel system to generate multilineage progenitor cells from normal human thyroid tissues. This seems to be achieved by dedifferentiation of thyroid follicular cells. The presently described culture system may be useful for regenerative medicine, but the primary importance will be as a tool to elucidate the mechanisms of thyroid diseases. PMID:21556376

P-HYDROXYPHENYLPYRUVATE DIOXYGENASE from Medicago sativa is involved in vitamin E biosynthesis and abscisic acid-mediated seed germination

PubMed Central

Jiang, Jishan; Chen, Zhihong; Ban, Liping; Wu, Yudi; Huang, Jianping; Chu, Jinfang; Fang, Shuang; Wang, Zan; Gao, Hongwen; Wang, Xuemin

2017-01-01

P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved in the biosynthesis of vitamin E, and is characterized by catalyzing the conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl, abscisic acid and salicylic acid were shown to significantly induce MsHPPD expression, especially in the cotyledons and root tissues. Overexpression of MsHPPD was found to significantly increase the level of β-tocotrienol and the total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic Arabidopsis seeds exhibited an accelerated germination time, compared with wild-type seeds under normal conditions, as well as under NaCl and ABA treatments. Meanwhile, the expression level of several genes associated with ABA biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3 and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic lines, as well as the total free ABA content. Taken together, these results demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway, but also plays a critical role in seed germination via affecting ABA biosynthesis and signaling. PMID:28084442

Capturing in vivo RNA transcriptional dynamics from the malaria parasite Plasmodium falciparum

PubMed Central

Painter, Heather J.; Carrasquilla, Manuela; Llinás, Manuel

2017-01-01

To capture the transcriptional dynamics within proliferating cells, methods to differentiate nascent transcription from preexisting mRNAs are desired. One approach is to label newly synthesized mRNA transcripts in vivo through the incorporation of modified pyrimidines. However, the human malaria parasite, Plasmodium falciparum, is incapable of pyrimidine salvage for mRNA biogenesis. To capture cellular mRNA dynamics during Plasmodium development, we engineered parasites that can salvage pyrimidines through the expression of a single bifunctional yeast fusion gene, cytosine deaminase/uracil phosphoribosyltransferase (FCU). We show that expression of FCU allows for the direct incorporation of thiol-modified pyrimidines into nascent mRNAs. Using developmental stage-specific promoters to express FCU-GFP enables the biosynthetic capture and in-depth analysis of mRNA dynamics from subpopulations of cells undergoing differentiation. We demonstrate the utility of this method by examining the transcriptional dynamics of the sexual gametocyte stage transition, a process that is essential to malaria transmission between hosts. Using the pfs16 gametocyte-specific promoter to express FCU-GFP in 3D7 parasites, we found that sexual stage commitment is governed by transcriptional reprogramming and stabilization of a subset of essential gametocyte transcripts. We also measured mRNA dynamics in F12 gametocyte-deficient parasites and demonstrate that the transcriptional program required for sexual commitment and maturation is initiated but likely aborted due to the absence of the PfAP2-G transcriptional regulator and a lack of gametocyte-specific mRNA stabilization. Biosynthetic labeling of Plasmodium mRNAs is incredibly versatile, can be used to measure transcriptional dynamics at any stage of parasite development, and will allow for future applications to comprehensively measure RNA-protein interactions in the malaria parasite. PMID:28416533

Stability and Multiattractor Dynamics of a Toggle Switch Based on a Two-Stage Model of Stochastic Gene Expression

PubMed Central

Strasser, Michael; Theis, Fabian J.; Marr, Carsten

2012-01-01

A toggle switch consists of two genes that mutually repress each other. This regulatory motif is active during cell differentiation and is thought to act as a memory device, being able to choose and maintain cell fate decisions. Commonly, this switch has been modeled in a deterministic framework where transcription and translation are lumped together. In this description, bistability occurs for transcription factor cooperativity, whereas autoactivation leads to a tristable system with an additional undecided state. In this contribution, we study the stability and dynamics of a two-stage gene expression switch within a probabilistic framework inspired by the properties of the Pu/Gata toggle switch in myeloid progenitor cells. We focus on low mRNA numbers, high protein abundance, and monomeric transcription-factor binding. Contrary to the expectation from a deterministic description, this switch shows complex multiattractor dynamics without autoactivation and cooperativity. Most importantly, the four attractors of the system, which only emerge in a probabilistic two-stage description, can be identified with committed and primed states in cell differentiation. To begin, we study the dynamics of the system and infer the mechanisms that move the system between attractors using both the quasipotential and the probability flux of the system. Next, we show that the residence times of the system in one of the committed attractors are geometrically distributed. We derive an analytical expression for the parameter of the geometric distribution, therefore completely describing the statistics of the switching process and elucidate the influence of the system parameters on the residence time. Moreover, we find that the mean residence time increases linearly with the mean protein level. This scaling also holds for a one-stage scenario and for autoactivation. Finally, we study the implications of this distribution for the stability of a switch and discuss the influence of the stability on a specific cell differentiation mechanism. Our model explains lineage priming and proposes the need of either high protein numbers or long-term modifications such as chromatin remodeling to achieve stable cell fate decisions. Notably, we present a system with high protein abundance that nevertheless requires a probabilistic description to exhibit multistability, complex switching dynamics, and lineage priming. PMID:22225794

Transcriptome and gene expression profile of ovarian follicle tissue of the triatomine bug Rhodnius prolixus

PubMed Central

Medeiros, Marcelo N.; Logullo, Raquel; Ramos, Isabela B.; Sorgine, Marcos H. F.; Paiva-Silva, Gabriela O.; Mesquita, Rafael D.; Machado, Ednildo Alcantara; Coutinho, Maria Alice; Masuda, Hatisaburo; Capurro, Margareth L.; Ribeiro, José M.C.; Cardoso Braz, Glória Regina; Oliveira, Pedro L

2013-01-01

Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types. PMID:21736942

Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor

PubMed Central

Enuka, Yehoshua; Lauriola, Mattia; Feldman, Morris E.; Sas-Chen, Aldema; Ulitsky, Igor; Yarden, Yosef

2016-01-01

Circular RNAs (circRNAs) are widespread circles of non-coding RNAs with largely unknown function. Because stimulation of mammary cells with the epidermal growth factor (EGF) leads to dynamic changes in the abundance of coding and non-coding RNA molecules, and culminates in the acquisition of a robust migratory phenotype, this cellular model might disclose functions of circRNAs. Here we show that circRNAs of EGF-stimulated mammary cells are stably expressed, while mRNAs and microRNAs change within minutes. In general, the circRNAs we detected are relatively long-lived and weakly expressed. Interestingly, they are almost ubiquitously co-expressed with the corresponding linear transcripts, and the respective, shared promoter regions are more active compared to genes producing linear isoforms with no detectable circRNAs. These findings imply that altered abundance of circRNAs, unlike changes in the levels of other RNAs, might not play critical roles in signaling cascades and downstream transcriptional networks that rapidly commit cells to specific outcomes. PMID:26657629

Competing Death Programs in Poliovirus-Infected Cells: Commitment Switch in the Middle of the Infectious Cycle

PubMed Central

Agol, Vadim I.; Belov, George A.; Bienz, Kurt; Egger, Denise; Kolesnikova, Marina S.; Romanova, Lyudmila I.; Sladkova, Larissa V.; Tolskaya, Elena A.

2000-01-01

Productive poliovirus infection of HeLa cells leads to the canonical cytopathic effect (CPE), whereas certain types of abortive infection result in apoptosis. To define the time course of commitment to the different types of poliovirus-induced death, inhibitors of viral replication (guanidine HCl) or translation (cycloheximide) were added at different times postinfection (p.i.). Early in the infection (during the first ∼2 h p.i.), predominantly proapoptotic viral function was expressed, rendering the cells committed to apoptosis, which developed several hours after viral expression was arrested. In the middle of infection, concomitantly with the onset of fast generation of viral progeny, the implementation of the viral apoptotic program was abruptly interrupted. In particular, activation of an Asp-Glu-Val-Asp (DEVD)-specific caspase(s) occurring in the apoptosis-committed cells was prevented by the ongoing productive infection. Simultaneously, the cells retaining normal or nearly normal morphology became committed to CPE, which eventually developed regardless of whether or not further viral expression was allowed to proceed. The implementation of the poliovirus-induced apoptotic program was suppressed in HeLa cells overexpressing the Bcl-2 protein, indicating that the fate of poliovirus-infected cells depends on the balance of host and viral pro- and antiapoptotic factors. PMID:10823859

Myostatin genetic inactivation inhibits myogenesis by muscle-derived stem cells in vitro but not when implanted in the mdx mouse muscle

PubMed Central

2013-01-01

Introduction Stimulating the commitment of implanted dystrophin+ muscle-derived stem cells (MDSCs) into myogenic, as opposed to lipofibrogenic lineages, is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). Methods To examine whether counteracting myostatin, a negative regulator of muscle mass and a pro-lipofibrotic factor, would help this process, we compared the in vitro myogenic and fibrogenic capacity of MDSCs from wild-type (WT) and myostatin knockout (Mst KO) mice under various modulators, the expression of key stem cell and myogenic genes, and the capacity of these MDSCs to repair the injured gastrocnemius in aged dystrophic mdx mice with exacerbated lipofibrosis. Results Surprisingly, the potent in vitro myotube formation by WT MDSCs was refractory to modulators of myostatin expression or activity, and the Mst KO MDSCs failed to form myotubes under various conditions, despite both MDSC expressing Oct 4 and various stem cell genes and differentiating into nonmyogenic lineages. The genetic inactivation of myostatin in MDSCs was associated with silencing of critical genes for early myogenesis (Actc1, Acta1, and MyoD). WT MDSCs implanted into the injured gastrocnemius of aged mdx mice significantly improved myofiber repair and reduced fat deposition and, to a lesser extent, fibrosis. In contrast to their in vitro behavior, Mst KO MDSCs in vivo also significantly improved myofiber repair, but had few effects on lipofibrotic degeneration. Conclusions Although WT MDSCs are very myogenic in culture and stimulate muscle repair after injury in the aged mdx mouse, myostatin genetic inactivation blocks myotube formation in vitro, but the myogenic capacity is recovered in vivo under the influence of the myostatin+ host-tissue environment, presumably by reactivation of key genes originally silenced in the Mst KO MDSCs. PMID:23295128

The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function.

PubMed

Gu, Yue; Jones, Amanda E; Yang, Wei; Liu, Shanrun; Dai, Qian; Liu, Yudong; Swindle, C Scott; Zhou, Dewang; Zhang, Zhuo; Ryan, Thomas M; Townes, Tim M; Klug, Christopher A; Chen, Dongquan; Wang, Hengbin

2016-01-05

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

Systematically labeling developmental stage-specific genes for the study of pancreatic β-cell differentiation from human embryonic stem cells.

PubMed

Liu, Haisong; Yang, Huan; Zhu, Dicong; Sui, Xin; Li, Juan; Liang, Zhen; Xu, Lei; Chen, Zeyu; Yao, Anzhi; Zhang, Long; Zhang, Xi; Yi, Xing; Liu, Meng; Xu, Shiqing; Zhang, Wenjian; Lin, Hua; Xie, Lan; Lou, Jinning; Zhang, Yong; Xi, Jianzhong; Deng, Hongkui

2014-10-01

The applications of human pluripotent stem cell (hPSC)-derived cells in regenerative medicine has encountered a long-standing challenge: how can we efficiently obtain mature cell types from hPSCs? Attempts to address this problem are hindered by the complexity of controlling cell fate commitment and the lack of sufficient developmental knowledge for guiding hPSC differentiation. Here, we developed a systematic strategy to study hPSC differentiation by labeling sequential developmental genes to encompass the major developmental stages, using the directed differentiation of pancreatic β cells from hPSCs as a model. We therefore generated a large panel of pancreas-specific mono- and dual-reporter cell lines. With this unique platform, we visualized the kinetics of the entire differentiation process in real time for the first time by monitoring the expression dynamics of the reporter genes, identified desired cell populations at each differentiation stage and demonstrated the ability to isolate these cell populations for further characterization. We further revealed the expression profiles of isolated NGN3-eGFP(+) cells by RNA sequencing and identified sushi domain-containing 2 (SUSD2) as a novel surface protein that enriches for pancreatic endocrine progenitors and early endocrine cells both in human embryonic stem cells (hESC)-derived pancreatic cells and in the developing human pancreas. Moreover, we captured a series of cell fate transition events in real time, identified multiple cell subpopulations and unveiled their distinct gene expression profiles, among heterogeneous progenitors for the first time using our dual reporter hESC lines. The exploration of this platform and our new findings will pave the way to obtain mature β cells in vitro.

Altered Expression of Wnt Signaling Pathway Components in Osteogenesis of Mesenchymal Stem Cells in Osteoarthritis Patients.

PubMed

Tornero-Esteban, Pilar; Peralta-Sastre, Ascensión; Herranz, Eva; Rodríguez-Rodríguez, Luis; Mucientes, Arkaitz; Abásolo, Lydia; Marco, Fernando; Fernández-Gutiérrez, Benjamín; Lamas, José Ramón

2015-01-01

Osteoarthritis (OA) is characterized by altered homeostasis of joint cartilage and bone, whose functional properties rely on chondrocytes and osteoblasts, belonging to mesenchymal stem cells (MSCs). WNT signaling acts as a hub integrating and crosstalking with other signaling pathways leading to the regulation of MSC functions. The aim of this study was to evaluate the existence of a differential signaling between Healthy and OA-MSCs during osteogenesis. MSCs of seven OA patients and six healthy controls were isolated, characterised and expanded. During in vitro osteogenesis, cells were recovered at days 1, 10 and 21. RNA and protein content was obtained. Expression of WNT pathway genes was evaluated using RT-qPCR. Functional studies were also performed to study the MSC osteogenic commitment and functional and post-traslational status of β-catenin and several receptor tyrosine kinases. Several genes were downregulated in OA-MSCs during osteogenesis in vitro. These included soluble Wnts, inhibitors, receptors, co-receptors, several kinases and transcription factors. Basal levels of β-catenin were higher in OA-MSCs, but calcium deposition and expression of osteogenic genes was similar between Healthy and OA-MSCs. Interestingly an increased phosphorylation of p44/42 MAPK (ERK1/2) signaling node was present in OA-MSCs. Our results point to the existence in OA-MSCs of alterations in expression of Wnt pathway components during in vitro osteogenesis that are partially compensated by post-translational mechanisms modulating the function of other pathways. We also point the relevance of other signaling pathways in OA pathophysiology suggesting their role in the maintenance of joint homeostasis through modulation of MSC osteogenic potential.

Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive Gs-G protein signaling in osteoblasts

PubMed Central

Wattanachanya, Lalita; Wang, Liping; Millard, Susan M.; Lu, Wei-Dar; O’Carroll, Dylan; Hsiao, Edward C.; Conklin, Bruce R.; Nissenson, Robert A.

2015-01-01

G protein–coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active Gs-coupled GPCR, under the control of the 2.3-kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced Gs signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1(2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated Gs G protein–coupled receptor signaling, at the cellular level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3-kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of Gs-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of Gs signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to Gs signaling in mature OBs. PMID:25704759

Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

PubMed

Li, Yin-Ji; Kukita, Akiko; Kyumoto-Nakamura, Yukari; Kukita, Toshio

2016-09-01

Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

A Novel View of the Adult Stem Cell Compartment From the Perspective of a Quiescent Population of Very Small Embryonic-Like Stem Cells.

PubMed

Ratajczak, Mariusz Z; Ratajczak, Janina; Suszynska, Malwina; Miller, Donald M; Kucia, Magda; Shin, Dong-Myung

2017-01-06

Evidence has accumulated that adult hematopoietic tissues and other organs contain a population of dormant stem cells (SCs) that are more primitive than other, already restricted, monopotent tissue-committed SCs (TCSCs). These observations raise several questions, such as the developmental origin of these cells, their true pluripotent or multipotent nature, which surface markers they express, how they can be efficiently isolated from adult tissues, and what role they play in the adult organism. The phenotype of these cells and expression of some genes characteristic of embryonic SCs, epiblast SCs, and primordial germ cells suggests their early-embryonic deposition in developing tissues as precursors of adult SCs. In this review, we will critically discuss all these questions and the concept that small dormant SCs related to migratory primordial germ cells, described as very small embryonic-like SCs, are deposited during embryogenesis in bone marrow and other organs as a backup population for adult tissue-committed SCs and are involved in several processes related to tissue or organ rejuvenation, aging, and cancerogenesis. The most recent results on successful ex vivo expansion of human very small embryonic-like SC in chemically defined media free from feeder-layer cells open up new and exciting possibilities for their application in regenerative medicine. © 2017 American Heart Association, Inc.

Mineralogenic characteristics of osteogenic lineage-committed human dental pulp stem cells following their exposure to a discoloration-free calcium aluminosilicate cement.

PubMed

Niu, Li-Na; Pei, Dan-Dan; Morris, Matthew; Jiao, Kai; Huang, Xue-Qing; Primus, Carolyn M; Susin, Lisiane F; Bergeron, Brian E; Pashley, David H; Tay, Franklin R

2016-10-01

An experimental discoloration-free calcium aluminosilicate cement has been developed with the intention of maximizing the beneficial attributes of tricalcium silicate cements and calcium aluminate cements. The present study examined the effects of this experimental cement (Quick-Set2) on the mineralogenic characteristics of osteogenic lineage-committed human dental pulp stem cells (hDPSCs), by comparing the cellular responses with a commercially available tricalcium silicate cement (white mineral trioxide aggregate (ProRoot(®) MTA); WMTA). The osteogenic potential of hDPSCs exposed to the cements was examined using qRT-PCR for osteogenic gene expressions, Western blot for osteogenic-related protein expressions, alkaline phosphatase enzyme activity, Alizarin red S staining, Fourier transform infrared spectroscopy and transmission electron microscopy of extracellular calcium deposits. Results of the six assays indicated that osteogenic differentiation of hDPSCs was significantly enhanced after exposure to the tricalcium silicate cement or the experimental calcium aluminosilicate cement, with the former demonstrating better mineralogenic stimulation capacity. The better osteogenic stimulating effect of the tricalcium silicate cement on hDPSCs may be due to its relatively higher silicate content, or higher OH(-) and Ca(2+) release. Further investigations with the use of in vivo animal models are required to validate the potential augmenting osteogenic effects of the experimental discoloration-free calcium aluminosilicate cement. Published by Elsevier Ltd.

Fetal liver contains committed NK progenitors, but is not a site for development of CD34+ cells into T cells.

PubMed

Jaleco, A C; Blom, B; Res, P; Weijer, K; Lanier, L L; Phillips, J H; Spits, H

1997-07-15

The presence of T and NK cells in the human fetal liver and the fact that fetal liver hemopoietic progenitor cells develop into T and NK cells suggest a role for the fetal liver compartment in T and NK cell development. In this work, we show that the capacity of fetal liver progenitors to develop into T cells, in a human/mouse fetal thymic organ culture system, is restricted to an immature subset of CD34+ CD38- cells. No T cell-committed precursors are contained within the more differentiated CD34+ CD38+ population. This conclusion is supported by the observations that no TCR-delta gene rearrangements and no pre-TCR-alpha expression can be detected in this population. However, NK cells were derived from CD34+ CD38- and CD34+ CD38+ fetal liver cells cultured in the presence of IL-15, IL-7, and Flt-3 ligand. Eighty to ninety percent of cells arising from the CD34+ CD38+ population expressed the NK cell-associated markers CD56, CD16, CD94, and NKR-P1A. Several subpopulations of NK cell precursors were identified by differential expression of these receptors. Based on the detection of populations with a similar antigenic profile in freshly isolated fetal liver cells, we propose a model of NK cell differentiation. Collectively, our findings suggest that CD34+ cells differentiate into NK cells, but not into mature T cells, in the human fetal liver.

Wild-type myoblasts rescue the ability of myogenin-null myoblasts to fuse in vivo.

PubMed

Myer, A; Wagner, D S; Vivian, J L; Olson, E N; Klein, W H

1997-05-15

Skeletal muscle is formed via a complex series of events during embryogenesis. These events include commitment of mesodermal precursor cells, cell migration, cell-cell recognition, fusion of myoblasts, activation of structural genes, and maturation. In mice lacking the bHLH transcription factor myogenin, myoblasts are specified and positioned correctly, but few fuse to form multinucleated fibers. This indicates that myogenin is critical for the fusion process and subsequent differentiation events of myogenesis. To further define the nature of the myogenic defects in myogenin-null mice, we investigated whether myogenin-null myoblasts are capable of fusing with wild-type myoblasts in vivo using chimeric mice containing mixtures of myogenin-null and wild-type cells. Chimeric embryos demonstrated that myogenin-null myoblasts readily fused in the presence of wild-type myoblasts. However, chimeric myofibers did not express wild-type levels of muscle-specific gene products, and myofibers with a high percentage of mutant nuclei appeared abnormal, suggesting that the wild-type nuclei could not fully rescue mutant nuclei in the myofibers. These data demonstrate that myoblast fusion can be uncoupled from complete myogenic differentiation and that myogenin regulates a specific subset of genes with diverse function. Thus, myogenin appears to control not only transcription of muscle structural genes but also the extracellular environment in which myoblast fusion takes place. We propose that myogenin regulates the expression of one or more extracellular or cell surface proteins required to initiate the muscle differentiation program.

Dissonance, resistance and commitment: a pilot analysis of moderated mediation relationships.

PubMed

Draycott, Simon

2012-07-01

Encouraging engagement is a challenge in forensic services. A model of behaviour change suggests that dissatisfaction with the self may express itself either as resistance to or commitment to engagement in treatment, and that this expression may be moderated by the individual's attributional style. It was hypothesised that there would be relationships between measures of dissonance, resistance to treatment and commitment to treatment, and that these relationships would be moderated by locus of control. Thirty-two patients from within a high secure dangerous and severe personality disorder service completed assessments measuring dissonance, locus of control, resistance and commitment. These measures were analysed for a moderated mediation using techniques outlined by Preacher et al. (2007). There were significant indirect relationships between dissonance and both resistance and commitment. These relationships were moderated by locus of control. However, the specifics of the relationships were not as expected - both internal and external locus of control interfered with therapy, but in different ways. A patient who experiences dissonance may translate this into either commitment to or resistance to treatment, depending on features of the patient such as attributional style. Copyright © 2012 John Wiley & Sons, Ltd.

A chalcone isomerase-like protein enhances flavonoid production and flower pigmentation.

PubMed

Morita, Yasumasa; Takagi, Kyoko; Fukuchi-Mizutani, Masako; Ishiguro, Kanako; Tanaka, Yoshikazu; Nitasaka, Eiji; Nakayama, Masayoshi; Saito, Norio; Kagami, Takashi; Hoshino, Atsushi; Iida, Shigeru

2014-04-01

Flavonoids are major pigments in plants, and their biosynthetic pathway is one of the best-studied metabolic pathways. Here we have identified three mutations within a gene that result in pale-colored flowers in the Japanese morning glory (Ipomoea nil). As the mutations lead to a reduction of the colorless flavonoid compound flavonol as well as of anthocyanins in the flower petal, the identified gene was designated enhancer of flavonoid production (EFP). EFP encodes a chalcone isomerase (CHI)-related protein classified as a type IV CHI protein. CHI is the second committed enzyme of the flavonoid biosynthetic pathway, but type IV CHI proteins are thought to lack CHI enzymatic activity, and their functions remain unknown. The spatio-temporal expression of EFP and structural genes encoding enzymes that produce flavonoids is very similar. Expression of both EFP and the structural genes is coordinately promoted by genes encoding R2R3-MYB and WD40 family proteins. The EFP gene is widely distributed in land plants, and RNAi knockdown mutants of the EFP homologs in petunia (Petunia hybrida) and torenia (Torenia hybrida) had pale-colored flowers and low amounts of anthocyanins. The flavonol and flavone contents in the knockdown petunia and torenia flowers, respectively, were also significantly decreased, suggesting that the EFP protein contributes in early step(s) of the flavonoid biosynthetic pathway to ensure production of flavonoid compounds. From these results, we conclude that EFP is an enhancer of flavonoid production and flower pigmentation, and its function is conserved among diverse land plant species. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

Cellular commitment in the developing cerebellum

PubMed Central

Marzban, Hassan; Del Bigio, Marc R.; Alizadeh, Javad; Ghavami, Saeid; Zachariah, Robby M.; Rastegar, Mojgan

2014-01-01

The mammalian cerebellum is located in the posterior cranial fossa and is critical for motor coordination and non-motor functions including cognitive and emotional processes. The anatomical structure of cerebellum is distinct with a three-layered cortex. During development, neurogenesis and fate decisions of cerebellar primordium cells are orchestrated through tightly controlled molecular events involving multiple genetic pathways. In this review, we will highlight the anatomical structure of human and mouse cerebellum, the cellular composition of developing cerebellum, and the underlying gene expression programs involved in cell fate commitments in the cerebellum. A critical evaluation of the cell death literature suggests that apoptosis occurs in ~5% of cerebellar cells, most shortly after mitosis. Apoptosis and cellular autophagy likely play significant roles in cerebellar development, we provide a comprehensive discussion of their role in cerebellar development and organization. We also address the possible function of unfolded protein response in regulation of cerebellar neurogenesis. We discuss recent advancements in understanding the epigenetic signature of cerebellar compartments and possible connections between DNA methylation, microRNAs and cerebellar neurodegeneration. Finally, we discuss genetic diseases associated with cerebellar dysfunction and their role in the aging cerebellum. PMID:25628535

Insulin antagonises pigment epithelium-derived factor (PEDF)-induced modulation of lineage commitment of myocytes and heterotrophic ossification.

PubMed

Carnagarin, Revathy; Elahy, Mina; Dharmarajan, Arun M; Dass, Crispin R

2017-12-16

Extensive bone defects arising as a result of trauma, infection and tumour resection and other bone pathologies necessitates the identification of effective strategies in the form of tissue engineering, gene therapy and osteoinductive agents to enhance the bone repair process. PEDF is a multifunctional glycoprotein which plays an important role in regulating osteoblastic differentiation and bone formation. PEDF treatment of mice and human skeletal myocytes at physiological concentration inhibited myogenic differentiation and activated Erk1/2 MAPK- dependent osteogenic transdifferentiation of myocytes. In mice, insulin, a promoter of bone regeneration, attenuated PEDF-induced expression of osteogenic markers such as osteocalcin, alkaline phosphatase and mineralisation for bone formation in the muscle and surrounding adipose tissue. These results provide new insights into the molecular aspects of the antagonising effect of insulin on PEDF-dependent modulation of the differentiation commitment of musculoskeletal environment into osteogenesis, and suggest that PEDF may be developed as an effective clinical therapy for bone regeneration as its heterotopic ossification can be controlled via co-administration of insulin. Copyright © 2017 Elsevier B.V. All rights reserved.

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Angiomodulin is required for cardiogenesis of embryonic stem cells and is maintained by a feedback loop network of p63 and Activin-A.

PubMed

Wolchinsky, Zohar; Shivtiel, Shoham; Kouwenhoven, Evelyn Nathalie; Putin, Daria; Sprecher, Eli; Zhou, Huiqing; Rouleau, Matthieu; Aberdam, Daniel

2014-01-01

The transcription factor p63, member of the p53 gene family, encodes for two main isoforms, TAp63 and ΔNp63 with distinct functions on epithelial homeostasis and cancer. Recently, we discovered that TAp63 is essential for in vitro cardiogenesis and heart development in vivo. TAp63 is expressed by embryonic endoderm and acts on cardiac progenitors by a cell-non-autonomous manner. In the present study, we search for cardiogenic secreted factors that could be regulated by TAp63 and, by ChIP-seq analysis, identified Angiomodulin (AGM), also named IGFBP7 or IGFBP-rP1. We demonstrate that AGM is necessary for cardiac commitment of embryonic stem cells (ESCs) and its regulation depends on TAp63 isoform. TAp63 directly activates both AGM and Activin-A during ESC cardiogenesis while these secreted factors modulate TAp63 gene expression by a feedback loop mechanism. The molecular circuitry controlled by TAp63 on AGM/Activin-A signaling pathway and thus on cardiogenesis emphasizes the importance of p63 during early cardiac development. © 2013.

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

Onopiuk, Marta; Wierzbicka, Katarzyna; Brutkowski, Wojciech

Activation of T-cells triggers store-operated Ca{sup 2+} entry, which begins a signaling cascade leading to induction of appropriate gene expression and eventually lymphocyte proliferation and differentiation. The simultaneous enhancement of Fas ligand gene expression in activated cells allows the immune response to be limited by committing the activated cells to apoptosis. In apoptotic cells the store-operated calcium entry is significantly inhibited. It has been documented that moderate activation of Fas receptor may cause reversible inhibition of store-operated channels by ceramide released from hydrolyzed sphingomyelin. Here we show that activation of Fas receptor in T-cells results in caspase-dependent decrease of cellularmore » STIM1 and Orai1 protein content. This effect may be responsible for the substantial inhibition of Ca{sup 2+} entry into Jurkat cells undergoing apoptosis. In turn, this inhibition might prevent overloading of cells with calcium and protect them against necrosis. -- Research highlights: {yields} Fas activation reduces STIM1 and Orai1 protein content in caspase dependent manner. {yields} Fas activation partially reduces mitochondrial potential in caspase dependent manner. {yields} Fas stimulation inhibits of store-operated Ca{sup 2+} entry in caspase dependent manner. {yields} Inhibition of Ca{sup 2+} entry in apoptotic cells may protect them from secondary necrosis.« less

Histone Deacetylase Inhibition Promotes Osteoblast Maturation by Altering the Histone H4 Epigenome and Reduces Akt Phosphorylation*

PubMed Central

Dudakovic, Amel; Evans, Jared M.; Li, Ying; Middha, Sumit; McGee-Lawrence, Meghan E.; van Wijnen, Andre J.; Westendorf, Jennifer J.

2013-01-01

Bone has remarkable regenerative capacity, but this ability diminishes during aging. Histone deacetylase inhibitors (HDIs) promote terminal osteoblast differentiation and extracellular matrix production in culture. The epigenetic events altered by HDIs in osteoblasts may hold clues for the development of new anabolic treatments for osteoporosis and other conditions of low bone mass. To assess how HDIs affect the epigenome of committed osteoblasts, MC3T3 cells were treated with suberoylanilide hydroxamic acid (SAHA) and subjected to microarray gene expression profiling and high-throughput ChIP-Seq analysis. As expected, SAHA induced differentiation and matrix calcification of osteoblasts in vitro. ChIP-Seq analysis revealed that SAHA increased histone H4 acetylation genome-wide and in differentially regulated genes, except for the 500 bp upstream of transcriptional start sites. Pathway analysis indicated that SAHA increased the expression of insulin signaling modulators, including Slc9a3r1. SAHA decreased phosphorylation of insulin receptor β, Akt, and the Akt substrate FoxO1, resulting in FoxO1 stabilization. Thus, SAHA induces genome-wide H4 acetylation and modulates the insulin/Akt/FoxO1 signaling axis, whereas it promotes terminal osteoblast differentiation in vitro. PMID:23940046

A new 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene encoding the committed-step enzyme in the MEP pathway from Rauvolfia verticillata.

PubMed

Liao, Zhihua; Chen, Rong; Chen, Min; Yang, Chunxian; Wang, Qiang; Gong, Yifu

2007-01-01

1-Deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes a committed step of the methylerythritol phosphate (MEP) pathway for the biosynthesis of pharmaceutical terpenoid indole alkaloid (TIA) precursors. The full-length cDNA sequence was cloned and characterized from a TIA-producing species, Rauvolfia verticillata, using rapid amplification of cDNA ends (RACE) technique. The new cDNA was named as RvDXR and submitted to GenBank to be assigned with an accession number (DQ779286). The full-length cDNA of RvDXR was 1804 bp containing a 1425 bp open reading frame (ORF) encoding a polypeptide of 474 amino acids with a calculated molecular mass of 51.3 kDa and an isoelectric point of 5.88. Comparative and bioinformatic analyses revealed that RvDXR showed extensive homology with DXRs from other plant species and contained a conserved transit peptide for plastids, an extended Pro-rich region and a highly conserved NADPH-binding motif in its N-terminal region owned by all plant DXRs. The phylogenetic analysis revealed that DXRs had two groups including a plant and bacterial group; RvDXR belonged to angiosperm DXRs that were obtained from Synechocystis through gene transfer according to the phylogenetic analysis. The structural modeling of RvDXR showed that RvDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that RvDXR expressed in all tissues including roots, stems, leaves, fruits and followers but at different levels. The lowest transcription level was observed in followers and the highest transcription was found in fruits of R. verticillata; the transcription level of RvDXR was a little higher in roots and stems than in leaves. The cloning and characterization of RvDXR will be helpful to understand more about the role of DXR involved in R. verticillata TIA biosynthesis at the molecular level and provides a candidate gene for metabolic engineering of the TIAs pathway in R. verticillata.

The interaction between Sertoli cells and luekemia inhibitory factor on the propagation and differentiation of spermatogonial stem cells in vitro

PubMed Central

Rastegar, Tayebeh; Habibi Roudkenar, Mehryar; Parvari, Soraya; Baazm, Maryam

2015-01-01

Background: Sertoli cells play a pivotal role in creating microenvironments essential for spermatogonial stem cells (SSCs) self-renewal and commitment to differentiation. Maintenance of SSCs and or induction of in vitro spermiogenesis may provide a therapeutic strategy to treat male infertility. Objective: This study investigated the role of luekemia inhibitory factor (LIF) on the propagation of SSCs and both functions of Sertoli cells on the proliferation and differentiation of these cells. Materials and Methods: SSCs were sorted from the testes of adult male mice by magnetic activated cell sorting and thymus cell antigen 1 antibody. On the other hand, isolated Sertoli cells were enriched using lectin coated plates. SSCs were cultured on Sertoli cells for 7 days in the absence or presence of LIF. The effects of these conditions were evaluated by microscopy and expression of meiotic and post meiotic transcripts by reverse transcriptase polymerase chain reaction. Results: Our data showed that SSCs co-cultured with Sertoli cells in the presence of LIF formed colonies on top of the Sertoli cells. These colonies had alkaline phosphatesase activity and expressed SSCs specific genes. SSCs were enjoyed limited development after the mere removal of LIF, and exhibiting expression of meiotic and postmeiotic transcript and loss of SSCs specific gene expression (p< 0.05). Conclusion: Our findings represent co-culture of SSCs with Sertoli cells provides conditions that may allow efficient proliferation and differentiation of SSCs for male infertility treatment. PMID:26730242

The interaction between Sertoli cells and luekemia inhibitory factor on the propagation and differentiation of spermatogonial stem cells in vitro.

PubMed

Rastegar, Tayebeh; Habibi Roudkenar, Mehryar; Parvari, Soraya; Baazm, Maryam

2015-11-01

Sertoli cells play a pivotal role in creating microenvironments essential for spermatogonial stem cells (SSCs) self-renewal and commitment to differentiation. Maintenance of SSCs and or induction of in vitro spermiogenesis may provide a therapeutic strategy to treat male infertility. This study investigated the role of luekemia inhibitory factor (LIF) on the propagation of SSCs and both functions of Sertoli cells on the proliferation and differentiation of these cells. SSCs were sorted from the testes of adult male mice by magnetic activated cell sorting and thymus cell antigen 1 antibody. On the other hand, isolated Sertoli cells were enriched using lectin coated plates. SSCs were cultured on Sertoli cells for 7 days in the absence or presence of LIF. The effects of these conditions were evaluated by microscopy and expression of meiotic and post meiotic transcripts by reverse transcriptase polymerase chain reaction. Our data showed that SSCs co-cultured with Sertoli cells in the presence of LIF formed colonies on top of the Sertoli cells. These colonies had alkaline phosphatesase activity and expressed SSCs specific genes. SSCs were enjoyed limited development after the mere removal of LIF, and exhibiting expression of meiotic and postmeiotic transcript and loss of SSCs specific gene expression (p< 0.05). Our findings represent co-culture of SSCs with Sertoli cells provides conditions that may allow efficient proliferation and differentiation of SSCs for male infertility treatment.

Phenotypic and Gene Expression Modification with Normal Brain Aging in GFAP-Positive Astrocytes and Neural Stem Cells

PubMed Central

Bernal, Giovanna M.; Peterson, Daniel A.

2011-01-01

Summary Astrocytes secrete growth factors that are both neuroprotective and supportive for the local environment. Identified by glial fibrillary acidic protein (GFAP) expression, astrocytes exhibit heterogeneity in morphology and in expression of phenotypic markers and growth factors throughout different adult brain regions. In adult neurogenic niches, astrocytes secrete vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) within the neurogenic niche, and are also a source of special GFAP-positive multipotent neural stem cells (NSCs). Normal aging is accompanied by a decline in CNS function and reduced neurogenesis. We asked if a decreased availability of astrocyte-derived factors may contribute to the age-related decline in neurogenesis. Determining alterations of astrocytic activity in the aging brain is crucial for understanding CNS homeostasis in aging and for assessing appropriate therapeutic targets for an aging population. We found region-specific alterations in gene expression of GFAP, VEGF and FGF-2 and their receptors in the aged brain corresponding to changes in astrocytic reactivity, supporting astrocytic heterogeneity and demonstrating a differential aging effect. We found that GFAP-positive NSCs uniquely coexpress both VEGF and its key mitotic receptor Flk-1 in both young and aged hippocampus, indicating a possible autocrine/paracrine signaling mechanism. VEGF expression is lost once NSCs commit to a neuronal fate, but Flk-1-mediated sensitivity to VEGF signaling is maintained. We propose that age-related astrocytic changes result in reduced VEGF and FGF-2 signaling, which in turn limits neural stem cell and progenitor cell maintenance and contributes to decreased neurogenesis. PMID:21385309

Precommitment low-level Neurog3 expression defines a long-lived mitotic endocrine-biased progenitor pool that drives production of endocrine-committed cells

PubMed Central

Bechard, Matthew E.; Bankaitis, Eric D.; Hipkens, Susan B.; Ustione, Alessandro; Piston, David W.; Yang, Yu-Ping; Magnuson, Mark A.; Wright, Christopher V.E.

2016-01-01

The current model for endocrine cell specification in the pancreas invokes high-level production of the transcription factor Neurogenin 3 (Neurog3) in Sox9+ bipotent epithelial cells as the trigger for endocrine commitment, cell cycle exit, and rapid delamination toward proto-islet clusters. This model posits a transient Neurog3 expression state and short epithelial residence period. We show, however, that a Neurog3TA.LO cell population, defined as Neurog3 transcriptionally active and Sox9+ and often containing nonimmunodetectable Neurog3 protein, has a relatively high mitotic index and prolonged epithelial residency. We propose that this endocrine-biased mitotic progenitor state is functionally separated from a pro-ductal pool and endows them with long-term capacity to make endocrine fate-directed progeny. A novel BAC transgenic Neurog3 reporter detected two types of mitotic behavior in Sox9+ Neurog3TA.LO progenitors, associated with progenitor pool maintenance or derivation of endocrine-committed Neurog3HI cells, respectively. Moreover, limiting Neurog3 expression dramatically increased the proportional representation of Sox9+ Neurog3TA.LO progenitors, with a doubling of its mitotic index relative to normal Neurog3 expression, suggesting that low Neurog3 expression is a defining feature of this cycling endocrine-biased state. We propose that Sox9+ Neurog3TA.LO endocrine-biased progenitors feed production of Neurog3HI endocrine-committed cells during pancreas organogenesis. PMID:27585590

Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cells.

PubMed

Gas-Pascual, Elisabet; Simonovik, Biljana; Schaller, Hubert; Bach, Thomas J

2015-08-01

Tobacco BY-2 cell suspensions are our preferred model for studying isoprenoid biosynthesis pathways, due to their easy genetic transformation and the efficient absorption of metabolic precursors, intermediates, and/or inhibitors. Using this model system, we have analyzed the effects of chemical and genetic blockage of cycloartenol synthase (CAS, EC 5.4.99.8), an oxidosqualene cyclase that catalyzes the first committed step in the sterol pathway of plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Short-term treatments (24 h) resulted in accumulation of oxidosqualene with no changes in the final sterol products. Interestingly, long-term treatments (6 days) induced down-regulation in gene expression not only of CAS but also of the SMT2 gene coding sterol methyltransferase 2 (EC 2.1.1.41). This explains some of the increase in 24-methyl sterols at the expense of the 24-ethyl sterols stigmasterol and sitosterol. In our alternative strategy, CAS gene expression was partially blocked by using an inducible artificial microRNA. The limited effectiveness of this approach might be explained by some dependence of the machinery for RNAi formation on an operating MVA/sterol pathway. For comparison we checked the effect of RO 48-8071 on a green cell suspension of Arabidopsis and on seedlings, containing a small spectrum of triterpenes besides phytosterols. Triterpenes remained essentially unaffected, but phytosterol accumulation was clearly diminished.

Characterization of somatic embryogenesis initiated from the Arabidopsis shoot apex.

PubMed

Kadokura, Satoshi; Sugimoto, Kaoru; Tarr, Paul; Suzuki, Takamasa; Matsunaga, Sachihiro

2018-04-28

Somatic embryogenesis is one of the best examples of the remarkable developmental plasticity of plants, in which committed somatic cells can dedifferentiate and acquire the ability to form an embryo and regenerate an entire plant. In Arabidopsis thaliana, the shoot apices of young seedlings have been reported as an alternative tissue source for somatic embryos (SEs) besides the widely studied zygotic embryos taken from siliques. Although SE induction from shoots demonstrates the plasticity of plants more clearly than the embryo-to-embryo induction system, the underlying developmental and molecular mechanisms involved are unknown. Here we characterized SE formation from shoot apex explants by establishing a system for time-lapse observation of explants during SE induction. We also established a method to distinguish SE-forming and non-SE-forming explants prior to anatomical SE formation, enabling us to identify distinct transcriptome profiles of these two explants at SE initiation. We show that embryonic fate commitment takes place at day 3 of SE induction and the SE arises directly, not through callus formation, from the base of leaf primordia just beside the shoot apical meristem (SAM), where auxin accumulates and shoot-root polarity is formed. The expression domain of a couple of key developmental genes for the SAM transiently expands at this stage. Our data demonstrate that SE-forming and non-SE-forming explants share mostly the same transcripts except for a limited number of embryonic genes and root genes that might trigger the SE-initiation program. Thus, SE-forming explants possess a mixed identity (SAM, root and embryo) at the time of SE specification. Copyright © 2018. Published by Elsevier Inc.

Differences in the ovine HSP90AA1 gene expression rates caused by two linked polymorphisms at its promoter affect rams sperm DNA fragmentation under environmental heat stress conditions.

PubMed

Salces-Ortiz, Judit; Ramón, Manuel; González, Carmen; Pérez-Guzmán, M Dolores; Garde, J Julián; García-Álvarez, Olga; Maroto-Morales, Alejandro; Calvo, Jorge H; Serrano, M Magdalena

2015-01-01

Heat shock (HS) is one of the best-studied exogenous cellular stresses. Almost all tissues, cell types, metabolic pathways and biochemical reactions are affected in greater or lesser extent by HS. However, there are some especially thermo sensible cellular types such as the mammalian male germ cells. The present study examined the role of three INDELs in conjunction with the -660G/C polymorphism located at the HSP90AA1 promoter region over the gene expression rate under HS. Specially, the -668insC INDEL, which is very close to the -660G/C transversion, is a good candidate to be implied in the transcriptional regulation of the gene by itself or in a cooperative way with this SNP. Animals carrying the genotype II-668 showed higher transcription rates than those with ID-668 (FC = 3.07) and DD-668 (FC = 3.40) genotypes for samples collected under HS. A linkage between gene expression and sperm DNA fragmentation was also found. When HS conditions were present along or in some stages of the spermatogenesis, alternative genotypes of the -668insC and -660G/C mutations are involved in the effect of HS over sperm DNA fragmentation. Thus, unfavorable genotypes in terms of gene expression induction (ID-668GC-660 and DD-668GG-660) do not produce enough mRNA (stored as messenger ribonucleoprotein particles) and Hsp90α protein to cope with future thermal stress which might occur in posterior stages when transcriptional activity is reduced and cell types and molecular processes are more sensible to heat (spermatocytes in pachytene and spermatids protamination). This would result in the impairment of DNA packaging and the consequent commitment of the events occurring shortly after fertilization and during embryonic development. In the short-term, the assessment of the relationship between sperm DNA fragmentation sensitivity and ram's fertility will be of interest to a better understanding of the mechanisms of response to HS and its consequences on animal production and reproduction performance.

A cascade of Ca2+/calmodulin-dependent protein kinases regulates the differentiation and functional activation of murine neutrophils

PubMed Central

Gaines, Peter; Lamoureux, James; Marisetty, Anantha; Chi, Jeffrey; Berliner, Nancy

2008-01-01

Objective The function of neutrophils as primary mediators of innate immunity depends on the activity of granule proteins and critical components of the NADPH oxidase complex. Expression of their cognate genes is regulated during neutrophil differentiation by a complex network of intracellular signaling pathways. In this study we have investigated the role of two members of the calcium/calmodulin-dependent protein kinase (CaMK) signaling cascade, CaMKI-like kinase (CKLiK) and CaMKKα, in regulating neutrophil differentiation and functional activation. Materials and Methods Mouse myeloid cell lines were used to examine the expression of a CaMK cascade in developing neutrophils and to examine the effects of constitutive activation versus inhibition of CaMKs on neutrophil maturation. Results Expression of CaMKKα was shown to increase during neutrophil differentiation in multiple cell lines, whereas expression of CKLiK increased as multipotent progenitors committed to promyelocytes but then decreased as cells differentiated into mature neutrophils. Expression of constitutively active CKLiKs did not affect morphologic maturation, but caused dramatic decreases in both respiratory burst responses and chemotaxis. This loss of neutrophil function was accompanied by reduced secondary granule and gp91phox gene expression. The CaMK inhibitor KN93 attenuated cytokine-stimulated proliferative responses in promyelocytic cell lines, and inhibited the respiratory burst. Similar data were observed with the CaMKKα inhibitor, STO-609. Conclusions Overactivation of a cascade of CaMKs inhibits neutrophil maturation, suggesting that these kinases play an antagonistic role during neutrophil differentiation, but at least one CaMK is required for myeloid cell expansion and functional activation. PMID:18400360

Reduced Notch signalling leads to postnatal skeletal muscle hypertrophy in Pofut1cax/cax mice.

PubMed

Al Jaam, Bilal; Heu, Katy; Pennarubia, Florian; Segelle, Alexandre; Magnol, Laetitia; Germot, Agnès; Legardinier, Sébastien; Blanquet, Véronique; Maftah, Abderrahman

2016-09-01

Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1(cax/cax) (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1(cax/cax) mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1(cax/cax) SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7(+)/MYOD(-) progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice. © 2016 The Authors.

Reduced Notch signalling leads to postnatal skeletal muscle hypertrophy in Pofut1cax/cax mice

PubMed Central

Al Jaam, Bilal; Heu, Katy; Pennarubia, Florian; Segelle, Alexandre; Magnol, Laetitia; Germot, Agnès; Blanquet, Véronique; Maftah, Abderrahman

2016-01-01

Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O-fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1cax/cax (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1cax/cax mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1cax/cax SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7+/MYOD− progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O-fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice. PMID:27628322

Disregulated expression of the transcription factor ThPOK during T-cell development leads to high incidence of T-cell lymphomas.

PubMed

Lee, Hyung-Ok; He, Xiao; Mookerjee-Basu, Jayati; Zhongping, Dai; Hua, Xiang; Nicolas, Emmanuelle; Sulis, Maria Luisa; Ferrando, Adolfo A; Testa, Joseph R; Kappes, Dietmar J

2015-06-23

The transcription factor T-helper-inducing POZ/Krueppel-like factor (ThPOK, encoded by the Zbtb7b gene) plays widespread and critical roles in T-cell development, particularly as the master regulator of CD4 commitment. Here we show that mice expressing a constitutive T-cell-specific ThPOK transgene (ThPOK(const) mice) develop thymic lymphomas. These tumors resemble human T-cell acute lymphoblastic leukemia (T-ALL), in that they predominantly exhibit activating Notch1 mutations. Lymphomagenesis is prevented if thymocyte development is arrested at the DN3 stage by recombination-activating gene (RAG) deficiency, but restored by introduction of a T-cell receptor (TCR) transgene or by a single injection of anti-αβTCR antibody into ThPOK(const) RAG-deficient mice, which promotes development to the CD4(+)8(+) (DP) stage. Hence, TCR signals and/or traversal of the DN (double negative) > DP (double positive) checkpoint are required for ThPOK-mediated lymphomagenesis. These results demonstrate a novel link between ThPOK, TCR signaling, and lymphomagenesis. Finally, we present evidence that ectopic ThPOK expression gives rise to a preleukemic and self-perpetuating DN4 lymphoma precursor population. Our results collectively define a novel role for ThPOK as an oncogene and precisely map the stage in thymopoiesis susceptible to ThPOK-dependent tumor initiation.

Biochemical and Expression Analyses of the Rice Cinnamoyl-CoA Reductase Gene Family.

PubMed

Park, Hye Lin; Bhoo, Seong Hee; Kwon, Mi; Lee, Sang-Won; Cho, Man-Ho

2017-01-01

Cinnamoyl-CoA reductase (CCR) is the first committed enzyme in the monolignol pathway for lignin biosynthesis and catalyzes the conversion of hydroxycinnamoyl-CoAs into hydroxycinnamaldehydes. In the rice genome, 33 genes are annotated as CCR and CCR-like genes, collectively called OsCCR s. To elucidate the functions of OsCCR s, their phylogenetic relationships, expression patterns at the transcription levels and biochemical characteristics were thoroughly analyzed. Of the 33 OsCCR s, 24 of them encoded polypeptides of lengths similar to those of previously identified plant CCRs. The other nine OsCCRs had much shorter peptide lengths. Phylogenetic tree and sequence similarities suggested OsCCR4, 5, 17, 18, 19, 20, and 21 as likely candidates for functional CCRs in rice. To elucidate biochemical functions, OsCCR1, 5, 17, 19, 20, 21, and 26 were heterologously expressed in Escherichia coli and the resulting recombinant OsCCRs were purified to apparent homogeneity. Activity assays of the recombinant OsCCRs with hydroxycinnamoyl-CoAs revealed that OsCCR17, 19, 20, and 21 were biochemically active CCRs, in which the NAD(P)-binding and NADP-specificity motifs as well as the CCR signature motif were fully conserved. The kinetic parameters of enzyme reactions revealed that feruloyl-CoA, a precursor for the guaiacyl (G)-unit of lignin, is the most preferred substrate of OsCCR20 and 21. This result is consistent with a high content (about 70%) of G-units in rice lignins. Phylogenetic analysis revealed that OsCCR19 and 20 were grouped with other plant CCRs involved in developmental lignification, whereas OsCCR17 and 21 were closely related to stress-responsible CCRs identified from other plant species. In agreement with the phylogenetic analysis, expression analysis demonstrated that OsCCR20 was constitutively expressed throughout the developmental stages of rice, showing particularly high expression levels in actively lignifying tissues, such as roots and stems. These results suggest that OsCCR20 is primarily involved in developmental deposition of lignins in secondary cell walls. As expected, the expressions of OsCCR17 and 21 were induced in response to biotic and abiotic stresses, such as Magnaporthe grisea and Xanthomonas oryzae pv. oryzae ( Xoo ) infections, UV-irradiation and high salinity, suggesting that these genes play a role in defense-related processes in rice.

Evolution and development of the vertebrate ear

NASA Technical Reports Server (NTRS)

Fritzsch, B.; Beisel, K. W.

2001-01-01

This review outlines major aspects of development and evolution of the ear, specifically addressing issues of cell fate commitment and the emerging molecular governance of these decisions. Available data support the notion of homology of subsets of mechanosensors across phyla (proprioreceptive mechanosensory neurons in insects, hair cells in vertebrates). It is argued that this conservation is primarily related to the specific transducing environment needed to achieve mechanosensation. Achieving this requires highly conserved transcription factors that regulate the expression of the relevant structural genes for mechanosensory transduction. While conserved at the level of some cell fate assignment genes (atonal and its mammalian homologue), the ear has also radically reorganized its development by implementing genes used for cell fate assignment in other parts of the developing nervous systems (e.g., neurogenin 1) and by evolving novel sets of genes specifically associated with the novel formation of sensory neurons that contact hair cells (neurotrophins and their receptors). Numerous genes have been identified that regulate morphogenesis, but there is only one common feature that emerges at the moment: the ear appears to have co-opted genes from a large variety of other parts of the developing body (forebrain, limbs, kidneys) and establishes, in combination with existing transcription factors, an environment in which those genes govern novel, ear-related morphogenetic aspects. The ear thus represents a unique mix of highly conserved developmental elements combined with co-opted and newly evolved developmental elements.

Lamin A/C Haploinsufficiency Modulates the Differentiation Potential of Mouse Embryonic Stem Cells

PubMed Central

Sehgal, Poonam; Chaturvedi, Pankaj; Kumaran, R. Ileng; Kumar, Satish; Parnaik, Veena K.

2013-01-01

Background Lamins are structural proteins that are the major determinants of nuclear architecture and play important roles in various nuclear functions including gene regulation and cell differentiation. Mutations in the human lamin A gene cause a spectrum of genetic diseases that affect specific tissues. Most available mouse models for laminopathies recapitulate disease symptoms for muscle diseases and progerias. However, loss of human lamin A/C also has highly deleterious effects on fetal development. Hence it is important to understand the impact of lamin A/C expression levels on embryonic differentiation pathways. Methodology and Principal Findings We have investigated the differentiation potential of mouse embryonic stem cells containing reduced levels of lamin A/C by detailed lineage analysis of embryoid bodies derived from these cells by in vitro culture. We initially carried out a targeted disruption of one allele of the mouse lamin A/C gene (Lmna). Undifferentiated wild-type and Lmna+/− embryonic stem cells showed similar expression of pluripotency markers and cell cycle profiles. Upon spontaneous differentiation into embryoid bodies, markers for visceral endoderm such as α-fetoprotein were highly upregulated in haploinsufficient cells. However, neuronal markers such as β-III tubulin and nestin were downregulated. Furthermore, we observed a reduction in the commitment of Lmna+/− cells into the myogenic lineage, but no discernible effects on cardiac, adipocyte or osteocyte lineages. In the next series of experiments, we derived embryonic stem cell clones expressing lamin A/C short hairpin RNA and examined their differentiation potential. These cells expressed pluripotency markers and, upon differentiation, the expression of lineage-specific markers was altered as observed with Lmna+/− embryonic stem cells. Conclusions We have observed significant effects on embryonic stem cell differentiation to visceral endoderm, neuronal and myogenic lineages upon depletion of lamin A/C. Hence our results implicate lamin A/C level as an important determinant of lineage-specific differentiation during embryonic development. PMID:23451281

Notch-Induced Expression of FZD7 Requires Noncanonical NOTCH3 Signaling in Human Breast Epithelial Cells.

PubMed

Bhat, Vasudeva; Sun, Yu Jia; Weger, Steve; Raouf, Afshin

2016-04-01

The evolutionarily conserved Notch and Wnt signaling pathways have demonstrated roles in normal mammary gland development and in breast carcinogenesis. We previously reported that in human mammary gland, signaling through NOTCH3 alone regulates the commitment of the undifferentiated bipotential progenitors to the luminal cell fate, indicating that NOTCH3 may regulate the expression of unique genes apart from the other Notch receptors. In this study, we used gain of function and loss of function experiments and found that a Wnt signaling receptor, Frizzled7 (FZD7), is a unique and nonredundant target of NOTCH3 in human breast epithelial cells. Interestingly, neither the constitutively active forms of NOTCH1-2, 4 nor loss of expression of these receptors were able to alter expression of FZD7 in human breast epithelial cells. We further show that FZD7-expressing cells are found more frequently in the luminal progenitor-enriched subpopulation of cells obtained from breast reduction samples compared with the undifferentiated bipotent progenitors. Also, we show that NOTCH3-induced expression of FZD7 occurs in the absence of CSL (CBF1-Suppressor of Hairless-Lag-1). Our data suggest that noncanonical Notch signaling through NOTCH3 could modulate Wnt signaling via FZD7 and in this way, might be involved in luminal cell differentiation.

Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate

PubMed Central

Gomez, Danielle L.; O’Driscoll, Marci; Sheets, Timothy P.; Hruban, Ralph H.; Oberholzer, Jose; McGarrigle, James J.; Shamblott, Michael J.

2015-01-01

Neurogenin 3 (NGN3) is necessary and sufficient for endocrine differentiation during pancreatic development and is expressed by a population of progenitor cells that give rise exclusively to hormone-secreting cells within islets. NGN3 protein can be detected in the adult rodent pancreas only following certain types of injury, when it is transiently expressed by exocrine cells undergoing reprogramming to an endocrine cell fate. Here, NGN3 protein can be detected in 2% of acinar and duct cells in living biopsies of histologically normal adult human pancreata and 10% in cadaveric biopsies of organ donor pancreata. The percentage and total number of NGN3+ cells increase during culture without evidence of proliferation or selective cell death. Isolation of highly purified and viable NGN3+ cell populations can be achieved based on coexpression of the cell surface glycoprotein CD133. Transcriptome and targeted expression analyses of isolated CD133+ / NGN3+ cells indicate that they are distinct from surrounding exocrine tissue with respect to expression phenotype and Notch signaling activity, but retain high level mRNA expression of genes indicative of acinar and duct cell function. NGN3+ cells have an mRNA expression profile that resembles that of mouse early endocrine progenitor cells. During in vitro differentiation, NGN3+ cells express genes in a pattern characteristic of endocrine development and result in cells that resemble beta cells on the basis of coexpression of insulin C-peptide, chromogranin A and pancreatic and duodenal homeobox 1. NGN3 expression in the adult human exocrine pancreas marks a dedifferentiating cell population with the capacity to take on an endocrine cell fate. These cells represent a potential source for the treatment of diabetes either through ex vivo manipulation, or in vivo by targeting mechanisms controlling their population size and endocrine cell fate commitment. PMID:26288179

Epigenetic Plasticity Drives Adipogenic and Osteogenic Differentiation of Marrow-derived Mesenchymal Stem Cells*

PubMed Central

Meyer, Mark B.; Benkusky, Nancy A.; Sen, Buer; Rubin, Janet; Pike, J. Wesley

2016-01-01

Terminal differentiation of multipotent stem cells is achieved through a coordinated cascade of activated transcription factors and epigenetic modifications that drive gene transcription responsible for unique cell fate. Within the mesenchymal lineage, factors such as RUNX2 and PPARγ are indispensable for osteogenesis and adipogenesis, respectively. We therefore investigated genomic binding of transcription factors and accompanying epigenetic modifications that occur during osteogenic and adipogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (MSCs). As assessed by ChIP-sequencing and RNA-sequencing analyses, we found that genes vital for osteogenic identity were linked to RUNX2, C/EBPβ, retinoid X receptor, and vitamin D receptor binding sites, whereas adipocyte differentiation favored PPARγ, retinoid X receptor, C/EBPα, and C/EBPβ binding sites. Epigenetic marks were clear predictors of active differentiation loci as well as enhancer activities and selective gene expression. These marrow-derived MSCs displayed an epigenetic pattern that suggested a default preference for the osteogenic pathway; however, these patterns were rapidly altered near the Adipoq, Cidec, Fabp4, Lipe, Plin1, Pparg, and Cebpa genes during adipogenic differentiation. Surprisingly, we found that these cells also exhibited an epigenetic plasticity that enabled them to trans-differentiate from adipocytes to osteoblasts (and vice versa) after commitment, as assessed by staining, gene expression, and ChIP-quantitative PCR analysis. The osteogenic default pathway may be subverted during pathological conditions, leading to skeletal fragility and increased marrow adiposity during aging, estrogen deficiency, and skeletal unloading. Taken together, our data provide an increased mechanistic understanding of the epigenetic programs necessary for multipotent differentiation of MSCs that may prove beneficial in the development of therapeutic strategies. PMID:27402842

Proteomic Cornerstones of Hematopoietic Stem Cell Differentiation: Distinct Signatures of Multipotent Progenitors and Myeloid Committed Cells*

PubMed Central

Klimmeck, Daniel; Hansson, Jenny; Raffel, Simon; Vakhrushev, Sergey Y.; Trumpp, Andreas; Krijgsveld, Jeroen

2012-01-01

Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem/progenitor cells (HSPCs, LinnegSca-1+c-Kit+) or myeloid committed precursors (LinnegSca-1−c-Kit+). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical evaluation, 893 proteins were found differentially expressed between multipotent and myeloid committed cells. The differential protein content in these cell populations points to a distinct structural organization of the cytoskeleton including remodeling activity. In addition, we found a marked difference in the expression of metabolic enzymes, including a clear shift of specific protein isoforms of the glycolytic pathway. Proteins involved in translation showed a collective higher expression in myeloid progenitors, indicating an increased translational activity. Strikingly, the data uncover a unique signature related to immune defense mechanisms, centering on the RIG-I and type-1 interferon response systems, which are installed in multipotent progenitors but not evident in myeloid committed cells. This suggests that specific, and so far unrecognized, mechanisms protect these immature cells before they mature. In conclusion, this study indicates that the transition of hematopoietic stem/progenitors toward myeloid commitment is accompanied by a profound change in processing of cellular resources, adding novel insights into the molecular mechanisms at the interface between multipotency and lineage commitment. PMID:22454540

Recombinant yeast as a functional tool for understanding bitterness and cucurbitacin biosynthesis in watermelon (Citrullus spp.).

PubMed

Davidovich-Rikanati, Rachel; Shalev, Lior; Baranes, Nadine; Meir, Ayala; Itkin, Maxim; Cohen, Shahar; Zimbler, Kobi; Portnoy, Vitaly; Ebizuka, Yutaka; Shibuya, Masaaki; Burger, Yosef; Katzir, Nurit; Schaffer, Arthur A; Lewinsohn, Efraim; Tadmor, Ya'akov

2015-01-01

Cucurbitacins are a group of bitter-tasting oxygenated tetracyclic triterpenes that are produced in the family Cucurbitaceae and other plant families. The natural roles of cucurbitacins in plants are probably related to defence against pathogens and pests. Cucurbitadienol, a triterpene synthesized from oxidosqualene, is the first committed precursor to cucurbitacins produced by a specialized oxidosqualene cyclase termed cucurbitadienol synthase. We explored cucurbitacin accumulation in watermelon in relation to bitterness. Our findings show that cucurbitacins are accumulated in bitter-tasting watermelon, Citrullus lanatus var. citroides, as well as in their wild ancestor, C. colocynthis, but not in non-bitter commercial cultivars of sweet watermelon (C. lanatus var. lanatus). Molecular analysis of genes expressed in the roots of several watermelon accessions led to the isolation of three sequences (CcCDS1, CcCDS2 and ClCDS1), all displaying high similarity to the pumpkin CpCPQ, encoding a protein previously shown to possess cucurbitadienol synthase activity. We utilized the Saccharomyces cerevisiae strain BY4743, heterozygous for lanosterol synthase, to probe for possible encoded cucurbitadienol synthase activity of the expressed watermelon sequences. Functional expression of the two sequences isolated from C. colocynthis (CcCDS1 and CcCDS2) in yeast revealed that only CcCDS2 possessed cucurbitadienol synthase activity, while CcCDS1 did not display cucurbitadienol synthase activity in recombinant yeast. ClCDS1 isolated from C. lanatus var. lanatus is almost identical to CcCDS1. Our results imply that CcCDS2 plays a role in imparting bitterness to watermelon. Yeast has been an excellent diagnostic tool to determine the first committed step of cucurbitacin biosynthesis in watermelon. Copyright © 2014 John Wiley & Sons, Ltd.

The political economy of health promotion: part 2, national provision of the prerequisites of health.

PubMed

Raphael, Dennis

2013-03-01

Governmental authorities of wealthy developed nations differ in their professed commitments and activity related to the provision of the prerequisites of health through public policy action. Part 1 of this article showed how nations identified as social democratic or liberal welfare states were those where such commitments are present. Nations identified as conservative or Latin welfare states were less likely to express such commitments. However, the political economy literature suggests that despite their expressed commitments to provision of the prerequisites of health, liberal welfare states fare rather poorly in implementing these commitments. The opposite is seen for conservative welfare states. Social democratic welfare states show both commitments and public policy consistent with this objective. Part 2 of this article documents the extent to which public policy activity that provides the prerequisites of health through public policy action differs among varying welfare state regimes. Despite extensive rhetoric concerning the prerequisites of health, nations identified as liberal welfare states do a rather poor job of meeting these goals and show evidence of adverse health outcomes. In contrast, social democratic welfare states fare better in providing such prerequisites--consistent with their rhetorical statements--with better health outcomes. Interestingly, conservative--and to a lesser extent Latin--nations fare well in providing the prerequisites of health despite their lack of explicit commitment to such concepts. Findings suggest that health promoters have to concern themselves with the broad strokes of public policymaking whether or not these policy activities are identified as health promotion activities.

Microarray Analyses Reveal Marked Differences in Growth Factor and Receptor Expression Between 8-Cell Human Embryos and Pluripotent Stem Cells

PubMed Central

Vlismas, Antonis; Bletsa, Ritsa; Mavrogianni, Despina; Mamali, Georgina; Pergamali, Maria; Dinopoulou, Vasiliki; Partsinevelos, George; Drakakis, Peter; Loutradis, Dimitris

2016-01-01

Previous microarray analyses of RNAs from 8-cell (8C) human embryos revealed a lack of cell cycle checkpoints and overexpression of core circadian oscillators and cell cycle drivers relative to pluripotent human stem cells [human embryonic stem cells/induced pluripotent stem (hES/iPS)] and fibroblasts, suggesting growth factor independence during early cleavage stages. To explore this possibility, we queried our combined microarray database for expression of 487 growth factors and receptors. Fifty-one gene elements were overdetected on the 8C arrays relative to hES/iPS cells, including 14 detected at least 80-fold higher, which annotated to multiple pathways: six cytokine family (CSF1R, IL2RG, IL3RA, IL4, IL17B, IL23R), four transforming growth factor beta (TGFB) family (BMP6, BMP15, GDF9, ENG), one fibroblast growth factor (FGF) family [FGF14(FH4)], one epidermal growth factor member (GAB1), plus CD36, and CLEC10A. 8C-specific gene elements were enriched (73%) for reported circadian-controlled genes in mouse tissues. High-level detection of CSF1R, ENG, IL23R, and IL3RA specifically on the 8C arrays suggests the embryo plays an active role in blocking immune rejection and is poised for trophectoderm development; robust detection of NRG1, GAB1, -2, GRB7, and FGF14(FHF4) indicates novel roles in early development in addition to their known roles in later development. Forty-four gene elements were underdetected on the 8C arrays, including 11 at least 80-fold under the pluripotent cells: two cytokines (IFITM1, TNFRSF8), five TGFBs (BMP7, LEFTY1, LEFTY2, TDGF1, TDGF3), two FGFs (FGF2, FGF receptor 1), plus ING5, and WNT6. The microarray detection patterns suggest that hES/iPS cells exhibit suppressed circadian competence, underexpression of early differentiation markers, and more robust expression of generic pluripotency genes, in keeping with an artificial state of continual uncommitted cell division. In contrast, gene expression patterns of the 8C embryo suggest that it is an independent circadian rhythm-competent equivalence group poised to signal its environment, defend against maternal immune rejection, and begin the rapid commitment events of early embryogenesis. PMID:26493868

MicroRNA-132 targets HB-EGF upon IgE-mediated activation in murine and human mast cells.

PubMed

Molnár, Viktor; Érsek, Barbara; Wiener, Zoltán; Tömböl, Zsófia; Szabó, Péter M; Igaz, Péter; Falus, András

2012-03-01

MicroRNAs provide an additional layer in the regulation of gene expression acting as repressors with several targets at the posttranscriptional level. This study describes microRNA expression patterns during differentiation and activation of mast cells. The expression levels of 567 different mouse miRNAs were compared by microarray between c-Kit+ committed progenitors, mucosal mast cells, resting and IgE-crosslinked BMMCs in vitro. The strongest upregulation of miR-132 upon IgE-mediated activation was validated in human cord blood-derived mast cells as well. HB-EGF growth factor also upregulated upon activation and was ranked high by more prediction algorithms. Co-transfection of miR-132 mimicking precursor and the 3'UTR of human Hbegf-containing luciferase vector proves that the predicted binding site is functional. In line with this, neutralization of miR-132 by anti-miR inhibitor leads to sustained production of HB-EGF protein in activated mast cells. Our data provide a novel example for negative regulation of a growth factor by an upregulated miRNA. © Springer Basel AG 2011

Brief report: ectopic expression of NUP98-HOXA10 augments erythroid differentiation of human embryonic stem cells.

PubMed

Ji, Junfeng; Risueño, Ruth M; Hong, Seokho; Allan, David; Rosten, Patty; Humphries, Keith; Bhatia, Mickie

2011-04-01

Hox genes encode highly conserved transcription factors that have been implicated in hematopoietic development and self-renewal of hematopoietic stem cells (HSCs) and hematopoietic development. The potency of NUP98-HOXA10hd (NA10) on adult murine bone marrow HSC self-renewal prompted us to examine its effect on specification and proliferation of hematopoietic cells derived from human embryonic stem cells (hESCs). Here, we demonstrate that expression of NA10 in hESCs influences the hematopoietic differentiation program. The specific effect of NA10 is dependent on the developmental stage of hematopoietic emergence from hESCs. Overexpression of NA10 in either undifferentiated hESCs or early hemogenic precursors augmented the frequency of CD45(-) GlycophorinA(+) cells and erythroid progenitors (blast-forming unit-erythrocyte). In contrast, targeted NA10 expression in primitive CD34+ cells committed to the hematopoietic lineage had no effect on erythropoietic capacity but instead increased hematopoietic progenitor proliferation. Our study reveals a novel neomorphic effect of NA10 in early human erythroid development from pluripotent stem cells. Copyright © 2011 AlphaMed Press.

Staff nurse commitment, work relationships, and turnover intentions: a latent profile analysis.

PubMed

Gellatly, Ian R; Cowden, Tracy L; Cummings, Greta G

2014-01-01

The three-component model of organization commitment has typically been studied using a variable-centered rather than a person-centered approach, preventing a more complete understanding of how these forms of commitment are felt and expressed as a whole. Latent profile analysis was used to identify qualitatively distinct categories or profiles of staff nurses' commitment. Then, associations of the profiles with perceived work unit relations and turnover intentions were examined. Three hundred thirty-six registered nurses provided data on affective, normative, and continuance commitment, perceived work unit relations, and turnover intentions. Latent profile analysis of the nurses' commitment scores revealed six distinct profile groups. Work unit relations and turnover intentions were compared in the six profile-defined groups. Staff nurses with profiles characterized by high affective commitment and/or high normative commitment in relation to other components experienced stronger work unit relations and reported lower turnover intentions. Profiles characterized by high continuance commitment relative to other components or by low overall commitment experienced poorer work unit relations, and the turnover risk was higher. High continuance commitment in combination with high affective and normative commitment was experienced differently than high continuance commitment in combination with low affective and normative commitment. Healthcare organizations often foster commitment by using continuance commitment-enhancing strategies (e.g., offer high salaries and attractive benefits) that may inadvertently introduce behavioral risk. This work suggests the importance of changing the context in which continuance commitment occurs by strengthening the other two components.

Integrins engage mitochondrial function for signal transduction by a mechanism dependent on Rho GTPases

PubMed Central

Werner, Erica; Werb, Zena

2002-01-01

We show here the transient activation of the small GTPase Rac, followed by a rise in reactive oxygen species (ROS), as necessary early steps in a signal transduction cascade that lead to NFκB activation and collagenase-1 (CL-1)/matrix metalloproteinase-1 production after integrin-mediated cell shape changes. We show evidence indicating that this constitutes a new mechanism for ROS production mediated by small GTPases. Activated RhoA also induced ROS production and up-regulated CL-1 expression. A Rac mutant (L37) that prevents reorganization of the actin cytoskeleton prevented integrin-induced CL-1 expression, whereas mutations that abrogate Rac binding to the neutrophil NADPH membrane oxidase in vitro (H26 and N130) did not. Instead, ROS were produced by integrin-induced changes in mitochondrial function, which were inhibited by Bcl-2 and involved transient membrane potential loss. The cells showing this transient decrease in mitochondrial membrane potential were already committed to CL-1 expression. These results unveil a new molecular mechanism of signal transduction triggered by integrin engagement where a global mitochondrial metabolic response leads to gene expression rather than apoptosis. PMID:12119354

Integrins engage mitochondrial function for signal transduction by a mechanism dependent on Rho GTPases.

PubMed

Werner, Erica; Werb, Zena

2002-07-22

We show here the transient activation of the small GTPase Rac, followed by a rise in reactive oxygen species (ROS), as necessary early steps in a signal transduction cascade that lead to NFkappaB activation and collagenase-1 (CL-1)/matrix metalloproteinase-1 production after integrin-mediated cell shape changes. We show evidence indicating that this constitutes a new mechanism for ROS production mediated by small GTPases. Activated RhoA also induced ROS production and up-regulated CL-1 expression. A Rac mutant (L37) that prevents reorganization of the actin cytoskeleton prevented integrin-induced CL-1 expression, whereas mutations that abrogate Rac binding to the neutrophil NADPH membrane oxidase in vitro (H26 and N130) did not. Instead, ROS were produced by integrin-induced changes in mitochondrial function, which were inhibited by Bcl-2 and involved transient membrane potential loss. The cells showing this transient decrease in mitochondrial membrane potential were already committed to CL-1 expression. These results unveil a new molecular mechanism of signal transduction triggered by integrin engagement where a global mitochondrial metabolic response leads to gene expression rather than apoptosis.

Comparative epigenetic influence of autologous versus fetal bovine serum on mesenchymal stem cells through in vitro osteogenic and adipogenic differentiation

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

Fani, Nesa; Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran; Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran

Mesenchymal stem cells (MSCs) derived from bone marrow (BM) represents a useful source of adult stem cells for cell therapy and tissue engineering. MSCs are present at a low frequency in the BM; therefore expansion is necessary before performing clinical studies. Fetal bovine serum (FBS) as a nutritional supplement for in vitro culture of MSCs is a suitable additive for human cell culture, but not regarding subsequent use of these cells for clinical treatment of human patients due to the risk of viral and prion transmission as well as xenogeneic immune responses after transplantation. Recently, autologous serum (AS) has beenmore » as a supplement to replace FBS in culture medium. We compared the effect of FBS versus AS on the histone modification pattern of MSCs through in vitro osteogenesis and adipogenesis. Differentiation of stem cells under various serum conditions to a committed state involves global changes in epigenetic patterns that are critically determined by chromatin modifications. Chromatin immunoprecipitation (ChIP) coupled with real-time PCR showed significant changes in the acetylation and methylation patterns in lysine 9 (Lys9) of histone H3 on the regulatory regions of stemness (Nanog, Sox2, Rex1), osteogenic (Runx2, Oc, Sp7) and adipogenic (Ppar-γ, Lpl, adiponectin) marker genes in undifferentiated MSCs, FBS and AS. All epigenetic changes occurred in a serum dependent manner which resulted in higher expression level of stemness genes in undifferentiated MSCs compared to differentiated MSCs and increased expression levels of osteogenic genes in AS compared to FBS. Adipogenic genes showed greater expression in FBS compared to AS. These findings have demonstrated the epigenetic influence of serum culture conditions on differentiation potential of MSCs, which suggest that AS is possibly more efficient serum for osteogenic differentiation of MSCs in cell therapy purposes. - Highlights: • Bone marrow derived MSC could proliferate in AS as well as in FBS. • Different serum conditions influence epigentic patterns of genes. • Osteogenic genes specifically up-regulate in AS in response to differentiation signals.« less

Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism

PubMed Central

Chen, Julia C.; Hoey, David A.; Chua, Mardonn; Bellon, Raymond; Jacobs, Christopher R.

2016-01-01

It has long been suspected, but never directly shown, that bone formed to accommodate an increase in mechanical loading is related to the creation of osteoblasts from skeletal stem cells. Indeed, biophysical stimuli potently regulate osteogenic lineage commitment in vitro. In this study, we transplanted bone marrow cells expressing green fluorescent protein, to enable lineage tracing, and subjected mice to a biophysical stimulus, to elicit a bone-forming response. We detected cells derived from transplanted progenitors embedded within the bone matrix near active bone-forming surfaces in response to loading, demonstrating for the first time, that mechanical signals enhance the homing and attachment of bone marrow cells to bone surfaces and the commitment to an osteogenic lineage of these cells in vivo. Furthermore, we used an inducible Cre/Lox recombination system to delete kinesin family member 3A (Kif3a), a gene that is essential for primary cilia formation, at will in transplanted cells and their progeny, regardless of which tissue may have incorporated them. Disruption of the mechanosensing organelle, the primary cilium in a progenitor population, significantly decreased the amount of bone formed in response to mechanical stimulation. The collective results of our study directly demonstrate that, in a novel experimental stem cell mechanobiology model, mechanical signals enhance osteogenic lineage commitment in vivo and that the primary cilium contributes to this process.—Chen, J. C., Hoey, D. A., Chua, M., Bellon, R., Jacobs, C. R. Mechanical signals promote osteogenic fate through a primary cilia-mediated mechanism. PMID:26675708

The Obligation to Provide Assistive Technology: Enhancing the General Curriculum Access.

ERIC Educational Resources Information Center

Smith, Sean J.; Jones, Eric D.

1999-01-01

Describes public commitment (expressed in federal legislation) to using assistive technology devices and services in K-12 special education and discusses approaches for addressing obstacles in meeting this commitment. Reauthorization of the Individuals with Disabilities Act represents progress toward integrating assistive technology into disabled…

Impaired preadipocyte differentiation into adipocytes in subcutaneous abdominal adipose of PCOS-like female rhesus monkeys.

PubMed

Keller, Erica; Chazenbalk, Gregorio D; Aguilera, Paul; Madrigal, Vanessa; Grogan, Tristan; Elashoff, David; Dumesic, Daniel A; Abbott, David H

2014-07-01

Metabolic characteristics of polycystic ovary syndrome women and polycystic ovary syndrome-like, prenatally androgenized (PA) female monkeys worsen with age, with altered adipogenesis of sc abdominal adipose potentially contributing to age-related adverse effects on metabolism. This study examines whether adipocyte morphology and gene expression in sc abdominal adipose differ between late reproductive-aged PA female rhesus monkeys compared with age-matched controls (C). Subcutaneous abdominal adipose of both groups was obtained for histological imaging and mRNA determination of zinc finger protein 423 (Zfp423) as a marker of adipose stem cell commitment to preadipocytes, and CCAAT/enhancer binding protein (C/EBP)α/peroxisome proliferator-activated receptor (PPAR)δ as well as C/EBPα/PPARγ as respective markers of early- and late-stage differentiation of preadipocytes to adipocytes. In all females combined, serum testosterone (T) levels positively correlated with fasting serum levels of total free fatty acid (r(2) = 0.73, P < .002). PA females had a greater population of small adipocytes vs C (P < .001) in the presence of increased Zfp423 (P < .025 vs C females) and decreased C/EBPα (P < .003, vs C females) mRNA expression. Moreover, Zfp423 mRNA expression positively correlated with circulating total free fatty acid levels during iv glucose tolerance testing (P < .004, r(2) = 0.66), whereas C/EBPα mRNA expression negatively correlated with serum T levels (P < .02, r(2) = 0.43). Gene expression of PPARδ and PPARγ were comparable between groups (P = .723 and P = .18, respectively). Early-to-mid gestational T excess in female rhesus monkeys impairs adult preadipocyte differentiation to adipocytes in sc abdominal adipose and may constrain the ability of this adipose depot to safely store fat with age.

Phenotypic and gene expression modification with normal brain aging in GFAP-positive astrocytes and neural stem cells.

PubMed

Bernal, Giovanna M; Peterson, Daniel A

2011-06-01

Astrocytes secrete growth factors that are both neuroprotective and supportive for the local environment. Identified by glial fibrillary acidic protein (GFAP) expression, astrocytes exhibit heterogeneity in morphology and in the expression of phenotypic markers and growth factors throughout different adult brain regions. In adult neurogenic niches, astrocytes secrete vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) within the neurogenic niche and are also a source of special GFAP-positive multipotent neural stem cells (NSCs). Normal aging is accompanied by a decline in CNS function and reduced neurogenesis. We asked whether a decreased availability of astrocyte-derived factors may contribute to the age-related decline in neurogenesis. Determining alterations of astrocytic activity in the aging brain is crucial for understanding CNS homeostasis in aging and for assessing appropriate therapeutic targets for an aging population. We found region-specific alterations in the gene expression of GFAP, VEGF, and FGF-2 and their receptors in the aged brain corresponding to changes in astrocytic reactivity, supporting astrocytic heterogeneity and demonstrating a differential aging effect. We found that GFAP-positive NSCs uniquely coexpress both VEGF and its key mitotic receptor Flk-1 in both young and aged hippocampus, indicating a possible autocrine/paracrine signaling mechanism. VEGF expression is lost once NSCs commit to a neuronal fate, but Flk-1-mediated sensitivity to VEGF signaling is maintained. We propose that age-related astrocytic changes result in reduced VEGF and FGF-2 signaling, which in turn limits NSC and progenitor cell maintenance and contributes to decreased neurogenesis. © 2011 The Authors. Aging Cell © 2011 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

Characterization and Expression Analysis of Phytoene Synthase from Bread Wheat (Triticum aestivum L.)

PubMed Central

Flowerika; Alok, Anshu; Kumar, Jitesh; Thakur, Neha; Pandey, Ashutosh; Pandey, Ajay Kumar; Upadhyay, Santosh Kumar; Tiwari, Siddharth

2016-01-01

Phytoene synthase (PSY) regulates the first committed step of the carotenoid biosynthetic pathway in plants. The present work reports identification and characterization of the three PSY genes (TaPSY1, TaPSY2 and TaPSY3) in wheat (Triticum aestivum L.). The TaPSY1, TaPSY2, and TaPSY3 genes consisted of three homoeologs on the long arm of group 7 chromosome (7L), short arm of group 5 chromosome (5S), and long arm of group 5 chromosome (5L), respectively in each subgenomes (A, B, and D) with a similarity range from 89% to 97%. The protein sequence analysis demonstrated that TaPSY1 and TaPSY3 retain most of conserved motifs for enzyme activity. Phylogenetic analysis of all TaPSY revealed an evolutionary relationship among PSY proteins of various monocot species. TaPSY derived from A and D subgenomes shared proximity to the PSY of Triticum urartu and Aegilops tauschii, respectively. The differential expression of TaPSY1, TaPSY2, and TaPSY3 in the various tissues, seed development stages, and stress treatments suggested their role in plant development, and stress condition. TaPSY3 showed higher expression in all tissues, followed by TaPSY1. The presence of multiple stress responsive cis-regulatory elements in promoter region of TaPSY3 correlated with the higher expression during drought and heat stresses has suggested their role in these conditions. The expression pattern of TaPSY3 was correlated with the accumulation of β-carotene in the seed developmental stages. Bacterial complementation assay has validated the functional activity of each TaPSY protein. Hence, TaPSY can be explored in developing genetically improved wheat crop. PMID:27695116

Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling.

PubMed

Kukolj, Tamara; Trivanović, Drenka; Djordjević, Ivana Okić; Mojsilović, Slavko; Krstić, Jelena; Obradović, Hristina; Janković, Srdja; Santibanez, Juan Francisco; Jauković, Aleksandra; Bugarski, Diana

2018-01-01

Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast-like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF-β, fibronectin (FN), α-SMA, and NG2. LPS also increased protein and gene expression levels of anti-inflammatory COX-2 and pro-inflammatory IL-6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS-treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen-stimulated proliferation of CD4 + and the ratio of CD4 + CD25 high /CD4 + CD25 low lymphocytes. LPS-treated PDLSCs did not change the frequency of CD34 + and CD45 + cells, but decreased the frequency of CD33 + and CD14 + myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU-GM number. The results indicated that LPS-activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features. © 2017 Wiley Periodicals, Inc.

FOXM1 in sarcoma: role in cell cycle, pluripotency genes and stem cell pathways.

PubMed

Kelleher, Fergal C; O'Sullivan, Hazel

2016-07-05

FOXM1 is a pro-proliferative transcription factor that promotes cell cycle progression at the G1-S, and G2-M transitions. It is activated by phosphorylation usually mediated by successive cyclin - cyclin dependent kinase complexes, and is highly expressed in sarcoma. p53 down regulates FOXM1 and FOXM1 inhibition is also partly dependent on Rb and p21. Abnormalities of p53 or Rb are frequent in sporadic sarcomas with bone or soft tissue sarcoma, accounting for 36% of index cancers in the high penetrance TP53 germline disorder, Li-Fraumeni syndrome.FOXM1 stimulates transcription of pluripotency related genes including SOX2, KLF4, OCT4, and NANOG many of which are important in sarcoma, a disorder of mesenchymal stem cell/ partially committed progenitor cells. In a selected specific, SOX2 is uniformly expressed in synovial sarcoma. Embryonic pathways preferentially used in stem cell such as Hippo, Hedgehog, and Wnt dominate in FOXM1 stoichiometry to alter rates of FOXM1 production or degradation. In undifferentiated pleomorphic sarcoma, liposarcoma, and fibrosarcoma, dysregulation of the Hippo pathway increases expression of the effector co-transcriptional activator Yes-Associated Protein (YAP). A complex involving YAP and the transcription factor TEAD elevates FOXM1 in these sarcoma subtypes. In another scenario 80% of desmoid tumors have nuclear localization of β-catenin, the Wnt pathway effector molecule. Thiazole antibiotics inhibit FOXM1 and because they have an auto-regulator loop FOXM1 expression is also inhibited. Current systemic treatment of sarcoma is of limited efficacy and inhibiting FOXM1 represents a potential new strategy.

3D heterogeneous islet organoid generation from human embryonic stem cells using a novel engineered hydrogel platform.

PubMed

Candiello, Joseph; Grandhi, Taraka Sai Pavan; Goh, Saik Kia; Vaidya, Vimal; Lemmon-Kishi, Maya; Eliato, Kiarash Rahmani; Ros, Robert; Kumta, Prashant N; Rege, Kaushal; Banerjee, Ipsita

2018-05-25

Organoids, which exhibit spontaneous organ specific organization, function, and multi-cellular complexity, are in essence the in vitro reproduction of specific in vivo organ systems. Recent work has demonstrated human pluripotent stem cells (hPSCs) as a viable regenerative cell source for tissue-specific organoid engineering. This is especially relevant for engineering islet organoids, due to the recent advances in generating functional beta-like cells from human pluripotent stem cells. In this study, we report specific engineering of regenerative islet organoids of precise size and cellular heterogeneity, using a novel hydrogel system, Amikagel. Amikagel facilitated controlled and spontaneous aggregation of human embryonic stem cell derived pancreatic progenitor cells (hESC-PP) into robust homogeneous spheroids. This platform further allowed fine control over the integration of multiple cell populations to produce heterogeneous spheroids, which is a necessity for complex organoid engineering. Amikagel induced hESC-PP spheroid formation enhanced pancreatic islet-specific Pdx-1 and NKX6.1 gene and protein expression, while also increasing the percentage of committed population. hESC-PP spheroids were further induced towards mature beta-like cells which demonstrated increased Beta-cell specific INS1 gene and C-peptide protein expression along with functional insulin production in response to in vitro glucose challenge. Further integration of hESC-PP with biologically relevant supporting endothelial cells resulted in multicellular organoids which demonstrated spontaneous maturation towards islet-specific INS1 gene and C-peptide protein expression along with a significantly developed extracellular matrix support system. These findings establish Amikagel -facilitated platform ideal for islet organoid engineering. Copyright © 2018. Published by Elsevier Ltd.

The meta-epigenomic structure of purified human stem cell populations is defined at cis-regulatory sequences

PubMed Central

Zhao, Yong Mei; Golden, Aaron; Mar, Jessica C.; Einstein, Francine H.; Greally, John M.

2014-01-01

The mechanism and significance of epigenetic variability in the same cell type between healthy individuals are not clear. Here, we purify human CD34+ hematopoietic stem and progenitor cells (HSPCs) from different individuals and find that there is increased variability of DNA methylation at loci with properties of promoters and enhancers. The variability is especially enriched at candidate enhancers near genes transitioning between silent and expressed states, and encoding proteins with leukocyte differentiation properties. Our findings of increased variability at loci with intermediate DNA methylation values, at candidate “poised” enhancers, and at genes involved in HSPC lineage commitment suggest that CD34+ cell subtype heterogeneity between individuals is a major mechanism for the variability observed. Epigenomic studies performed on cell populations, even when purified, are testing collections of epigenomes, or meta-epigenomes. Our findings show that meta-epigenomic approaches to data analysis can provide insights into cell subpopulation structure. PMID:25327398

Cosuppression of limonene-3-hydroxylase in peppermint promotes accumulation of limonene in the essential oil.

PubMed

Mahmoud, Soheil S; Williams, Matthew; Croteau, Rodney

2004-03-01

cDNA clones encoding limonene synthase and limonene-3-hydroxylase, both driven by the CaMV 35S promoter, were independently transformed into peppermint (Menthaxpiperita) to alter the production and disposition of (-)-limonene, the first committed intermediate of essential oil biosynthesis in this species. Although both genes were constitutively expressed in leaves of transformed plants, the corresponding enzyme activities were not significantly increased in the glandular trichome sites of essential oil biosynthesis; thus, there was no effect on oil yield or composition in the regenerated plants. Cosuppression of the hydroxylase gene, however, resulted in the accumulation of limonene (up to 80% of the essential oil compared to about 2% of the oil in wild type plants), without influence on oil yield. These results indicate that limonene does not impose negative feedback on the synthase, or apparently influence other enzymes of monoterpene biosynthesis in peppermint, and suggests that pathway engineering can be employed to significantly alter essential oil composition without adverse metabolic consequences.

Generation of stomach tissue from mouse embryonic stem cells.

PubMed

Noguchi, Taka-aki K; Ninomiya, Naoto; Sekine, Mari; Komazaki, Shinji; Wang, Pi-Chao; Asashima, Makoto; Kurisaki, Akira

2015-08-01

Successful pluripotent stem cell differentiation methods have been developed for several endoderm-derived cells, including hepatocytes, β-cells and intestinal cells. However, stomach lineage commitment from pluripotent stem cells has remained a challenge, and only antrum specification has been demonstrated. We established a method for stomach differentiation from embryonic stem cells by inducing mesenchymal Barx1, an essential gene for in vivo stomach specification from gut endoderm. Barx1-inducing culture conditions generated stomach primordium-like spheroids, which differentiated into mature stomach tissue cells in both the corpus and antrum by three-dimensional culture. This embryonic stem cell-derived stomach tissue (e-ST) shared a similar gene expression profile with adult stomach, and secreted pepsinogen as well as gastric acid. Furthermore, TGFA overexpression in e-ST caused hypertrophic mucus and gastric anacidity, which mimicked Ménétrier disease in vitro. Thus, in vitro stomach tissue derived from pluripotent stem cells mimics in vivo development and can be used for stomach disease models.

Leukaemia cell of origin identified by chromatin landscape of bulk tumour cells

PubMed Central

George, Joshy; Uyar, Asli; Young, Kira; Kuffler, Lauren; Waldron-Francis, Kaiden; Marquez, Eladio; Ucar, Duygu; Trowbridge, Jennifer J.

2016-01-01

The precise identity of a tumour's cell of origin can influence disease prognosis and outcome. Methods to reliably define tumour cell of origin from primary, bulk tumour cell samples has been a challenge. Here we use a well-defined model of MLL-rearranged acute myeloid leukaemia (AML) to demonstrate that transforming haematopoietic stem cells (HSCs) and multipotent progenitors results in more aggressive AML than transforming committed progenitor cells. Transcriptome profiling reveals a gene expression signature broadly distinguishing stem cell-derived versus progenitor cell-derived AML, including genes involved in immune escape, extravasation and small GTPase signal transduction. However, whole-genome profiling of open chromatin reveals precise and robust biomarkers reflecting each cell of origin tested, from bulk AML tumour cell sampling. We find that bulk AML tumour cells exhibit distinct open chromatin loci that reflect the transformed cell of origin and suggest that open chromatin patterns may be leveraged as prognostic signatures in human AML. PMID:27397025

Three loci on mouse chromosome 5 and 10 modulate sex determination in XX Ods/+ mice.

PubMed

Poirier, Christophe; Moran, Jennifer L; Kovanci, Ertug; Petit, Deborah C; Beier, David R; Bishop, Colin E

2007-07-01

In mouse, XY embryos are committed to the male sex determination pathway after the transient expression of the Y-linked Sry gene in the Sertoli cell lineage between 10.5 and 12.5 dpc. In the C57BL/6J strain, male sex determination program can be modulated by some autosomal genes. The C57BL/6J alleles at these autosomal loci can antagonize male sex determination in combination with specific Sry alleles. In this report, the authors have identified an effect of these C57BL/6J specific alleles in combination with a mutated Sox9 allele, Sox9(Ods). Authors report the mapping of three of these genetic loci on mouse chromosome 5 and 10 in a backcross of the Ods mutation to the C57BL/6J background. Our study confirms the importance of the strain C57BL/6J for the investigation of the genetic mechanisms that control sex determination.

Motor neurons with limb-innervating character in the cervical spinal cord are sculpted by apoptosis based on the Hox code in chick embryo.

PubMed

Mukaigasa, Katsuki; Sakuma, Chie; Okada, Tomoaki; Homma, Shunsaku; Shimada, Takako; Nishiyama, Keiji; Sato, Noboru; Yaginuma, Hiroyuki

2017-12-15

In the developing chick embryo, a certain population of motor neurons (MNs) in the non-limb-innervating cervical spinal cord undergoes apoptosis between embryonic days 4 and 5. However, the characteristics of these apoptotic MNs remain undefined. Here, by examining the spatiotemporal profiles of apoptosis and MN subtype marker expression in normal or apoptosis-inhibited chick embryos, we found that this apoptotic population is distinguishable by Foxp1 expression. When apoptosis was inhibited, the Foxp1 + MNs survived and showed characteristics of lateral motor column (LMC) neurons, which are of a limb-innervating subtype, suggesting that cervical Foxp1 + MNs are the rostral continuation of the LMC. Knockdown and misexpression of Foxp1 did not affect apoptosis progression, but revealed the role of Foxp1 in conferring LMC identity on the cervical MNs. Furthermore, ectopic expression of Hox genes that are normally expressed in the brachial region prevented apoptosis, and directed Foxp1 + MNs to LMC neurons at the cervical level. These results indicate that apoptosis in the cervical spinal cord plays a role in sculpting Foxp1 + MNs committed to LMC neurons, depending on the Hox expression pattern. © 2017. Published by The Company of Biologists Ltd.

Students' Instructional Dissent and Relationships with Faculty Members' Burnout, Commitment, Satisfaction, and Efficacy

ERIC Educational Resources Information Center

Frisby, Brandi N.; Goodboy, Alan K.; Buckner, Marjorie M.

2015-01-01

Extending research on instructional dissent beyond student reports, this study examined the potential for students' expressed dissent to have deleterious effects on faculty members. Instructors (N = 113) completed surveys about students' instructional dissent regarding their classes and reported their own burnout, commitment, satisfaction, and…

10 CFR 609.8 - Term sheets and conditional commitments.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 10 Energy 4 2012-01-01 2012-01-01 false Term sheets and conditional commitments. 609.8 Section 609.8 Energy DEPARTMENT OF ENERGY (CONTINUED) ASSISTANCE REGULATIONS LOAN GUARANTEES FOR PROJECTS THAT... Project Sponsor and the Eligible Lender or other Holder express agreement with the terms and conditions...

10 CFR 609.8 - Term sheets and conditional commitments.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 10 Energy 4 2013-01-01 2013-01-01 false Term sheets and conditional commitments. 609.8 Section 609.8 Energy DEPARTMENT OF ENERGY (CONTINUED) ASSISTANCE REGULATIONS LOAN GUARANTEES FOR PROJECTS THAT... Project Sponsor and the Eligible Lender or other Holder express agreement with the terms and conditions...

10 CFR 609.8 - Term sheets and conditional commitments.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 4 2010-01-01 2010-01-01 false Term sheets and conditional commitments. 609.8 Section 609.8 Energy DEPARTMENT OF ENERGY (CONTINUED) ASSISTANCE REGULATIONS LOAN GUARANTEES FOR PROJECTS THAT... Project Sponsor and the Eligible Lender or other Holder express agreement with the terms and conditions...

10 CFR 609.8 - Term sheets and conditional commitments.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 10 Energy 4 2014-01-01 2014-01-01 false Term sheets and conditional commitments. 609.8 Section 609.8 Energy DEPARTMENT OF ENERGY (CONTINUED) ASSISTANCE REGULATIONS LOAN GUARANTEES FOR PROJECTS THAT... Project Sponsor and the Eligible Lender or other Holder express agreement with the terms and conditions...

10 CFR 609.8 - Term sheets and conditional commitments.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 4 2011-01-01 2011-01-01 false Term sheets and conditional commitments. 609.8 Section 609.8 Energy DEPARTMENT OF ENERGY (CONTINUED) ASSISTANCE REGULATIONS LOAN GUARANTEES FOR PROJECTS THAT... Project Sponsor and the Eligible Lender or other Holder express agreement with the terms and conditions...

[Cloning, expression and transcriptional analysis of biotin carboxyl carrier protein gene (accA) from Amycolatopsis mediterranei U32 ].

PubMed

Lu, Jie; Yao, Yufeng; Jiang, Weihong; Jiao, Ruishen

2003-02-01

Acetyl CoA carboxylase (EC 6.4.1.2, ACC) catalyzes the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA, which is the first committed step in fatty acid synthesis. A pair of degenerate PCR primers were designed according to the conserved amino acid sequence of AccA from M. tuberculosis and S. coelicolor. The product of the PCR amplification, a DNA fragment of 250bp was used as a probe for screening the U32 genomic cosmid library and its gene, accA, coding the biotinylated protein subunit of acetyl CoA carboxylase, was successfully cloned from U32. The accA ORF encodes a 598-amino-acid protein with the calculated molecular mass of 63.7kD, with 70.1% of G + C content. A typical Streptomyces RBS sequence, AGGAGG, was found at the - 6 position upstream of the start codon GTG. Analysis of the deduced amino acid sequence showed the presence of biotin-binding site and putative ATP-bicarbonate interaction region, which suggested the U32 AccA may act as a biotin carboxylase as well as a biotin carrier protein. Gene accA was then cloned into the pET28 (b) vector and expressed solubly in E. coli BL21 (DE3) by 0.1 mmol/L IPTG induction. Western blot confirmed the covalent binding of biotin with AccA. Northern blot analyzed transcriptional regulation of accA by 5 different nitrogen sources.

Wnt signaling maintains the notochord fate for progenitor cells and supports the posterior extension of the notochord.

PubMed

Ukita, Kanako; Hirahara, Shino; Oshima, Naoko; Imuta, Yu; Yoshimoto, Aki; Jang, Chuan-Wei; Oginuma, Masayuki; Saga, Yumiko; Behringer, Richard R; Kondoh, Hisato; Sasaki, Hiroshi

2009-10-01

The notochord develops from notochord progenitor cells (NPCs) and functions as a major signaling center to regulate trunk and tail development. NPCs are initially specified in the node by Wnt and Nodal signals at the gastrula stage. However, the underlying mechanism that maintains the NPCs throughout embryogenesis to contribute to the posterior extension of the notochord remains unclear. Here, we demonstrate that Wnt signaling in the NPCs is essential for posterior extension of the notochord. Genetic labeling revealed that the Noto-expressing cells in the ventral node contribute the NPCs that reside in the tail bud. Robust Wnt signaling in the NPCs was observed during posterior notochord extension. Genetic attenuation of the Wnt signal via notochord-specific beta-catenin gene ablation resulted in posterior truncation of the notochord. In the NPCs of such mutant embryos, the expression of notochord-specific genes was down-regulated, and an endodermal marker, E-cadherin, was observed. No significant alteration of cell proliferation or apoptosis of the NPCs was detected. Taken together, our data indicate that the NPCs are derived from Noto-positive node cells, and are not fully committed to a notochordal fate. Sustained Wnt signaling is required to maintain the NPCs' notochordal fate.

Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.

PubMed

Sviderskaya, Elena V; Easty, David J; Lawrence, Mark A; Sánchez, Daniel P; Negulyaev, Yuri A; Patel, Ricken H; Anand, Praveen; Korchev, Yuri E; Bennett, Dorothy C

2009-09-01

Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural-crest-related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest-like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage-gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.

In silico analysis of single-cell RNA sequencing data from 3 and 7 days old mouse spermatogonial stem cells to identify their differentially expressed genes and transcriptional regulators.

PubMed

Sisakhtnezhad, Sajjad

2018-05-11

Spermatogonial stem cells (SSCs), which are at the basis of spermatogenesis process, are valuable cells with different applications in biotechnology and regenerative medicine. Understanding the molecular basis of SSC self-renewal and differentiation at various developmental stages of the male organism is crucial to find key factors in the SSCs fate and function. Therefore, this study was aimed to use single-cell RNA-sequencing dataset analysis for identification of differentially expressed genes (DEGs) and their regulators in 3 and 7 days old mouse-derived single SSCs (mSSCs). Results showed 68 upregulated and 203 downregulated genes in 7 days old mouse-derived SSCs compared to 3 days old mSSCs, which were associated with 1493 and 3077 biological processes, respectively. It also found that DAZL, FKBP6, PAIP2, DDX4, H3F3B, TEX15, XRN2, MAEL, and SOD1 are important factors with the higher gene expression pattern, which may be pivotal for mSSCs fate and function during development of germ cells. Moreover, NR3C1, RXRA, NCOA, ESR1, PML, ATF2, BMI1, POU5F1, and CHD1 were the main central regulators for the upregulated DEGs, while HNF1A, C/EBPα, and NFATC1 were the master regulators for the downregulated DEGs. In this regard, two significant protein complexes were found in the protein-protein interactions network for the upregulated DEGs regulators. Furthermore, 24 protein kinases detected upstream of the main central regulators of DEGs. In conclusion, this study presents DEGs and their transcriptional regulators that are crucial for inducing and regulating SSCs commitment during development, and for developing efficient protocols to identify and isolate SSCs for different applications. © 2018 Wiley Periodicals, Inc.

Comparative analysis of single-cell RNA sequencing data from mouse spermatogonial and mesenchymal stem cells to identify differentially expressed genes and transcriptional regulators of germline cells.

PubMed

Sisakhtnezhad, Sajjad; Heshmati, Parvin

2018-07-01

Identifying effective internal factors for regulating germline commitment during development and for maintaining spermatogonial stem cells (SSCs) self-renewal is important to understand the molecular basis of spermatogenesis process, and to develop new protocols for the production of the germline cells from other cell sources. Therefore, this study was designed to investigate single-cell RNA-sequencing data for identification of differentially expressed genes (DEGs) in 12 mouse-derived single SSCs (mSSCs) in compare with 16 mouse-derived single mesenchymal stem cells. We also aimed to find transcriptional regulators of DEGs. Collectively, 1,584 up-regulated DEGs were identified that are associated with 32 biological processes. Moreover, investigation of the expression profiles of genes including in spermatogenesis process revealed that Dazl, Ddx4, Sall4, Fkbp6, Tex15, Tex19.1, Rnf17, Piwil2, Taf7l, Zbtb16, and Cadm1 are presented in the first 30 up-regulated DEGs. We also found 12 basal transcription factors (TFs) and three sequence-specific TFs that control the expression of DEGs. Our findings also indicated that MEIS1, SMC3, TAF1, KAT2A, STAT3, GTF3C2, SIN3A, BDP1, PHC1, and EGR1 are the main central regulators of DEGs in mSSCs. In addition, we collectively detected two significant protein complexes in the protein-protein interactions network for DEGs regulators. Finally, this study introduces the major upstream kinases for the main central regulators of DEGs and the components of core protein complexes. In conclusion, this study provides a molecular blueprint to uncover the molecular mechanisms behind the biology of SSCs and offers a list of candidate factors for cell type conversion approaches and production of germ cells. © 2017 Wiley Periodicals, Inc.

Enhancer of Zeste Homolog 2 Inhibition Stimulates Bone Formation and Mitigates Bone Loss Caused by Ovariectomy in Skeletally Mature Mice*

PubMed Central

Dudakovic, Amel; Camilleri, Emily T.; Riester, Scott M.; Paradise, Christopher R.; Gluscevic, Martina; O'Toole, Thomas M.; Thaler, Roman; Evans, Jared M.; Yan, Huihuang; Subramaniam, Malayannan; Hawse, John R.; Stein, Gary S.; Montecino, Martin A.; McGee-Lawrence, Meghan E.; Westendorf, Jennifer J.; van Wijnen, Andre J.

2016-01-01

Perturbations in skeletal development and bone degeneration may result in reduced bone mass and quality, leading to greater fracture risk. Bone loss is mitigated by bone protective therapies, but there is a clinical need for new bone-anabolic agents. Previous work has demonstrated that Ezh2 (enhancer of zeste homolog 2), a histone 3 lysine 27 (H3K27) methyltransferase, suppressed differentiation of osteogenic progenitors. Here, we investigated whether inhibition of Ezh2 can be leveraged for bone stimulatory applications. Pharmacologic inhibition and siRNA knockdown of Ezh2 enhanced osteogenic commitment of MC3T3 preosteoblasts. Next generation RNA sequencing of mRNAs and real time quantitative PCR profiling established that Ezh2 inactivation promotes expression of bone-related gene regulators and extracellular matrix proteins. Mechanistically, enhanced gene expression was linked to decreased H3K27 trimethylation (H3K27me3) near transcriptional start sites in genome-wide sequencing of chromatin immunoprecipitations assays. Administration of an Ezh2 inhibitor modestly increases bone density parameters of adult mice. Furthermore, Ezh2 inhibition also alleviated bone loss in an estrogen-deficient mammalian model for osteoporosis. Ezh2 inhibition enhanced expression of Wnt10b and Pth1r and increased the BMP-dependent phosphorylation of Smad1/5. Thus, these data suggest that inhibition of Ezh2 promotes paracrine signaling in osteoblasts and has bone-anabolic and osteoprotective potential in adults. PMID:27758858

A Pitx transcription factor controls the establishment and maintenance of the serotonergic lineage in planarians.

PubMed

März, Martin; Seebeck, Florian; Bartscherer, Kerstin

2013-11-01

In contrast to adult vertebrates, which have limited capacities for neurogenesis, adult planarians undergo constitutive cellular turnover during homeostasis and are even able to regenerate a whole brain after decapitation. This enormous plasticity derives from pluripotent stem cells residing in the planarian body in large numbers. It is still obscure how these stem cells are programmed for differentiation into specific cell lineages and how lineage identity is maintained. Here we identify a Pitx transcription factor of crucial importance for planarian regeneration. In addition to patterning defects that are co-dependent on the LIM homeobox transcription factor gene islet1, which is expressed with pitx at anterior and posterior regeneration poles, RNAi against pitx results in islet1-independent specific loss of serotonergic (SN) neurons during regeneration. Besides its expression in terminally differentiated SN neurons we found pitx in stem cell progeny committed to the SN fate. Also, intact pitx RNAi animals gradually lose SN markers, a phenotype that depends neither on increased apoptosis nor on stem cell-based turnover or transdifferentiation into other neurons. We propose that pitx is a terminal selector gene for SN neurons in planarians that controls not only their maturation but also their identity by regulating the expression of the Serotonin production and transport machinery. Finally, we made use of this function of pitx and compared the transcriptomes of regenerating planarians with and without functional SN neurons, identifying at least three new neuronal targets of Pitx.

Light and abiotic stresses regulate the expression of GDP-L-galactose phosphorylase and levels of ascorbic acid in two kiwifruit genotypes via light-responsive and stress-inducible cis-elements in their promoters.

PubMed

Li, Juan; Liang, Dong; Li, Mingjun; Ma, Fengwang

2013-09-01

Ascorbic acid (AsA) plays an essential role in plants by protecting cells against oxidative damage. GDP-L-galactose phosphorylase (GGP) is the first committed gene for AsA synthesis. Our research examined AsA levels, regulation of GGP gene expression, and how these are related to abiotic stresses in two species of Actinidia (kiwifruit). When leaves were subjected to continuous darkness or light, ABA or MeJA, heat, or a hypoxic environment, we found some correlation between the relative levels of GGP mRNA and AsA concentrations. In transformed tobacco plants, activity of the GGP promoter was induced by all of these treatments. However, the degree of inducibility in the two kiwifruit species differed among the GGP promoter deletions. We deduced that the G-box motif, a light-responsive element, may have an important function in regulating GGP transcripts under various light conditions in both A. deliciosa and A. eriantha. Other elements such as ABRE, the CGTCA motif, and HSE might also control the promoter activities of GGP in kiwifruit. Altogether, these data suggest that GGP expression in the two kiwifruit species is regulated by light or abiotic stress via the relative cis-elements in their promoters. Furthermore, GGP has a critical role in modulating AsA concentrations in kiwifruit species under abiotic stresses.

Formation and specification of a Drosophila dopaminergic precursor cell.

PubMed

Watson, Joseph D; Crews, Stephen T

2012-09-01

Dopaminergic neurons play important roles in animal behavior, including motivation, reward and locomotion. The Drosophila dopaminergic H-cell interneuron is an attractive system for studying the genetics of neural development because analysis is focused on a single neuronal cell type. Here we provide a mechanistic understanding of how MP3, the precursor to the H-cell, forms and acquires its identity. We show that the gooseberry/gooseberry-neuro (gsb/gsb-n) transcription factor genes act to specify MP3 cell fate. It is proposed that single-minded commits neuroectodermal cells to a midline fate, followed by a series of signaling events that result in the formation of a single gsb(+)/gsb-n(+) MP3 cell per segment. The wingless signaling pathway establishes a midline anterior domain by activating expression of the forkhead transcription factors sloppy paired 1 and sloppy paired 2. This is followed by hedgehog signaling that activates gsb/gsb-n expression in a subgroup of anterior cells. Finally, Notch signaling results in the selection of a single MP3, with the remaining cells becoming midline glia. In MP3, gsb/gsb-n direct H-cell development, in large part by activating expression of the lethal of scute and tailup H-cell regulatory genes. Thus, a series of signaling and transcriptional events result in the specification of a unique dopaminergic precursor cell. Additional genetic experiments indicate that the molecular mechanisms that govern MP3/H-cell development might also direct the development of non-midline dopaminergic neurons.

Formation and specification of a Drosophila dopaminergic precursor cell

PubMed Central

Watson, Joseph D.; Crews, Stephen T.

2012-01-01

Dopaminergic neurons play important roles in animal behavior, including motivation, reward and locomotion. The Drosophila dopaminergic H-cell interneuron is an attractive system for studying the genetics of neural development because analysis is focused on a single neuronal cell type. Here we provide a mechanistic understanding of how MP3, the precursor to the H-cell, forms and acquires its identity. We show that the gooseberry/gooseberry-neuro (gsb/gsb-n) transcription factor genes act to specify MP3 cell fate. It is proposed that single-minded commits neuroectodermal cells to a midline fate, followed by a series of signaling events that result in the formation of a single gsb+/gsb-n+ MP3 cell per segment. The wingless signaling pathway establishes a midline anterior domain by activating expression of the forkhead transcription factors sloppy paired 1 and sloppy paired 2. This is followed by hedgehog signaling that activates gsb/gsb-n expression in a subgroup of anterior cells. Finally, Notch signaling results in the selection of a single MP3, with the remaining cells becoming midline glia. In MP3, gsb/gsb-n direct H-cell development, in large part by activating expression of the lethal of scute and tailup H-cell regulatory genes. Thus, a series of signaling and transcriptional events result in the specification of a unique dopaminergic precursor cell. Additional genetic experiments indicate that the molecular mechanisms that govern MP3/H-cell development might also direct the development of non-midline dopaminergic neurons. PMID:22874915

Human dental pulp stem cells cultured in serum-free supplemented medium

PubMed Central

Bonnamain, Virginie; Thinard, Reynald; Sergent-Tanguy, Solène; Huet, Pascal; Bienvenu, Géraldine; Naveilhan, Philippe; Farges, Jean-Christophe; Alliot-Licht, Brigitte

2013-01-01

Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. Methodology: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR. Results: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers β-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, β-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. Conclusion: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH-DPSCs. PMID:24376422

Tracking Single Cells in Live Animals Using a Photoconvertible Near-Infrared Cell Membrane Label

PubMed Central

Wu, Juwell; Runnels, Judith M.; Turcotte, Raphaël; Celso, Cristina Lo; Scadden, David T.; Strom, Terry B.; Lin, Charles P.

2013-01-01

We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4+ T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution. PMID:23990881

Tracking single cells in live animals using a photoconvertible near-infrared cell membrane label.

PubMed

Carlson, Alicia L; Fujisaki, Joji; Wu, Juwell; Runnels, Judith M; Turcotte, Raphaël; Spencer, Joel A; Celso, Cristina Lo; Scadden, David T; Strom, Terry B; Lin, Charles P

2013-01-01

We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4(+) T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution.

Commitment language and homework completion in a behavioral employment program for gang-affiliated youth.

PubMed

Smith, Caitlin; Huey, Stanley J; McDaniel, Dawn D

2015-05-01

Research with substance-abusing samples suggests that eliciting commitment language during treatment may improve motivation to change, increase treatment engagement, and promote positive treatment outcomes. However, the relationship between in-session client language and treatment success is not well-understood for youth offender populations. This study evaluated the relationship between commitment language, treatment engagement (i.e., homework completion), and weekly employment outcomes for six gang-affiliated juvenile offenders participating in an employment counseling intervention. Weekly counseling sessions were audio-recorded, transcribed, and coded for commitment language strength. Multilevel models were fit to the data to examine the relationship between commitment language and counseling homework or employment outcomes within participants over time. Commitment language strength predicted subsequent homework completion but not weekly employment. These findings imply that gang-affiliated delinquent youth who express motivation to change during employment counseling will be more likely to comply with counselor-initiated homework. Further research on counselor techniques for promoting commitment language among juvenile gang offenders is needed. © The Author(s) 2013.

Transcriptome analyses of rhesus monkey preimplantation embryos reveal a reduced capacity for DNA double-strand break repair in primate oocytes and early embryos

PubMed Central

Wang, Xinyi; Liu, Denghui; He, Dajian; Suo, Shengbao; Xia, Xian; He, Xiechao; Han, Jing-Dong J.; Zheng, Ping

2017-01-01

Preimplantation embryogenesis encompasses several critical events including genome reprogramming, zygotic genome activation (ZGA), and cell-fate commitment. The molecular basis of these processes remains obscure in primates in which there is a high rate of embryo wastage. Thus, understanding the factors involved in genome reprogramming and ZGA might help reproductive success during this susceptible period of early development and generate induced pluripotent stem cells with greater efficiency. Moreover, explaining the molecular basis responsible for embryo wastage in primates will greatly expand our knowledge of species evolution. By using RNA-seq in single and pooled oocytes and embryos, we defined the transcriptome throughout preimplantation development in rhesus monkey. In comparison to archival human and mouse data, we found that the transcriptome dynamics of monkey oocytes and embryos were very similar to those of human but very different from those of mouse. We identified several classes of maternal and zygotic genes, whose expression peaks were highly correlated with the time frames of genome reprogramming, ZGA, and cell-fate commitment, respectively. Importantly, comparison of the ZGA-related network modules among the three species revealed less robust surveillance of genomic instability in primate oocytes and embryos than in rodents, particularly in the pathways of DNA damage signaling and homology-directed DNA double-strand break repair. This study highlights the utility of monkey models to better understand the molecular basis for genome reprogramming, ZGA, and genomic stability surveillance in human early embryogenesis and may provide insights for improved homologous recombination-mediated gene editing in monkey. PMID:28223401

The Maize Gene terpene synthase 1 Encodes a Sesquiterpene Synthase Catalyzing the Formation of (E)-β-Farnesene, (E)-Nerolidol, and (E,E)-Farnesol after Herbivore Damage1

PubMed Central

Schnee, Christiane; Köllner, Tobias G.; Gershenzon, Jonathan; Degenhardt, Jörg

2002-01-01

Maize (Zea mays) emits a mixture of volatile compounds upon attack by the Egyptian cotton leafworm (Spodoptera littoralis). These substances, primarily mono- and sesquiterpenes, are used by parasitic wasps to locate the lepidopteran larvae, which are their natural hosts. This interaction among plant, lepidopteran larvae, and hymenopteran parasitoids benefits the plant and has been termed indirect defense. The committed step in the biosynthesis of the different skeletal types of mono- and sesquiterpenes is catalyzed by terpene synthases, a class of enzymes that forms a large variety of mono- and sesquiterpene products from prenyl diphosphate precursors. We isolated a terpene synthase gene, terpene synthase 1 (tps1), from maize that exhibits only a low degree of sequence identity to previously identified terpene synthases. Upon expression in a bacterial system, the encoded enzyme produced the acyclic sesquiterpenes, (E)-β-farnesene, (E,E)-farnesol, and (3R)-(E)-nerolidol, the last an intermediate in the formation of (3E)-4,8-dimethyl-1,3,7-nonatriene. Both (E)-β-farnesene and (3E)-4,8-dimethyl-1,3,7-nonatriene are prominent compounds of the maize volatile blend that is emitted after herbivore damage. The biochemical characteristics of the encoded enzyme are similar to those of terpene synthases from both gymnosperms and dicotyledonous angiosperms, suggesting that catalysis involves a similar electrophilic reaction mechanism. The transcript level of tps1 in the maize cv B73 was elevated after herbivory, mechanical damage, and treatment with elicitors. In contrast, the increase in the transcript level of the tps1 gene or gene homolog in the maize cv Delprim after herbivory was less pronounced, suggesting that the regulation of terpene synthase expression may vary among maize varieties. PMID:12481088

Multiple intracellular signaling pathways orchestrate adipocytic differentiation of human bone marrow stromal stem cells.

PubMed

Ali, Dalia; Abuelreich, Sarah; Alkeraishan, Nora; Shwish, Najla Bin; Hamam, Rimi; Kassem, Moustapha; Alfayez, Musaad; Aldahmash, Abdullah; Alajez, Nehad M

2018-02-28

Bone marrow adipocyte formation plays a role in bone homeostasis and whole body energy metabolism. However, the transcriptional landscape and signaling pathways associated with adipocyte lineage commitment and maturation are not fully delineated. Thus, we performed global gene expression profiling during adipocyte differentiation of human bone marrow stromal (mesenchymal) stem cells (hMSCs) and identified 2,589 up-regulated and 2,583 down-regulated mRNA transcripts. Pathway analysis on the up-regulated gene list untraveled enrichment in multiple signaling pathways including insulin receptor signaling, focal Adhesion, metapathway biotransformation, a number of metabolic pathways e.g. selenium metabolism, Benzo(a)pyrene metabolism, fatty acid, triacylglycerol, ketone body metabolism, tryptophan metabolism, and catalytic cycle of mammalian flavin-containing monooxygenase (FMOs). On the other hand, pathway analysis on the down-regulated genes revealed significant enrichment in pathways related to cell cycle regulation. Based on these data, we assessed the effect of pharmacological inhibition of FAK signaling using PF-573228, PF-562271, and InsR/IGF-1R using NVP-AEW541 and GSK-1904529A on adipocyte differentiation. hMSCs exposed to FAK or IGF-1R/InsR inhibitors exhibited fewer adipocyte formation (27-58% inhibition, P <0005). Concordantly, the expression of adipocyte-specific genes AP2, AdipoQ, and CEBPα was significantly reduced. On the other hand, we did not detect significant effects on cell viability as a result of FAK or IGF-1R/InsR inhibition. Our data identified FAK and insulin signaling as important intracellular signaling pathways relevant to bone marrow adipogenesis. © 2018 The Author(s).

Identification of novel genes potentially involved in somatic embryogenesis in chicory (Cichorium intybus L.)

PubMed Central

2010-01-01

Background In our laboratory we use cultured chicory (Cichorium intybus) explants as a model to investigate cell reactivation and somatic embryogenesis and have produced 2 chicory genotypes (K59, C15) sharing a similar genetic background. K59 is a responsive genotype (embryogenic) capable of undergoing complete cell reactivation i.e. cell de- and re-differentiation leading to somatic embryogenesis (SE), whereas C15 is a non-responsive genotype (non-embryogenic) and is unable to undergo SE. Previous studies [1] showed that the use of the β-D-glucosyl Yariv reagent (β-GlcY) that specifically binds arabinogalactan-proteins (AGPs) blocked somatic embryo production in chicory root explants. This observation indicates that β-GlcY is a useful tool for investigating somatic embryogenesis (SE) in chicory. In addition, a putative AGP (DT212818) encoding gene was previously found to be significantly up-regulated in the embryogenic K59 chicory genotype as compared to the non-embryogenic C15 genotype suggesting that this AGP could be involved in chicory re-differentiation [2]. In order to improve our understanding of the molecular and cellular regulation underlying SE in chicory, we undertook a detailed cytological study of cell reactivation events in K59 and C15 genotypes, and used microarray profiling to compare gene expression in these 2 genotypes. In addition we also used β-GlcY to block SE in order to identify genes potentially involved in this process. Results Microscopy confirmed that only the K59, but not the C15 genotype underwent complete cell reactivation leading to SE formation. β-GlcY-treatment of explants blocked in vitro SE induction, but not cell reactivation, and induced cell wall modifications. Microarray analyses revealed that 78 genes were differentially expressed between induced K59 and C15 genotypes. The expression profiles of 19 genes were modified by β-GlcY-treatment. Eight genes were both differentially expressed between K59 and C15 genotypes during SE induction and transcriptionally affected by β-GlcY-treatment: AGP (DT212818), 26 S proteasome AAA ATPase subunit 6 (RPT6), remorin (REM), metallothionein-1 (MT1), two non-specific lipid transfer proteins genes (SDI-9 and DEA1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), and snakin 2 (SN2). These results suggest that the 8 genes, including the previously-identified AGP gene (DT212818), could be involved in cell fate determination events leading to SE commitment in chicory. Conclusion The use of two different chicory genotypes differing in their responsiveness to SE induction, together with β-GlcY-treatment represented an efficient tool to discriminate cell reactivation from the SE morphogenetic pathway. Such an approach, together with microarray analyses, permitted us to identify several putative key genes related to the SE morphogenetic pathway in chicory. PMID:20565992

[Core principles for the regulation of placement subject to public law in psychiatric hospitals - with explanations].

PubMed

2016-03-01

While the provisions of the highest courts concerning the involuntary commitment and treatment in psychiatric hospitals of people unable to give their consent are being implemented, in many federal states corresponding adjustments to the rules governing involuntary commitment in accordance with the mental health laws and laws on involuntary commitment are still pending. In states where new regulations do exist, legal experts express doubts that they conform to the Constitution and the UN Convention on the Rights of Persons with Disabilities. The DGPPN has formulated key parameters for involuntary commitment from a clinical perspective, which should be taken into account in the new regulations of the individual federal states.

A transcriptional switch underlies commitment to sexual development in malaria parasites.

PubMed

Kafsack, Björn F C; Rovira-Graells, Núria; Clark, Taane G; Bancells, Cristina; Crowley, Valerie M; Campino, Susana G; Williams, April E; Drought, Laura G; Kwiatkowski, Dominic P; Baker, David A; Cortés, Alfred; Llinás, Manuel

2014-03-13

The life cycles of many parasites involve transitions between disparate host species, requiring these parasites to go through multiple developmental stages adapted to each of these specialized niches. Transmission of malaria parasites (Plasmodium spp.) from humans to the mosquito vector requires differentiation from asexual stages replicating within red blood cells into non-dividing male and female gametocytes. Although gametocytes were first described in 1880, our understanding of the molecular mechanisms involved in commitment to gametocyte formation is extremely limited, and disrupting this critical developmental transition remains a long-standing goal. Here we show that expression levels of the DNA-binding protein PfAP2-G correlate strongly with levels of gametocyte formation. Using independent forward and reverse genetics approaches, we demonstrate that PfAP2-G function is essential for parasite sexual differentiation. By combining genome-wide PfAP2-G cognate motif occurrence with global transcriptional changes resulting from PfAP2-G ablation, we identify early gametocyte genes as probable targets of PfAP2-G and show that their regulation by PfAP2-G is critical for their wild-type level expression. In the asexual blood-stage parasites pfap2-g appears to be among a set of epigenetically silenced loci prone to spontaneous activation. Stochastic activation presents a simple mechanism for a low baseline of gametocyte production. Overall, these findings identify PfAP2-G as a master regulator of sexual-stage development in malaria parasites and mark the first discovery of a transcriptional switch controlling a differentiation decision in protozoan parasites.

A model of early human embryonic stem cell differentiation reveals inter- and intracellular changes on transition to squamous epithelium.

PubMed

Galat, Vasiliy; Malchenko, Sergey; Galat, Yekaterina; Ishkin, Alex; Nikolsky, Yuri; Kosak, Steven T; Soares, Bento Marcelo; Iannaccone, Philip; Crispino, John D; Hendrix, Mary J C

2012-05-20

The molecular events leading to human embryonic stem cell (hESC) differentiation are the subject of considerable scrutiny. Here, we characterize an in vitro model that permits analysis of the earliest steps in the transition of hESC colonies to squamous epithelium on basic fibroblast growth factor withdrawal. A set of markers (GSC, CK18, Gata4, Eomes, and Sox17) point to a mesendodermal nature of the epithelial cells with subsequent commitment to definitive endoderm (Sox17, Cdx2, nestin, and Islet1). We assayed alterations in the transcriptome in parallel with the distribution of immunohistochemical markers. Our results indicate that the alterations of tight junctions in pluripotent culture precede the beginning of differentiation. We defined this cell population as "specified," as it is committed toward differentiation. The transitional zone between "specified" pluripotent and differentiated cells displays significant up-regulation of keratin-18 (CK18) along with a decrease in the functional activity of gap junctions and the down-regulation of 2 gap junction proteins, connexin 43 (Cx43) and connexin 45 (Cx45), which is coincidental with substantial elevation of intracellular Ca2+ levels. These findings reveal a set of cellular changes that may represent the earliest markers of in vitro hESC transition to an epithelial phenotype, before the induction of gene expression networks that guide hESC differentiation. Moreover, we hypothesize that these events may be common during the primary steps of hESC commitment to functionally varied epithelial tissue derivatives of different embryological origins.

A Model of Early Human Embryonic Stem Cell Differentiation Reveals Inter- and Intracellular Changes on Transition to Squamous Epithelium

PubMed Central

Malchenko, Sergey; Galat, Yekaterina; Ishkin, Alex; Nikolsky, Yuri; Kosak, Steven T.; Soares, Bento Marcelo; Iannaccone, Philip; Crispino, John D.; Hendrix, Mary J.C.

2012-01-01

The molecular events leading to human embryonic stem cell (hESC) differentiation are the subject of considerable scrutiny. Here, we characterize an in vitro model that permits analysis of the earliest steps in the transition of hESC colonies to squamous epithelium on basic fibroblast growth factor withdrawal. A set of markers (GSC, CK18, Gata4, Eomes, and Sox17) point to a mesendodermal nature of the epithelial cells with subsequent commitment to definitive endoderm (Sox17, Cdx2, nestin, and Islet1). We assayed alterations in the transcriptome in parallel with the distribution of immunohistochemical markers. Our results indicate that the alterations of tight junctions in pluripotent culture precede the beginning of differentiation. We defined this cell population as “specified,” as it is committed toward differentiation. The transitional zone between “specified” pluripotent and differentiated cells displays significant up-regulation of keratin-18 (CK18) along with a decrease in the functional activity of gap junctions and the down-regulation of 2 gap junction proteins, connexin 43 (Cx43) and connexin 45 (Cx45), which is coincidental with substantial elevation of intracellular Ca2+ levels. These findings reveal a set of cellular changes that may represent the earliest markers of in vitro hESC transition to an epithelial phenotype, before the induction of gene expression networks that guide hESC differentiation. Moreover, we hypothesize that these events may be common during the primary steps of hESC commitment to functionally varied epithelial tissue derivatives of different embryological origins. PMID:21861759

Identification of Loci and Functional Characterization of Trichothecene Biosynthesis Genes in Filamentous Fungi of the Genus Trichoderma▿†

PubMed Central

Cardoza, R. E.; Malmierca, M. G.; Hermosa, M. R.; Alexander, N. J.; McCormick, S. P.; Proctor, R. H.; Tijerino, A. M.; Rumbero, A.; Monte, E.; Gutiérrez, S.

2011-01-01

Trichothecenes are mycotoxins produced by Trichoderma, Fusarium, and at least four other genera in the fungal order Hypocreales. Fusarium has a trichothecene biosynthetic gene (TRI) cluster that encodes transport and regulatory proteins as well as most enzymes required for the formation of the mycotoxins. However, little is known about trichothecene biosynthesis in the other genera. Here, we identify and characterize TRI gene orthologues (tri) in Trichoderma arundinaceum and Trichoderma brevicompactum. Our results indicate that both Trichoderma species have a tri cluster that consists of orthologues of seven genes present in the Fusarium TRI cluster. Organization of genes in the cluster is the same in the two Trichoderma species but differs from the organization in Fusarium. Sequence and functional analysis revealed that the gene (tri5) responsible for the first committed step in trichothecene biosynthesis is located outside the cluster in both Trichoderma species rather than inside the cluster as it is in Fusarium. Heterologous expression analysis revealed that two T. arundinaceum cluster genes (tri4 and tri11) differ in function from their Fusarium orthologues. The Tatri4-encoded enzyme catalyzes only three of the four oxygenation reactions catalyzed by the orthologous enzyme in Fusarium. The Tatri11-encoded enzyme catalyzes a completely different reaction (trichothecene C-4 hydroxylation) than the Fusarium orthologue (trichothecene C-15 hydroxylation). The results of this study indicate that although some characteristics of the tri/TRI cluster have been conserved during evolution of Trichoderma and Fusarium, the cluster has undergone marked changes, including gene loss and/or gain, gene rearrangement, and divergence of gene function. PMID:21642405

New insights into Blimp-1 in T lymphocytes: a divergent regulator of cell destiny and effector function.

PubMed

Fu, Shin-Huei; Yeh, Li-Tzu; Chu, Chin-Chen; Yen, B Lin-Ju; Sytwu, Huey-Kang

2017-07-21

B lymphocyte-induced maturation protein-1 (Blimp-1) serves as a master regulator of the development and function of antibody-producing B cells. Given that its function in T lymphocytes has been identified within the past decade, we review recent findings with emphasis on its role in coordinated control of gene expression during the development, differentiation, and function of T cells. Expression of Blimp-1 is mainly confined to activated T cells and is essential for the production of interleukin (IL)-10 by a subset of forkhead box (Fox)p3 + regulatory T cells with an effector phenotype. Blimp-1 is also required to induce cell elimination in the thymus and critically modulates peripheral T cell activation and proliferation. In addition, Blimp-1 promotes T helper (Th) 2 lineage commitment and limits Th1, Th17 and follicular helper T cell differentiation. Furthermore, Blimp-1 coordinates with other transcription factors to regulate expression of IL-2, IL-21 and IL-10 in effector T lymphocytes. In CD8 + T cells, Blimp-1 expression is distinct in heterogeneous populations at the stages of clonal expansion, differentiation, contraction and memory formation when they encounter antigens. Moreover, Blimp-1 plays a fundamental role in coordinating cytokine receptor signaling networks and transcriptional programs to regulate diverse aspects of the formation and function of effector and memory CD8 + T cells and their exhaustion. Blimp-1 also functions as a gatekeeper of T cell activation and suppression to prevent or dampen autoimmune disease, antiviral responses and antitumor immunity. In this review, we discuss the emerging roles of Blimp-1 in the complex regulation of gene networks that regulate the destiny and effector function of T cells and provide a Blimp-1-dominated transcriptional framework for T lymphocyte homeostasis.

Activation of the Endoplasmic Reticulum Stress-Associated Transcription Factor X Box-Binding Protein-1 Occurs in a Subset of Normal Germinal-Center B Cells and in Aggressive B-Cell Lymphomas with Prognostic Implications

PubMed Central

Balague, Olga; Mozos, Ana; Martinez, Daniel; Hernandez, Luis; Colomo, Lluis; Mate, Jose Luis; Teruya-Feldstein, Julie; Lin, Oscar; Campo, Elias; Lopez-Guillermo, Armando; Martinez, Antonio

2009-01-01

X box-binding protein 1 (Xbp-1) is a transcription factor that is required for the terminal differentiation of B lymphocytes into plasma cells. The Xbp-1 gene is activated in response to endoplasmic reticulum stress signals, which generate a 50-kDa nuclear protein that acts as a potent transactivator and regulates the expression of genes related to the unfolded protein response. Activated Xbp-1 is essential for cell survival in plasma-cell tumors but its role in B-cell lymphomas is unknown. We analyzed the expression of activated Xbp-1 in reactive lymphoid tissues, 411 lymphomas and plasma-cell neoplasms, and 24 B-cell lines. In reactive tissues, Xbp-1 was only found in nuclear extracts. Nuclear expression of Xbp-1 was observed in occasional reactive plasma cells and in a subpopulation of Irf-4+/Bcl-6−/Pax-5− B cells in the light zones of reactive germinal centers, probably representing cells committed to plasma-cell differentiation. None of the low-grade lymphomas showed evidence of Xbp-1 activation; however, Xbp-1 activation was found in 28% of diffuse large B-cell lymphomas, independent of germinal or postgerminal center phenotype, as well as in 48% of plasmablastic lymphomas and 69% of plasma-cell neoplasms. Diffuse large B-cell lymphomas with nuclear Xbp-1 expression had a significantly worse response to therapy and shorter overall survival compared with negative tumors. These findings suggest that Xbp-1 activation may play a role in the pathogenesis of aggressive B-cell lymphomas. PMID:19389935

FOXM1 in sarcoma: role in cell cycle, pluripotency genes and stem cell pathways

PubMed Central

Kelleher, Fergal C.; O'sullivan, Hazel

2016-01-01

FOXM1 is a pro-proliferative transcription factor that promotes cell cycle progression at the G1-S, and G2-M transitions. It is activated by phosphorylation usually mediated by successive cyclin – cyclin dependent kinase complexes, and is highly expressed in sarcoma. p53 down regulates FOXM1 and FOXM1 inhibition is also partly dependent on Rb and p21. Abnormalities of p53 or Rb are frequent in sporadic sarcomas with bone or soft tissue sarcoma, accounting for 36% of index cancers in the high penetrance TP53 germline disorder, Li-Fraumeni syndrome. FOXM1 stimulates transcription of pluripotency related genes including SOX2, KLF4, OCT4, and NANOG many of which are important in sarcoma, a disorder of mesenchymal stem cell/ partially committed progenitor cells. In a selected specific, SOX2 is uniformly expressed in synovial sarcoma. Embryonic pathways preferentially used in stem cell such as Hippo, Hedgehog, and Wnt dominate in FOXM1 stoichiometry to alter rates of FOXM1 production or degradation. In undifferentiated pleomorphic sarcoma, liposarcoma, and fibrosarcoma, dysregulation of the Hippo pathway increases expression of the effector co-transcriptional activator Yes-Associated Protein (YAP). A complex involving YAP and the transcription factor TEAD elevates FOXM1 in these sarcoma subtypes. In another scenario 80% of desmoid tumors have nuclear localization of β-catenin, the Wnt pathway effector molecule. Thiazole antibiotics inhibit FOXM1 and because they have an auto-regulator loop FOXM1 expression is also inhibited. Current systemic treatment of sarcoma is of limited efficacy and inhibiting FOXM1 represents a potential new strategy. PMID:27074562

Transient Cnp expression by early progenitors causes Cre-Lox-based reporter lines to map profoundly different fates.

PubMed

Tognatta, Reshmi; Sun, Wenjing; Goebbels, Sandra; Nave, Klaus-Armin; Nishiyama, Akiko; Schoch, Susanne; Dimou, Leda; Dietrich, Dirk

2017-02-01

NG2 expressing oligodendroglial precursor cells are ubiquitous in the central nervous system and the only cell type cycling throughout life. Previous fate mapping studies have remained inconsistent regarding the question whether NG2 cells are capable of generating certain types of neurons. Here, we use CNP-Cre mice to map the fate of a sub-population of NG2 cells assumed to be close to differentiation. When crossing these mice with the ROSA26/YFP Cre-reporter line we discovered large numbers of reporter-expressing pyramidal neurons in the piriform and dorsal cortex. In contrast, when using Z/EG reporter mice to track the fate of Cnp-expressing NG2 cells only oligodendroglial cells were found reporter positive. Using BrdU-based birth dating protocols and inducible NG2CreER:ROSA26/YFP mice we show that YFP positive neurons are generated from radial glial cells and that these radial glial cells display temporary and low level activity of certain oligodendroglial genes sufficient to recombine the Cre-inducible reporter gene in ROSA26/YFP but not in Z/EG mice. Taken together, we did not obtain evidence for generation of neurons from NG2 cells. Our results suggest that with an appropriate reporter system Cnp activity can be used to define a proliferative subpopulation of NG2 cells committed to generate oligodendrocytes. However, the strikingly different results obtained from ROSA26/YFP versus Z/EG mice demonstrate that the choice of Cre-reporter line can be of crucial importance for fate mapping studies and other applications of the Cre-lox technology. GLIA 2017;65:342-359. © 2016 Wiley Periodicals, Inc.

Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation “Memories”

PubMed Central

Tompkins, Joshua D.; Jung, Marc; Chen, Chang-yi; Lin, Ziguang; Ye, Jingjing; Godatha, Swetha; Lizhar, Elizabeth; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

2016-01-01

The directed differentiation of human cardiomyocytes (CMs) from pluripotent cells provides an invaluable model for understanding mechanisms of cell fate determination and offers considerable promise in cardiac regenerative medicine. Here, we utilize a human embryonic stem cell suspension bank, produced according to a good manufacturing practice, to generate CMs using a fully defined and small molecule-based differentiation strategy. Primitive and cardiac mesoderm purification was used to remove non-committing and multi-lineage populations and this significantly aided the identification of key transcription factors, lncRNAs, and essential signaling pathways that define cardiomyogenesis. Global methylation profiles reflect CM development and we report on CM exon DNA methylation “memories” persisting beyond transcription repression and marking the expression history of numerous developmentally regulated genes, especially transcription factors. PMID:26981572

Reciprocal patterns of allergen-induced GATA-3 expression in peripheral blood mononuclear cells from atopics vs. non-atopics.

PubMed

Macaubas, C; Lee, P T; Smallacombe, T B; Holt, B J; Wee, C; Sly, P D; Holt, P G

2002-01-01

T helper (Th)2 cytokines are considered to play a central role in the induction and expression of allergic disease. However, the relative importance of individual cytokines is unclear, and overall disease pathogenesis appears to involve the coordinate activities of a range of Th2 cytokines acting in sequence or in parallel. The present study examines an alternative approach to the study of cytokine gene function in atopy, focusing instead upon T cell transcription factors (TFs) which play a role in the regulation of multiple cytokine genes. To investigate the allergen-induced expression of the TF GATA-3 and c-Maf in peripheral blood mononuclear cells (PBMCs) and in cytokine-driven Th polarization. PBMC from house dust mite (HDM)-atopic and non-atopics were stimulated in vitro with allergen or anti-CD3/IL-2. TF expression was analysed by semiquantitative RT-PCR and major findings were validated by real-time PCR. Cell separations were performed to analyse the contribution of CD45RO+ cells. CD4+ cord blood cells were Th1 or Th2 polarized in vitro by exogenous cytokines and TF expression analysed by Northern blot and real-time PCR. Results We demonstrate for the first time that during differentiation of CD4+ CD45RA+ naïve human T cells towards Th2 commitment, and during allergen-specific reactivation of peripheral CD4+ CD45RO+ Th2 memory cells in established atopics, expression of the Th2-associated TF GATA-3 is rapidly up-regulated, whereas T cells from non-atopics display equally rapid GATA-3 down-regulation under identical conditions of allergen stimulation. These findings identify Th2-associated TFs as key determinants of the atopic phenotype, suggesting their unique potential as therapeutic targets for disease control.

Conversion of adult endothelium to immunocompetent haematopoietic stem cells.

PubMed

Lis, Raphael; Karrasch, Charles C; Poulos, Michael G; Kunar, Balvir; Redmond, David; Duran, Jose G Barcia; Badwe, Chaitanya R; Schachterle, William; Ginsberg, Michael; Xiang, Jenny; Tabrizi, Arash Rafii; Shido, Koji; Rosenwaks, Zev; Elemento, Olivier; Speck, Nancy A; Butler, Jason M; Scandura, Joseph M; Rafii, Shahin

2017-05-25

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1 + FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.

Conversion of adult endothelium to immunocompetent haematopoietic stem cells

PubMed Central

Lis, Raphael; Karrasch, Charles C.; Poulos, Michael G.; Kunar, Balvir; Redmond, David; Barcia Duran, Jose G.; Badwe, Chaitanya R.; Schachterle, Will; Ginsberg, Michael; Xiang, Jenny; Tabrizi, Arash Rafii; Shido, Koji; Rosenwaks, Zev; Elemento, Olivier; Speck, Nancy; Butler, Jason M.; Scandura, Joseph M.; Rafii, Shahin

2018-01-01

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully converting adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of genes encoding the transcription factors Fosb, Gfi1, Runx1, and Spi1 (also known as Fgrs) and vascular-niche-derived angiocrine factors. The induction phase (day 0–8) of conversion is initiated by expression of Fgrs in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (day 8–20), Runx1+ Fgrs-transduced endothelial cells commit to a haematopoietic fate yielding rEC-HSCs that no longer require Fgrs expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (at day 20–28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, are competent for clonal engraftment and serial primary and secondary multi-lineage reconstituting potential, including antigen-dependent adaptive immune function. Inhibition of TGF-β and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders. PMID:28514438

When Do Personality and Emotion Predict Destructive Behavior During Relationship Conflict? The Role of Perceived Commitment Asymmetry.

PubMed

Lemay, Edward P; Dobush, Sarah

2015-10-01

The current research examined whether perceived asymmetries in relationship commitment moderate the associations of personality traits and emotional states with enactment of hostile behavior during relationship conflicts. Participants included both members of 53 heterosexual romantic couples (Mage  = 25.5 years). Participants completed questionnaire measures assessing personality traits, emotional states, relationship commitment, and perceptions of their partner's commitment. Participants then had an observed conflict discussion with their partner, which was rated by a panel of objective observers for hostile behavior. When participants perceived that they were less committed than their partners, their enactment of hostile behavior was predicted by traits and states that are associated with antisocial and pro-social orientations (i.e., agreeableness, trait anger, chronic jealousy, and state negative emotion). In contrast, participants who perceived that they were more committed than their partners tended to refrain from hostile behavior, despite traits or states that may suggest hostile inclinations. These results suggest that perceiving that one is less committed than one's partner promotes behavioral expression of interpersonal dispositions and emotions, whereas perceiving that one is more committed than one's partner motivates inhibition of hostile behavior. © 2014 Wiley Periodicals, Inc.

microRNA-184 Induces a Commitment Switch to Epidermal Differentiation.

PubMed

Nagosa, Sara; Leesch, Friederike; Putin, Daria; Bhattacharya, Swarnabh; Altshuler, Anna; Serror, Laura; Amitai-Lange, Aya; Nasser, Waseem; Aberdam, Edith; Rouleau, Matthieu; Tattikota, Sudhir G; Poy, Matthew N; Aberdam, Daniel; Shalom-Feuerstein, Ruby

2017-12-12

miR-184 is a highly evolutionary conserved microRNA (miRNA) from fly to human. The importance of miR-184 was underscored by the discovery that point mutations in miR-184 gene led to corneal/lens blinding disease. However, miR-184-related function in vivo remained unclear. Here, we report that the miR-184 knockout mouse model displayed increased p63 expression in line with epidermal hyperplasia, while forced expression of miR-184 by stem/progenitor cells enhanced the Notch pathway and induced epidermal hypoplasia. In line, miR-184 reduced clonogenicity and accelerated differentiation of human epidermal cells. We showed that by directly repressing cytokeratin 15 (K15) and FIH1, miR-184 induces Notch activation and epidermal differentiation. The disease-causing miR-184 C57U mutant failed to repress K15 and FIH1 and to induce Notch activation, suggesting a loss-of-function mechanism. Altogether, we propose that, by targeting K15 and FIH1, miR-184 regulates the transition from proliferation to early differentiation, while mis-expression or mutation in miR-184 results in impaired homeostasis. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

75 FR 72686 - Express Mail Open and Distribute and Priority Mail Open and Distribute

Federal Register 2010, 2011, 2012, 2013, 2014

2010-11-26

... POSTAL SERVICE 39 CFR Part 111 Express Mail Open and Distribute and Priority Mail Open and... ``DB'' prefix along with new Tag 257, Tag 267, or Label 257S, on all Express Mail[supreg] Open and Distribute containers. The Postal Service is also revising the service commitment for Express Mail Open and...

Conceptual and methodological challenges to measuring political commitment to respond to HIV

PubMed Central

2011-01-01

Background Researchers have long recognized the importance of a central government’s political “commitment” in order to mount an effective response to HIV. The concept of political commitment remains ill-defined, however, and little guidance has been given on how to measure this construct and its relationship with HIV-related outcomes. Several countries have experienced declines in HIV infection rates, but conceptual difficulties arise in linking these declines to political commitment as opposed to underlying social and behavioural factors. Methods This paper first presents a critical review of the literature on existing efforts to conceptualize and measure political commitment to respond to HIV and the linkages between political commitment and HIV-related outcomes. Based on the elements identified in this review, the paper then develops and presents a framework to assist researchers in making choices about how to assess a government's level of political commitment to respond to HIV and how to link political commitment to HIV-related outcomes. Results The review of existing studies identifies three components of commitment (expressed, institutional and budgetary commitment) as different dimensions along which commitment can be measured. The review also identifies normative and ideological aspects of commitment and a set of variables that mediate and moderate political commitment that need to be accounted for in order to draw valid inferences about the relationship between political commitment and HIV-related outcomes. The framework summarizes a set of steps that researchers can follow in order to assess a government's level of commitment to respond to HIV and suggests ways to apply the framework to country cases. Conclusions Whereas existing studies have adopted a limited and often ambiguous conception of political commitment, we argue that conceiving of political commitment along a greater number of dimensions will allow researchers to draw a more complete picture of political commitment to respond to HIV that avoids making invalid inferences about the relationship between political commitment and HIV outcomes. PMID:21968231

Deciphering the genomic structure, function and evolution of carotenogenesis related phytoene synthases in grasses

PubMed Central

2012-01-01

Background Carotenoids are isoprenoid pigments, essential for photosynthesis and photoprotection in plants. The enzyme phytoene synthase (PSY) plays an essential role in mediating condensation of two geranylgeranyl diphosphate molecules, the first committed step in carotenogenesis. PSY are nuclear enzymes encoded by a small gene family consisting of three paralogous genes (PSY1-3) that have been widely characterized in rice, maize and sorghum. Results In wheat, for which yellow pigment content is extremely important for flour colour, only PSY1 has been extensively studied because of its association with QTLs reported for yellow pigment whereas PSY2 has been partially characterized. Here, we report the isolation of bread wheat PSY3 genes from a Renan BAC library using Brachypodium as a model genome for the Triticeae to develop Conserved Orthologous Set markers prior to gene cloning and sequencing. Wheat PSY3 homoeologous genes were sequenced and annotated, unravelling their novel structure associated with intron-loss events and consequent exonic fusions. A wheat PSY3 promoter region was also investigated for the presence of cis-acting elements involved in the response to abscisic acid (ABA), since carotenoids also play an important role as precursors of signalling molecules devoted to plant development and biotic/abiotic stress responses. Expression of wheat PSYs in leaves and roots was investigated during ABA treatment to confirm the up-regulation of PSY3 during abiotic stress. Conclusions We investigated the structural and functional determinisms of PSY genes in wheat. More generally, among eudicots and monocots, the PSY gene family was found to be associated with differences in gene copy numbers, allowing us to propose an evolutionary model for the entire PSY gene family in Grasses. PMID:22672222

The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish

PubMed Central

Hall, Chris; Flores, Maria Vega; Storm, Thilo; Crosier, Kathy; Crosier, Phil

2007-01-01

Background How different immune cell compartments contribute to a successful immune response is central to fully understanding the mechanisms behind normal processes such as tissue repair and the pathology of inflammatory diseases. However, the ability to observe and characterize such interactions, in real-time, within a living vertebrate has proved elusive. Recently, the zebrafish has been exploited to model aspects of human disease and to study specific immune cell compartments using fluorescent reporter transgenic lines. A number of blood-specific lines have provided a means to exploit the exquisite optical clarity that this vertebrate system offers and provide a level of insight into dynamic inflammatory processes previously unavailable. Results We used regulatory regions of the zebrafish lysozyme C (lysC) gene to drive enhanced green fluorescent protein (EGFP) and DsRED2 expression in a manner that completely recapitulated the endogenous expression profile of lysC. Labeled cells were shown by co-expression studies and FACS analysis to represent a subset of macrophages and likely also granulocytes. Functional assays within transgenic larvae proved that these marked cells possess hallmark traits of myelomonocytic cells, including the ability to migrate to inflammatory sources and phagocytose bacteria. Conclusion These reporter lines will have utility in dissecting the genetic determinants of commitment to the myeloid lineage and in further defining how lysozyme-expressing cells participate during inflammation. PMID:17477879

A strategy for isolation of cDNAs encoding proteins affecting human intestinal epithelial cell growth and differentiation: characterization of a novel gut-specific N-myristoylated annexin.

PubMed

Wice, B M; Gordon, J I

1992-01-01

The human intestinal epithelium is rapidly and perpetually renewed as the descendants of multipotent stem cells located in crypts undergo proliferation, differentiation, and eventual exfoliation during a very well organized migration along the crypt to villus axis. The mechanisms that establish and maintain this balance between proliferation and differentiation are largely unknown. We have utilized HT-29 cells, derived from a human colon adenocarcinoma, as a model system for identifying gene products that may regulate these processes. Proliferating HT-29 cells cultured in the absence of glucose (e.g., using inosine as the carbon source) have some of the characteristics of undifferentiated but committed crypt epithelial cells while postconfluent cells cultured in the absence of glucose resemble terminally differentiated enterocytes or goblet cells. A cDNA library, constructed from exponentially growing HT-29 cells maintained in inosine-containing media, was sequentially screened with a series of probes depleted of sequences encoding housekeeping functions and enriched for intestine-specific sequences that are expressed in proliferating committed, but not differentiated, epithelial cells. Of 100,000 recombinant phage surveyed, one was found whose cDNA was derived from an apparently gut-specific mRNA. It encodes a 316 residue, 35,463-D protein that is a new member of the annexin/lipocortin family. Other family members have been implicated in regulation of cellular growth and in signal transduction pathways. RNA blot and in situ hybridization studies indicate that the gene encoding this new annexin exhibits region-specific expression along both axes of the human gut: (a) highest levels of mRNA are present in the jejunum with marked and progressive reductions occurring distally; (b) its mRNA appears in crypt-associated epithelial cells and increases in concentration as they exit the crypt. Villus-associated epithelial cells continue to transcribe this gene during their differentiation/translocation up the villus. Immunocytochemical studies reveal that the intestine-specific annexin (ISA) is associated with the plasma membrane of undifferentiated, proliferating crypt epithelial cells as well as differentiated villus enterocytes. In polarized enterocytes, the highest concentrations of ISA are found at the apical compared to basolateral membrane. In vitro studies using an octapeptide derived from residues 2-9 of the primary translation product of ISA mRNA and purified myristoyl-CoA:protein N-myristoyltransferase suggested that it is N-myristoylated. In vivo labeling studies confirmed that myristate is covalently attached to ISA via a hydroxylamine resistant amide linkage. The restricted cellular expression and acylation of ISA distinguish it from other known annexins.(ABSTRACT TRUNCATED AT 400 WORDS)

Uncoupling the Trade-Off between Somatic Proteostasis and Reproduction in Caenorhabditis elegans Models of Polyglutamine Diseases

PubMed Central

Shemesh, Netta; Shai, Nadav; Meshnik, Lana; Katalan, Rotem; Ben-Zvi, Anat

2017-01-01

Caenorhabditis elegans somatic protein homeostasis (proteostasis) is actively remodeled at the onset of reproduction. This proteostatic collapse is regulated cell-nonautonomously by signals from the reproductive system that transmit the commitment to reproduction to somatic cells. Here, we asked whether the link between the reproductive system and somatic proteostasis could be uncoupled by activating downstream effectors in the gonadal longevity cascade. Specifically, we examined whether over-expression of lipl-4 (lipl-4(oe)), a target gene of the gonadal longevity pathway, or increase in arachidonic acid (AA) levels, associated with lipl-4(oe), modulated proteostasis and reproduction. We found that lipl-4(oe) rescued somatic proteostasis and postponed the onset of aggregation and toxicity in C. elegans models of polyglutamine (polyQ) diseases. However, lipl-4(oe) also disrupted fatty acid transport into developing oocytes and reduced reproductive success. In contrast, diet supplementation of AA recapitulated lipl-4(oe)-mediated proteostasis enhancement in wild type animals but did not affect the reproductive system. Thus, the gonadal longevity pathway mediates a trade-off between somatic maintenance and reproduction, in part by regulating the expression of genes, such as lipl-4, with inverse effects on somatic maintenance and reproduction. We propose that AA could uncouple such germline to soma crosstalk, with beneficial implications protein misfolding diseases. PMID:28503130

Influence of three-dimensional hyaluronic acid microenvironments on mesenchymal stem cell chondrogenesis.

PubMed

Chung, Cindy; Burdick, Jason A

2009-02-01

Mesenchymal stem cells (MSCs) are multipotent progenitor cells whose plasticity and self-renewal capacity have generated significant interest for applications in tissue engineering. The objective of this study was to investigate MSC chondrogenesis in photo-cross-linked hyaluronic acid (HA) hydrogels. Because HA is a native component of cartilage, and MSCs may interact with HA via cell surface receptors, these hydrogels could influence stem cell differentiation. In vitro and in vivo cultures of MSC-laden HA hydrogels permitted chondrogenesis, measured by the early gene expression and production of cartilage-specific matrix proteins. For in vivo culture, MSCs were encapsulated with and without transforming growth factor beta-3 (TGF-beta3) or pre-cultured for 2 weeks in chondrogenic medium before implantation. Up-regulation of type II collagen, aggrecan, and sox 9 was observed for all groups over MSCs at the time of encapsulation, and the addition of TGF-beta3 further enhanced the expression of these genes. To assess the influence of scaffold chemistry on chondrogenesis, HA hydrogels were compared with relatively inert poly(ethylene glycol) (PEG) hydrogels and showed enhanced expression of cartilage-specific markers. Differences between HA and PEG hydrogels in vivo were most noticeable for MSCs and polymer alone, indicating that hydrogel chemistry influences the commitment of MSCs to undergo chondrogenesis (e.g., approximately 43-fold up-regulation of type II collagen of MSCs in HA over PEG hydrogels). Although this study investigated only early markers of tissue regeneration, these results emphasize the importance of material cues in MSC differentiation microenvironments, potentially through interactions between scaffold materials and cell surface receptors.

The Role of a Novel Long Noncoding RNA TUC40- in Cardiomyocyte Induction and Maturation in P19 Cells.

PubMed

Li, Huijuan; Jiang, Li; Yu, Zhangbin; Han, Shuping; Liu, Xuehua; Li, Mengmeng; Zhu, Chun; Qiao, Lixing; Huang, Li

2017-12-01

In previous studies, TUC40-, a new long noncoding RNA, was found to be overexpressed in human ventricular septal defect (VSD) embryonic heart samples. In this article, we carried out experiments on the P19 cell line to elucidate the effects of TUC40- overexpression on cardiomyocyte development relevant to VSD pathogenesis. We established the overexpression cell model by plasmid transfection, and explored the expression profile of Pbx1, the sense gene of TUC40-, and the marker genes of cardiomyocyte linage commitment (Nkx2.5 and GATA4) and maturation (cardiac troponin T). In addition, we combined cell cycle and Cell Counting Kit-8 analysis to detect cell proliferation and used flow cytometry and caspase-3 assays to test apoptosis. At last, bioinformatics analysis was performed to show the possible role of TUC40-. In the control group, Pbx1 elevated steadily during cardiomyocyte induction; whereas in the overexpression group, it showed significantly lower expression at day 6, 8 and 10 of induction. Cells in the overexpression group failed to induce cardiomyocytes indicated by GATA4 and cardiac troponin T. Proliferation was inhibited possibly owing to G2/M cell cycle arrest and the induced apoptosis rate was higher in the overexpression group. TUC40- overexpression reduced Pbx1 expression, cardiomyocyte induction and differentiation, inhibited proliferation and promoted apoptosis. Combining the results and previous studies, we propose TUC40- as a potential pathologic factor for VSD. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

PubMed

Saury, Charlotte; Lardenois, Aurélie; Schleder, Cindy; Leroux, Isabelle; Lieubeau, Blandine; David, Laurent; Charrier, Marine; Guével, Laëtitia; Viau, Sabrina; Delorme, Bruno; Rouger, Karl

2018-05-02

Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL). hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3'-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL. HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives. Our findings indicate that HS and hPL are efficient and viable alternatives to FBS for the preparation of hMuStem cell batches in compliance with GMPs.

Transient upregulation of CBFA1 in response to bone morphogenetic protein-2 and transforming growth factor beta1 in C2C12 myogenic cells coincides with suppression of the myogenic phenotype but is not sufficient for osteoblast differentiation.

PubMed

Lee, M H; Javed, A; Kim, H J; Shin, H I; Gutierrez, S; Choi, J Y; Rosen, V; Stein, J L; van Wijnen, A J; Stein, G S; Lian, J B; Ryoo, H M

1999-04-01

The bone morphogenetic protein (BMP)-2 is a potent osteoinductive signal, inducing bone formation in vivo and osteoblast differentiation from non-osseous cells in vitro. The runt domain-related protein Cbfa1/PEBP2alphaA/AML-3 is a critical component of bone formation in vivo and transcriptional regulator of osteoblast differentiation. To investigate the relationship between the extracellular BMP-2 signal, Cbfa1, and osteogenesis, we examined expression of Cbfa1 and osteoblastic genes during the BMP-2 induced osteogenic transdifferentiation of the myoblastic cell line C2C12. BMP-2 treatment completely blocked myotube formation and transiently induced expression of Cbfa1 and the bone-related homeodomain protein Msx-2 concomitant with loss of the myoblast phenotype. While induction of collagen type I and alkaline phosphatase (AP) expression coincided with Cbfa1 expression, Cbfa1 mRNA was strikingly downregulated at the onset of expression of osteopontin (OPN) and osteocalcin (OCN) genes, reflecting the mature osteoblast phenotype. TGF-beta1 treatment effectively suppressed myogenesis and induced Cbfa1 expression but was insufficient to support osteoblast differentiation reflected by the absence of ALP, OPN, and OCN. We addressed whether induction of Cbfa1 in response to BMP-2 results in the transcriptional activation of the OC promoter which contains three enhancer Cbfa1 elements. Transfection studies show BMP-2 suppresses OC promoter activity in C2C12, but not in osteoblastic ROS 17/2.8 cells. Maximal suppression of OC promoter activity in response to BMP-2 requires sequences in the proximal promoter (up to nt -365) and may occur independent of the three Cbfa sites. Taken together, our results demonstrate a dissociation of Cbfa1 expression from development of the osteoblast phenotype. Our findings suggest that Cbfal may function transiently to divert a committed myoblast to a potentially osteogenic cell. However, other factors induced by BMP-2 appear to be necessary for complete expression of the osteoblast phenotype.

Deciphering the Epigenetic Code in Embryonic and Dental Pulp Stem Cells

PubMed Central

Bayarsaihan, Dashzeveg

2016-01-01

A close cooperation between chromatin states, transcriptional modulation, and epigenetic modifications is required for establishing appropriate regulatory circuits underlying self-renewal and differentiation of adult and embryonic stem cells. A growing body of research has established that the epigenome topology provides a structural framework for engaging genes in the non-random chromosomal interactions to orchestrate complex processes such as cell-matrix interactions, cell adhesion and cell migration during lineage commitment. Over the past few years, the functional dissection of the epigenetic landscape has become increasingly important for understanding gene expression dynamics in stem cells naturally found in most tissues. Adult stem cells of the human dental pulp hold great promise for tissue engineering, particularly in the skeletal and tooth regenerative medicine. It is therefore likely that progress towards pulp regeneration will have a substantial impact on the clinical research. This review summarizes the current state of knowledge regarding epigenetic cues that have evolved to regulate the pluripotent differentiation potential of embryonic stem cells and the lineage determination of developing dental pulp progenitors. PMID:28018144

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification.

PubMed

Vicinanza, Carla; Aquila, Iolanda; Scalise, Mariangela; Cristiano, Francesca; Marino, Fabiola; Cianflone, Eleonora; Mancuso, Teresa; Marotta, Pina; Sacco, Walter; Lewis, Fiona C; Couch, Liam; Shone, Victoria; Gritti, Giulia; Torella, Annalaura; Smith, Andrew J; Terracciano, Cesare Mn; Britti, Domenico; Veltri, Pierangelo; Indolfi, Ciro; Nadal-Ginard, Bernardo; Ellison-Hughes, Georgina M; Torella, Daniele

2017-12-01

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kit pos ) cells. The adult heart indeed contains a heterogeneous mixture of c-kit pos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kit pos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kit pos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kit pos sorting. The blood/endothelial lineage-committed (Lineage pos ) CD45 pos c-kit pos cardiac cells were compared to CD45 neg (Lineage neg /Lin neg ) c-kit pos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kit pos cardiac cells are blood/endothelial lineage-committed CD45 pos CD31 pos c-kit pos cells. In contrast, the Lin neg CD45 neg c-kit pos cardiac cell cohort, which represents ⩽10% of the total c-kit pos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kit neg and the blood/endothelial lineage-committed c-kit pos cardiac cells. Single Lin neg c-kit pos cell-derived clones, which represent only 1-2% of total c-kit pos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Lin neg c-kit pos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kit pos cardiac cells were injected. Thus, among the cardiac c-kit pos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

Gastrulation and pre-gastrulation morphogenesis, inductions, and gene expression: similarities and dissimilarities between urodelean and anuran embryos.

PubMed

Kaneda, Teruo; Motoki, Jun-ya Doi

2012-09-01

Studies of meso-endoderm and neural induction and subsequent body plan formation have been analyzed using mainly amphibians as the experimental model. Xenopus is currently the predominant model, because it best enables molecular analysis of these induction processes. However, much of the embryological information on these inductions (e.g., those of the Spemann-Mangold organizer), and on the morphogenetic movements of inductively interacting tissues, derives from research on non-model amphibians, especially urodeles. Although the final body pattern is strongly conserved in vertebrates, and although many of the same developmental genes are expressed, it has become evident that there are individually diverse modes of morphogenesis and timing of developmental events. Whether or not this diversity represents essential differences in the early induction processes remains unclear. The aim of this review is to compare the gastrulation process, induction processes, and gene expressions between a urodele, mainly Cynops pyrrhogaster, and an anura, Xenopus laevis, thereby to clarify conserved and diversified aspects. Cynops gastrulation differs significantly from that of Xenopus in that specification of the regions of the Xenopus dorsal marginal zone (DMZ) are specified before the onset of gastrulation, as marked by blastopore formation, whereas the equivalent state of specification does not occur in Cynops until the middle of gastrulation. Detailed comparison of the germ layer structure and morphogenetic movements during the pre-gastrula and gastrula stages shows that the entire gastrulation process should be divided into two phases of notochord induction and neural induction. Cynops undergoes these processes sequentially after the onset of gastrulation, whereas Xenopus undergoes notochord induction during a series of pre-gastrulation movements, and its traditionally defined period of gastrulation only includes the neural induction phase. Comparing the structure, fate, function and state of commitment of each domain of the DMZ of Xenopus and Cynops has revealed that the true form of the Spemann-Mangold organizer is suprablastoporal gsc-expressing endoderm that has notochord-inducing activity. Gsc-expressing deep endoderm and/or superficial endoderm in Xenopus is involved in inducing notochord during pre-gastrulation morphogenesis, rather than both gsc- and bra-expressing tissues being induced at the same time. Copyright © 2012 Elsevier Inc. All rights reserved.

The human lipodystrophy gene product Berardinelli-Seip congenital lipodystrophy 2/seipin plays a key role in adipocyte differentiation.

PubMed

Chen, Weiqin; Yechoor, Vijay K; Chang, Benny Hung-Junn; Li, Ming V; March, Keith L; Chan, Lawrence

2009-10-01

Mutations in the Berardinelli-Seip congenital lipodystrophy 2 gene (BSCL2) are the underlying defect in patients with congenital generalized lipodystrophy type 2. BSCL2 encodes a protein called seipin, whose function is largely unknown. In this study, we investigated the role of Bscl2 in the regulation of adipocyte differentiation. Bscl2 mRNA is highly up-regulated during standard hormone-induced adipogenesis in 3T3-L1 cells in vitro. However, this up-regulation does not occur during mesenchymal stem cell (C3H10T1/2 cells) commitment to the preadipocyte lineage. Knockdown of Bscl2 by short hairpin RNA in C3H10T1/2 cells has no effect on bone morphogenetic protein-4-induced preadipocyte commitment. However, knockdown in 3T3-L1 cells prevents adipogenesis induced by a standard hormone cocktail, but adipogenesis can be rescued by the addition of peroxisome proliferator-activated receptor-gamma agonist pioglitazone at an early stage of differentiation. Interestingly, pioglitazone-induced differentiation in the absence of standard hormone is not associated with up-regulated Bscl2 expression. On the other hand, short hairpin RNA-knockdown of Bscl2 largely blocks pioglitazone-induced adipose differentiation. These experiments suggest that Bscl2 may be essential for normal adipogenesis; it works upstream or at the level of peroxisome proliferator-activated receptor-gamma, enabling the latter to exert its full activity during adipogenesis. Loss of Bscl2 function thus interferes with the normal transcriptional cascade of adipogenesis during fat cell differentiation, resulting in near total loss of fat or lipodystrophy.

Dynamics and function of distal regulatory elements during neurogenesis and neuroplasticity

PubMed Central

Thakurela, Sudhir; Sahu, Sanjeeb Kumar; Garding, Angela; Tiwari, Vijay K.

2015-01-01

Gene regulation in mammals involves a complex interplay between promoters and distal regulatory elements that function in concert to drive precise spatiotemporal gene expression programs. However, the dynamics of the distal gene regulatory landscape and its function in the transcriptional reprogramming that underlies neurogenesis and neuronal activity remain largely unknown. Here, we performed a combinatorial analysis of genome-wide data sets for chromatin accessibility (FAIRE-seq) and the enhancer mark H3K27ac, revealing the highly dynamic nature of distal gene regulation during neurogenesis, which gets progressively restricted to distinct genomic regions as neurons acquire a post-mitotic, terminally differentiated state. We further find that the distal accessible and active regions serve as target sites for distinct transcription factors that function in a stage-specific manner to contribute to the transcriptional program underlying neuronal commitment and maturation. Mature neurons respond to a sustained activity of NMDA receptors by epigenetic reprogramming at a large number of distal regulatory regions as well as dramatic reorganization of super-enhancers. Such massive remodeling of the distal regulatory landscape in turn results in a transcriptome that confers a transient loss of neuronal identity and gain of cellular plasticity. Furthermore, NMDA receptor activity also induces many novel prosurvival genes that function in neuroprotective pathways. Taken together, these findings reveal the dynamics of the distal regulatory landscape during neurogenesis and uncover novel regulatory elements that function in concert with epigenetic mechanisms and transcription factors to generate the transcriptome underlying neuronal development and activity. PMID:26170447

The Convergence of Counseling and Psychiatric Genetics: An Essential Role for Counselors

ERIC Educational Resources Information Center

Douthit, Kathryn Z.

2006-01-01

Research in psychiatric genetics shows that many mental disorders commonly seen by counselors are associated with the presence of specific genes. Although findings in gene research call for some changes in the way counselors approach intervention, in most cases, the importance of the counseling profession's core commitments is underscored. This…

Emotional labour, job satisfaction and organizational commitment amongst clinical nurses: a questionnaire survey.

PubMed

Yang, Feng-Hua; Chang, Chen-Chieh

2008-06-01

According to Hochschild's (1983. The Managed Heart. Berkeley: University of California Press) classification of emotional labour, nursing staff express high emotional labour. This paper investigates how nursing staff influence job satisfaction and organizational commitment when they perform emotional labour. This paper examines the relationship between emotional labour, job satisfaction, and organizational commitment from the perspective of nursing staff. A questionnaire survey was carried out to explore these interrelationships. Teaching hospital in Taiwan. Questionnaires were distributed to 500 nursing staff; 295 valid questionnaires were collected and analysed-a 59% response rate. The questionnaires contained items on emotional labour, job satisfaction, and organizational commitment as well as some basic socio-demographics. In addition, descriptive statistics, correlation and linear structure relation (LISREL) were computed. Emotional display rule (EDR) was significantly and negatively related to job satisfaction. Surface acting (SA) was not significantly related to job satisfaction but demonstrated a significantly negative relationship with organizational commitment. Deep acting (DA) significantly and positively correlated with job satisfaction but demonstrated no significance with organizational commitment. The variety of emotions required (VER) was not significantly related to job satisfaction; frequency and duration of interaction (FDI) and negatively related to job satisfaction; and job satisfaction significantly and positively correlated with organizational commitment. We found that some dimensions of emotional labour significantly relate to job satisfaction. Job satisfaction positively affects organizational commitment and has an intervening effect on DA and organizational commitment.

The Wish to Die and the Wish to Commit Suicide in the Adolescent: Two Different Matters?

ERIC Educational Resources Information Center

Manor, Iris; Vincent, Michel; Tyano, Sam

2004-01-01

We shall try to demonstrate the difference between two wishes--the wish to die and the wish to commit suicide -- as they express themselves during adolescence. First, death is seen as irreversible, while the suicidal act, at least during adolescence, is seen as reversible. While thoughts of suicide may be a part of normal adolescence, and the…

Complexities and sequence similarities of mRNA populations of cholinergic (NS20-Y) and adrenergic (N1E-115) murine neuroblastoma cell lines.

PubMed

Strauss, W L

1990-07-01

The clonal murine neuroblastoma cell lines NS20-Y and N1E-115 have been proposed as models for examining the commitment of neural crest cells to either the cholinergic or adrenergic phenotype, respectively. The validity of this model depends in part on the extent to which these two cell lines have diverged as a result of their transformed, rather than neuronal properties. In order to quantitate differences in gene expression between NS20-Y and N1E-115 cells, the mRNA complexity of each cell type was determined. An analysis of the kinetics of hybridization of NS20-Y cell mRNA with cDNA prepared from NS20-Y cell mRNA demonstrated the presence of approximately 11,700 mRNA species assuming an average length of 1900 nucleotides. A similar analysis using mRNA isolated from N1E-115 cells and cDNA prepared from N1E-115 cell mRNA demonstrated that the adrenergic cell line expressed approximately 11,600 mRNA species. The species of mRNA expressed by each cell line were resolved into high, intermediate, and low abundance populations. In order to determine whether mRNAs were expressed by the cholinergic, but not by the adrenergic cell line, NS20-Y cDNA was hybridized to an excess of N1E-115 cell mRNA. An analysis of the solution hybridization kinetics from this procedure demonstrated that the two cell lines do not differ significantly in the nucleotide complexity of their mRNA populations. The extensive similarity between the two mRNA populations suggests that only a small number of genes are expressed differentially between the two cell lines and supports their use as models for the differentiation of cholinergic and adrenergic neurons.

Abnormal expression and functional characteristics of cyclic adenosine monophosphate response element binding protein in postmortem brain of suicide subjects.

PubMed

Dwivedi, Yogesh; Rao, Jagadeesh Sridhara; Rizavi, Hooriyah S; Kotowski, Jacek; Conley, Robert R; Roberts, Rosalinda C; Tamminga, Carol A; Pandey, Ghanshyam N

2003-03-01

Cyclic adenosine monophosphate response element binding protein (CREB) is a transcription factor that, on phosphorylation by protein kinases, is activated, and in response, regulates the transcription of many neuronally expressed genes. In view of the recent observations that catalytic properties and/or expression of many kinases that mediate their physiological responses through the activation of CREB are altered in the postmortem brain of subjects who commit suicide (hereafter referred to as suicide subjects), we examined the status of CREB in suicidal behavior. These studies were performed in Brodmann area (BA) 9 and hippocampus obtained from 26 suicide subjects and 20 nonpsychiatric healthy control subjects. Messenger RNA levels of CREB and neuron-specific enolase were determined in total RNA by means of quantitative reverse transcriptase-polymerase chain reaction. Protein levels and the functional characteristics of CREB were determined in nuclear fractions by means of Western blot and cyclic adenosine monophosphate response element (CRE)-DNA binding activity, respectively. In the same nuclear fraction, we determined the catalytic activity of cyclic adenosine monophosphate-stimulated protein kinase A by means of enzymatic assay. We observed a significant reduction in messenger RNA and protein levels of CREB, CRE-DNA binding activity, and basal and cyclic adenosine monophosphate-stimulated protein kinase A activity in BA 9 and hippocampus of suicide subjects, without any change in messenger RNA levels of neuron-specific enolase in BA 9. Except for protein kinase A activity, changes in CREB expression and CRE-DNA binding activity were present in all suicide subjects, irrespective of diagnosis. These changes were unrelated to postmortem intervals, age, sex, or antidepressant treatment. Given the significance of CREB in mediating various physiological functions through gene transcription, our results of decreased expression and functional characteristics of CREB in postmortem brain of suicide subjects suggest that CREB may play an important role in suicidal behavior.

Galectin-3 Shapes Antitumor Immune Responses by Suppressing CD8+ T Cells via LAG-3 and Inhibiting Expansion of Plasmacytoid Dendritic Cells.

PubMed

Kouo, Theodore; Huang, Lanqing; Pucsek, Alexandra B; Cao, Minwei; Solt, Sara; Armstrong, Todd; Jaffee, Elizabeth

2015-04-01

Galectin-3 is a 31-kDa lectin that modulates T-cell responses through several mechanisms, including apoptosis, T-cell receptor (TCR) cross-linking, and TCR downregulation. We found that patients with pancreatic ductal adenocarcinoma (PDA) who responded to a granulocyte-macrophage colony-stimulating factor-secreting allogeneic PDA vaccine developed neutralizing antibodies to galectin-3 after immunization. We show that galectin-3 binds activated antigen-committed CD8(+) T cells only in the tumor microenvironment. Galectin-3-deficient mice exhibit improved CD8(+) T-cell effector function and increased expression of several inflammatory genes. Galectin-3 binds to LAG-3, and LAG-3 expression is necessary for galectin-3-mediated suppression of CD8(+) T cells in vitro. Lastly, galectin-3-deficient mice have elevated levels of circulating plasmacytoid dendritic cells, which are superior to conventional dendritic cells in activating CD8(+) T cells. Thus, inhibiting galectin-3 in conjunction with CD8(+) T-cell-directed immunotherapies should enhance the tumor-specific immune response. ©2015 American Association for Cancer Research.

c-Myc-induced apoptosis in fibroblasts is inhibited by specific cytokines.

PubMed Central

Harrington, E A; Bennett, M R; Fanidi, A; Evan, G I

1994-01-01

We have investigated the mechanism by which deregulated expression of c-Myc induces death by apoptosis in serum-deprived fibroblasts. We demonstrate that Myc-induced apoptosis in low serum is inhibited by a restricted group of cytokines, principally the insulin-like growth factors and PDGF. Cytokine-mediated protection from apoptosis is not linked to the cytokines' abilities to promote growth. Protection from apoptosis is evident in the post-commitment (mitogen-independent) S/G2/M phases of the cell cycle and also in cells that are profoundly blocked in cell cycle progression by drugs. Moreover, IGF-I inhibition of apoptosis occurs in the absence of protein synthesis, and so does not require immediate early gene expression. We conclude that c-Myc-induced apoptosis does not result from a conflict of growth signals but appears to be a normal physiological aspect of c-Myc function whose execution is regulated by the availability of survival factors. We discuss the possible implications of these findings for models of mammalian cell growth in vivo. Images PMID:8045259

CCR6 Defines Memory B Cell Precursors in Mouse and Human Germinal Centers, Revealing Light-Zone Location and Predominant Low Antigen Affinity.

PubMed

Suan, Dan; Kräutler, Nike J; Maag, Jesper L V; Butt, Danyal; Bourne, Katherine; Hermes, Jana R; Avery, Danielle T; Young, Clara; Statham, Aaron; Elliott, Michael; Dinger, Marcel E; Basten, Antony; Tangye, Stuart G; Brink, Robert

2017-12-19

Memory B cells (MBCs) and plasma cells (PCs) constitute the two cellular outputs of germinal center (GC) responses that together facilitate long-term humoral immunity. Although expression of the transcription factor BLIMP-1 identifies cells undergoing PC differentiation, no such marker exists for cells committed to the MBC lineage. Here, we report that the chemokine receptor CCR6 uniquely marks MBC precursors in both mouse and human GCs. CCR6 + GC B cells were highly enriched within the GC light zone (LZ), were the most quiescent of all GC B cells, exhibited a cell-surface phenotype and gene expression signature indicative of an MBC transition, and possessed the augmented response characteristics of MBCs. MBC precursors within the GC LZ predominantly possessed a low affinity for antigen but also included cells from within the high-affinity pool. These data indicate a fundamental dichotomy between the processes that drive MBC and PC differentiation during GC responses. Copyright © 2017 Elsevier Inc. All rights reserved.

Reduced TCR signaling potential impairs negative selection but does not result in autoimmune disease

PubMed Central

Hwang, SuJin; Song, Ki-Duk; Lesourne, Renaud; Lee, Jan; Pinkhasov, Julia; Li, LiQi; El-Khoury, Dalal

2012-01-01

Negative selection and regulatory T (T reg) cell development are two thymus-dependent processes necessary for the enforcement of self-tolerance, and both require high-affinity interactions between the T cell receptor (TCR) and self-ligands. However, it remains unclear if they are similarly impacted by alterations in TCR signaling potential. We generated a knock-in allele (6F) of the TCR ζ chain gene encoding a mutant protein lacking signaling capability whose expression is controlled by endogenous ζ regulatory sequences. Although negative selection was defective in 6F/6F mice, leading to the survival of autoreactive T cells, 6F/6F mice did not develop autoimmune disease. We found that 6F/6F mice generated increased numbers of thymus-derived T reg cells. We show that attenuation of TCR signaling potential selectively impacts downstream signaling responses and that this differential effect favors Foxp3 expression and T reg cell lineage commitment. These results identify a potential compensatory pathway for the enforcement of immune tolerance in response to defective negative selection caused by reduced TCR signaling capability. PMID:22945921

Epigenetic Alterations in Cellular Immunity: New Insights into Autoimmune Diseases.

PubMed

Wang, Zijun; Lu, Qianjin; Wang, Zhihui

2017-01-01

Epigenetic modification is an additional regulator in immune responses as the genome-wide profiling somehow fails to explain the sophisticated mechanisms in autoimmune diseases. The effect of epigenetic modifications on adaptive immunity derives from their regulations to induce a permissive or negative gene expression. Epigenetic events, such as DNA methylation, histone modifications and microRNAs (miRNAs) are often found in T cell activation, differentiation and commitment which are the major parts in cellular immunity. Recognizing the complexity of interactions between epigenetic mechanisms and immune disturbance in autoimmune diseases is essential for the exploration of efficient therapeutic targets. In this review, we summarize a list of studies that indicate the significance of dysregulated epigenetic modifications in autoimmune diseases while focusing on T cell immunity. © 2017 The Author(s)Published by S. Karger AG, Basel.

Natural gene therapy in monozygotic twins with Fanconi anemia.

PubMed

Mankad, Anuj; Taniguchi, Toshiyasu; Cox, Barbara; Akkari, Yassmine; Rathbun, R Keaney; Lucas, Lora; Bagby, Grover; Olson, Susan; D'Andrea, Alan; Grompe, Markus

2006-04-15

Monozygotic twin sisters, with nonhematologic symptoms of Fanconi anemia (FA), were discovered to be somatic mosaics for mutations in the FANCA gene. Skin fibroblasts, but not lymphocytes or committed hematopoietic progenitors, were sensitive to DNA cross-linking agents. Molecular analysis revealed, in skin cells of both twins, a frameshift causing deletion in exon 27 (2555deltaT) and an exon 28 missense mutation (2670G>A/R880Q). The latter resulted in primarily cytoplasmic expression and reduced function of the mutant FANCA (R880Q) protein. Surprisingly, the same acquired exon 30 missense change (2927G>A/E966K) was detected in the hematopoietic cells of both sisters, but not in their fibroblasts, nor in either parent. This compensatory mutation existed in cis with the maternal exon 28 mutation, and it restored function and nuclear localization of the resulting protein. Both sisters have been free of hematologic symptoms for more than 2 decades, suggesting that this de novo mutation occurred prenatally in a single hematopoietic stem cell (HSC) in one twin and that descendants of this functionally corrected HSC, via intra-uterine circulation, repopulated the blood lineages of both sisters. This finding suggests that treating FA patients with gene therapy might require transduction of only a few hematopoietic stem cells.

Uhrf1 controls the self-renewal versus differentiation of hematopoietic stem cells by epigenetically regulating the cell-division modes

PubMed Central

Zhao, Jingyao; Chen, Xufeng; Song, Guangrong; Zhang, Jiali; Liu, Haifeng; Liu, Xiaolong

2017-01-01

Hematopoietic stem cells (HSCs) are able to both self-renew and differentiate. However, how individual HSC makes the decision between self-renewal and differentiation remains largely unknown. Here we report that ablation of the key epigenetic regulator Uhrf1 in the hematopoietic system depletes the HSC pool, leading to hematopoietic failure and lethality. Uhrf1-deficient HSCs display normal survival and proliferation, yet undergo erythroid-biased differentiation at the expense of self-renewal capacity. Notably, Uhrf1 is required for the establishment of DNA methylation patterns of erythroid-specific genes during HSC division. The expression of these genes is enhanced in the absence of Uhrf1, which disrupts the HSC-division modes by promoting the symmetric differentiation and suppressing the symmetric self-renewal. Moreover, overexpression of one of the up-regulated genes, Gata1, in HSCs is sufficient to phenocopy Uhrf1-deficient HSCs, which show impaired HSC symmetric self-renewal and increased differentiation commitment. Taken together, our findings suggest that Uhrf1 controls the self-renewal versus differentiation of HSC through epigenetically regulating the cell-division modes, thus providing unique insights into the relationship among Uhrf1-mediated DNA methylation, cell-division mode, and HSC fate decision. PMID:27956603

75 FR 56920 - Express Mail Open and Distribute and Priority Mail Open and Distribute

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-17

... POSTAL SERVICE 39 CFR Part 111 Express Mail Open and Distribute and Priority Mail Open and...] Open and Distribute containers. The Postal Service also proposes to revise the service commitment for Express Mail Open and Distribute as a guaranteed end of day product; and to add a five-pound minimum...

Cognitive simplicity and self-deception are crucial in martyrdom and suicide terrorism.

PubMed

Fink, Bernhard; Trivers, Robert

2014-08-01

Suicide attacks and terrorism are characterized by cognitive simplicity, which is related to self-deception. In justifying violence in pursuit of ideologically and/or politically driven commitment, people with high religious commitment may be particularly prone to mechanisms of self-deception. Related megalomania and glorious self-perception are typical of self-deception, and are thus crucial in the emergence and expression of (suicide) terrorism.

Assessment of 'cancer-prone personality' characteristics in healthy study subjects and in patients with breast disease and breast cancer using the commitment questionnaire: a prospective case-control study in Finland.

PubMed

Eskelinen, Matti; Ollonen, Paula

2011-11-01

The findings of a repressed expression of emotions in cancer patients contributed to the hypothesis developed by Lydia Temoshok of a type C personality ('cancer-prone'). To the Authors' knowledge, the associations between the 'cancer-prone personality' characteristics in commitment test and the risk of breast cancer (BC) have rarely been considered together in a prospective study. In an extension of the Kuopio Breast Cancer Study, 115 women with breast symptoms were evaluated for commitment test before any diagnostic procedures were carried out. The clinical examination and biopsy showed BC in 34 patients, benign breast disease (BBD) in 53 patients and 28 individuals were shown to be healthy study subjects (HSS). The BC group reported significantly more commitment to own children (Function A) (mean Commitment score, 3.14) than the patients in the BBD group (mean Commitment score, 3.51) and in the HSS group (mean Commitment score, 3.77) (p=0.05). The women in the BC group also reported more commitment to own husband (Function B) (mean Commitment score, 3.30) than the patients in the BBD group (mean Commitment score, 3.83) and the patients in the HSS group (mean Commitment score, 3.76). The BC group reported significantly more commitment to own work and own body (Function D and G) (mean Commitment scores, 3.20 and 3.50) than the patients in the BBD group (mean Commitment scores, 3.75 and 3.71) or HSS group (mean Commitment scores, 3.46 and 3.50). The mean sum (mean, SD) of the scores were significantly lower in the BC group (31.1, 5.8) than in the BBD (35.2, 6.9) and HSS group (36.4, 5.6) (p=0.02), showing more commitment in the BC group. In summary, patients with BC tended to have an increased risk for bearing the 'high commitment' characteristic and this pattern could contribute to cancer risk through immune and hormonal pathways.

Hippo Cascade Controls Lineage Commitment of Liver Tumors in Mice and Humans.

PubMed

Zhang, Shanshan; Wang, Jingxiao; Wang, Haichuan; Fan, Lingling; Fan, Biao; Zeng, Billy; Tao, Junyan; Li, Xiaolei; Che, Li; Cigliano, Antonio; Ribback, Silvia; Dombrowski, Frank; Chen, Bin; Cong, Wenming; Wei, Lixin; Calvisi, Diego F; Chen, Xin

2018-04-01

Primary liver cancer consists mainly of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). A subset of human HCCs expresses a ICC-like gene signature and is classified as ICC-like HCC. The Hippo pathway is a critical regulator of normal and malignant liver development. However, the precise function(s) of the Hippo cascade along liver carcinogenesis remain to be fully delineated. The role of the Hippo pathway in a murine mixed HCC/ICC model induced by activated forms of AKT and Ras oncogenes (AKT/Ras) was investigated. The authors demonstrated the inactivation of Hippo in AKT/Ras liver tumors leading to nuclear localization of Yap and TAZ. Coexpression of AKT/Ras with Lats2, which activates Hippo, or the dominant negative form of TEAD2 (dnTEAD2), which blocks Yap/TAZ activity, resulted in delayed hepatocarcinogenesis and elimination of ICC-like lesions in the liver. Mechanistically, Notch2 expression was found to be down-regulated by the Hippo pathway in liver tumors. Overexpression of Lats2 or dnTEAD2 in human HCC cell lines inhibited their growth and led to the decreased expression of ICC-like markers, as well as Notch2 expression. Altogether, this study supports the key role of the Hippo cascade in regulating the differentiation status of liver tumors. Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

How Quorum Sensing Connects Sporulation to Necrotrophism in Bacillus thuringiensis

PubMed Central

Poncet, Sandrine; Lazar, Noureddine; Li de la Sierra-Gallay, Inès; Gohar, Michel; Lereclus, Didier; Nessler, Sylvie

2016-01-01

Bacteria use quorum sensing to coordinate adaptation properties, cell fate or commitment to sporulation. The infectious cycle of Bacillus thuringiensis in the insect host is a powerful model to investigate the role of quorum sensing in natural conditions. It is tuned by communication systems regulators belonging to the RNPP family and directly regulated by re-internalized signaling peptides. One such RNPP regulator, NprR, acts in the presence of its cognate signaling peptide NprX as a transcription factor, regulating a set of genes involved in the survival of these bacteria in the insect cadaver. Here, we demonstrate that, in the absence of NprX and independently of its transcriptional activator function, NprR negatively controls sporulation. NprR inhibits expression of Spo0A-regulated genes by preventing the KinA-dependent phosphorylation of the phosphotransferase Spo0F, thus delaying initiation of the sporulation process. This NprR function displays striking similarities with the Rap proteins, which also belong to the RNPP family, but are devoid of DNA-binding domain and indirectly control gene expression via protein-protein interactions in Bacilli. Conservation of the Rap residues directly interacting with Spo0F further suggests a common inhibition of the sporulation phosphorelay. The crystal structure of apo NprR confirms that NprR displays a highly flexible Rap-like structure. We propose a molecular regulatory mechanism in which key residues of the bifunctional regulator NprR are directly and alternatively involved in its two functions. NprX binding switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. NprR thus tightly coordinates sporulation and necrotrophism, ensuring survival and dissemination of the bacteria during host infection. PMID:27483473

How Quorum Sensing Connects Sporulation to Necrotrophism in Bacillus thuringiensis.

PubMed

Perchat, Stéphane; Talagas, Antoine; Poncet, Sandrine; Lazar, Noureddine; Li de la Sierra-Gallay, Inès; Gohar, Michel; Lereclus, Didier; Nessler, Sylvie

2016-08-01

Bacteria use quorum sensing to coordinate adaptation properties, cell fate or commitment to sporulation. The infectious cycle of Bacillus thuringiensis in the insect host is a powerful model to investigate the role of quorum sensing in natural conditions. It is tuned by communication systems regulators belonging to the RNPP family and directly regulated by re-internalized signaling peptides. One such RNPP regulator, NprR, acts in the presence of its cognate signaling peptide NprX as a transcription factor, regulating a set of genes involved in the survival of these bacteria in the insect cadaver. Here, we demonstrate that, in the absence of NprX and independently of its transcriptional activator function, NprR negatively controls sporulation. NprR inhibits expression of Spo0A-regulated genes by preventing the KinA-dependent phosphorylation of the phosphotransferase Spo0F, thus delaying initiation of the sporulation process. This NprR function displays striking similarities with the Rap proteins, which also belong to the RNPP family, but are devoid of DNA-binding domain and indirectly control gene expression via protein-protein interactions in Bacilli. Conservation of the Rap residues directly interacting with Spo0F further suggests a common inhibition of the sporulation phosphorelay. The crystal structure of apo NprR confirms that NprR displays a highly flexible Rap-like structure. We propose a molecular regulatory mechanism in which key residues of the bifunctional regulator NprR are directly and alternatively involved in its two functions. NprX binding switches NprR from a dimeric inhibitor of sporulation to a tetrameric transcriptional activator involved in the necrotrophic lifestyle of B. thuringiensis. NprR thus tightly coordinates sporulation and necrotrophism, ensuring survival and dissemination of the bacteria during host infection.

Ancient Antimicrobial Peptides Kill Antibiotic-Resistant Pathogens: Australian Mammals Provide New Options

PubMed Central

Wang, Jianghui; Wong, Emily S. W.; Whitley, Jane C.; Li, Jian; Stringer, Jessica M.; Short, Kirsty R.; Renfree, Marilyn B.

2011-01-01

Background To overcome the increasing resistance of pathogens to existing antibiotics the 10×'20 Initiative declared the urgent need for a global commitment to develop 10 new antimicrobial drugs by the year 2020. Naturally occurring animal antibiotics are an obvious place to start. The recently sequenced genomes of mammals that are divergent from human and mouse, including the tammar wallaby and the platypus, provide an opportunity to discover novel antimicrobials. Marsupials and monotremes are ideal potential sources of new antimicrobials because they give birth to underdeveloped immunologically naïve young that develop outside the sterile confines of a uterus in harsh pathogen-laden environments. While their adaptive immune system develops innate immune factors produced either by the mother or by the young must play a key role in protecting the immune-compromised young. In this study we focus on the cathelicidins, a key family of antimicrobial peptide genes. Principal Finding We identified 14 cathelicidin genes in the tammar wallaby genome and 8 in the platypus genome. The tammar genes were expressed in the mammary gland during early lactation before the adaptive immune system of the young develops, as well as in the skin of the pouch young. Both platypus and tammar peptides were effective in killing a broad range of bacterial pathogens. One potent peptide, expressed in the early stages of tammar lactation, effectively killed multidrug-resistant clinical isolates of Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. Conclusions and Significance Marsupial and monotreme young are protected by antimicrobial peptides that are potent, broad spectrum and salt resistant. The genomes of our distant relatives may hold the key for the development of novel drugs to combat multidrug-resistant pathogens. PMID:21912615

The Effect of Perceiving a Calling on Pakistani Nurses' Organizational Commitment, Organizational Citizenship Behavior, and Job Stress.

PubMed

Afsar, Bilal; Shahjehan, Asad; Cheema, Sadia; Javed, Farheen

2018-03-01

People differ considerably in the way in which they express and experience their nursing careers. The positive effects associated with having a calling may differ substantially based on individuals' abilities to live out their callings. In a working world where many individuals have little to no choice in their type of employment and thus are unable to live out a calling even if they have one, the current study examined how perceiving a calling and living a calling interacted to predict organizational commitment, organizational citizenship behavior, and job stress with career commitment mediating the effect of the interactions on the three outcome variables. The purpose of the study is to investigate the mediating effect of career commitment between the relationships of calling and (a) nurses' attitudes (organizational commitment), (b) behaviors (organizational citizenship behavior), and (c) subjective experiences regarding work (job stress). Using a descriptive exploratory design, data were collected from 332 registered nurses working in Pakistani hospitals. Descriptive analysis and hierarchical regression analysis were used for data analysis. Living a calling moderated the effect of calling on career commitment, organizational citizenship behavior, and job stress, and career commitment fully mediated the effect of calling on organizational commitment, organizational citizenship behavior, and job stress. Increasing the understanding of calling, living a calling, and career commitment may increase nurses' organizational commitment and organizational citizenship behavior and decrease job stress. The study provided evidence to help nursing managers and health policy makers integrate knowledge and skills related to calling into career interventions and help nurses discover their calling.

Signal transduction in the carnivorous plant Sarracenia purpurea. Regulation of secretory hydrolase expression during development and in response to resources.

PubMed Central

Gallie, D R; Chang, S C

1997-01-01

Carnivory in plants has developed as an evolutionary adaptation to nutrient-poor environments. A significant investment of the resources of a carnivorous plant is committed to producing the traps, attractants, and digestive enzymes needed for the carnivory. The cost:benefit ratio of carnivory can be improved by either maximizing the prey capture rate or by reducing the metabolic commitment toward carnivory. Using the pitcher plant Sarracenia purpurea, we have investigated whether the expression of the hydrolytic enzymes needed for digestion is regulated in response to the presence of prey. Expression of protease, RNase, nuclease, and phosphatase activities could be induced in the fluid of nonactive traps by the addition of nucleic acids, protein, or reduced nitrogen, suggesting that hydrolase expression is induced upon perception of the appropriate chemical signal. Hydrolase expression was also developmentally controlled since expression commenced upon opening of a trap, increased for several days, and in the absence of prey largely ceased within 2 weeks. Nevertheless, the traps remained competent to induce expression in response to the appropriate signals. These data suggest that in young traps hydrolase expression is developmentally regulated, which is later replaced by a signal transduction mechanism, and they demonstrate the ability of a carnivorous species to respond to the availability of resources. PMID:9414556

Signal transduction in the carnivorous plant Sarracenia purpurea. Regulation of secretory hydrolase expression during development and in response to resources.

PubMed

Gallie, D R; Chang, S C

1997-12-01

Carnivory in plants has developed as an evolutionary adaptation to nutrient-poor environments. A significant investment of the resources of a carnivorous plant is committed to producing the traps, attractants, and digestive enzymes needed for the carnivory. The cost:benefit ratio of carnivory can be improved by either maximizing the prey capture rate or by reducing the metabolic commitment toward carnivory. Using the pitcher plant Sarracenia purpurea, we have investigated whether the expression of the hydrolytic enzymes needed for digestion is regulated in response to the presence of prey. Expression of protease, RNase, nuclease, and phosphatase activities could be induced in the fluid of nonactive traps by the addition of nucleic acids, protein, or reduced nitrogen, suggesting that hydrolase expression is induced upon perception of the appropriate chemical signal. Hydrolase expression was also developmentally controlled since expression commenced upon opening of a trap, increased for several days, and in the absence of prey largely ceased within 2 weeks. Nevertheless, the traps remained competent to induce expression in response to the appropriate signals. These data suggest that in young traps hydrolase expression is developmentally regulated, which is later replaced by a signal transduction mechanism, and they demonstrate the ability of a carnivorous species to respond to the availability of resources.

CD200-expressing human basal cell carcinoma cells initiate tumor growth.

PubMed

Colmont, Chantal S; Benketah, Antisar; Reed, Simon H; Hawk, Nga V; Telford, William G; Ohyama, Manabu; Udey, Mark C; Yee, Carole L; Vogel, Jonathan C; Patel, Girish K

2013-01-22

Smoothened antagonists directly target the genetic basis of human basal cell carcinoma (BCC), the most common of all cancers. These drugs inhibit BCC growth, but they are not curative. Although BCC cells are monomorphic, immunofluorescence microscopy reveals a complex hierarchical pattern of growth with inward differentiation along hair follicle lineages. Most BCC cells express the transcription factor KLF4 and are committed to terminal differentiation. A small CD200(+) CD45(-) BCC subpopulation that represents 1.63 ± 1.11% of all BCC cells resides in small clusters at the tumor periphery. By using reproducible in vivo xenograft growth assays, we determined that tumor initiating cell frequencies approximate one per 1.5 million unsorted BCC cells. The CD200(+) CD45(-) BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200(+) CD45(-) cells, representing ~1,500-fold enrichment. CD200(-) CD45(-) BCC cells were unable to form tumors. These findings establish a platform to study the effects of Smoothened antagonists on BCC tumor initiating cell and also suggest that currently available anti-CD200 therapy be considered, either as monotherapy or an adjunct to Smoothened antagonists, in the treatment of inoperable BCC.

An Efficient Electroporation Protocol for the Genetic Modification of Mammalian Cells

PubMed Central

Chicaybam, Leonardo; Barcelos, Camila; Peixoto, Barbara; Carneiro, Mayra; Limia, Cintia Gomez; Redondo, Patrícia; Lira, Carla; Paraguassú-Braga, Flávio; Vasconcelos, Zilton Farias Meira De; Barros, Luciana; Bonamino, Martin Hernán

2017-01-01

Genetic modification of cell lines and primary cells is an expensive and cumbersome approach, often involving the use of viral vectors. Electroporation using square-wave generating devices, like Lonza’s Nucleofector, is a widely used option, but the costs associated with the acquisition of electroporation kits and the transient transgene expression might hamper the utility of this methodology. In the present work, we show that our in-house developed buffers, termed Chicabuffers, can be efficiently used to electroporate cell lines and primary cells from murine and human origin. Using the Nucleofector II device, we electroporated 14 different cell lines and also primary cells, like mesenchymal stem cells and cord blood CD34+, providing optimized protocols for each of them. Moreover, when combined with sleeping beauty-based transposon system, long-term transgene expression could be achieved in all types of cells tested. Transgene expression was stable and did not interfere with CD34+ differentiation to committed progenitors. We also show that these buffers can be used in CRISPR-mediated editing of PDCD1 gene locus in 293T and human peripheral blood mononuclear cells. The optimized protocols reported in this study provide a suitable and cost-effective platform for the genetic modification of cells, facilitating the widespread adoption of this technology. PMID:28168187

Congenital myasthenic syndromes due to mutations in ALG2 and ALG14.

PubMed

Cossins, Judith; Belaya, Katsiaryna; Hicks, Debbie; Salih, Mustafa A; Finlayson, Sarah; Carboni, Nicola; Liu, Wei Wei; Maxwell, Susan; Zoltowska, Katarzyna; Farsani, Golara Torabi; Laval, Steven; Seidhamed, Mohammed Zain; Donnelly, Peter; Bentley, David; McGowan, Simon J; Müller, Juliane; Palace, Jacqueline; Lochmüller, Hanns; Beeson, David

2013-03-01

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed linkage analysis, whole-exome and whole-genome sequencing to determine the underlying defect in patients with an inherited limb-girdle pattern of myasthenic weakness. We identify ALG14 and ALG2 as novel genes in which mutations cause a congenital myasthenic syndrome. Through analogy with yeast, ALG14 is thought to form a multiglycosyltransferase complex with ALG13 and DPAGT1 that catalyses the first two committed steps of asparagine-linked protein glycosylation. We show that ALG14 is concentrated at the muscle motor endplates and small interfering RNA silencing of ALG14 results in reduced cell-surface expression of muscle acetylcholine receptor expressed in human embryonic kidney 293 cells. ALG2 is an alpha-1,3-mannosyltransferase that also catalyses early steps in the asparagine-linked glycosylation pathway. Mutations were identified in two kinships, with mutation ALG2p.Val68Gly found to severely reduce ALG2 expression both in patient muscle, and in cell cultures. Identification of DPAGT1, ALG14 and ALG2 mutations as a cause of congenital myasthenic syndrome underscores the importance of asparagine-linked protein glycosylation for proper functioning of the neuromuscular junction. These syndromes form part of the wider spectrum of congenital disorders of glycosylation caused by impaired asparagine-linked glycosylation. It is likely that further genes encoding components of this pathway will be associated with congenital myasthenic syndromes or impaired neuromuscular transmission as part of a more severe multisystem disorder. Our findings suggest that treatment with cholinesterase inhibitors may improve muscle function in many of the congenital disorders of glycosylation.

Expression of a GALACTINOL SYNTHASE Gene in Tomato Seeds Is Up-Regulated before Maturation Desiccation and Again after Imbibition whenever Radicle Protrusion Is Prevented1

PubMed Central

Downie, Bruce; Gurusinghe, Sunitha; Dahal, Petambar; Thacker, Richard R.; Snyder, John C.; Nonogaki, Hiroyuki; Yim, Kyuock; Fukanaga, Keith; Alvarado, Veria; Bradford, Kent J.

2003-01-01

Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance. PMID:12644684

Single-cell RNA-seq and computational analysis using temporal mixture modelling resolves Th1/Tfh fate bifurcation in malaria

PubMed Central

Lönnberg, Tapio; Svensson, Valentine; James, Kylie R.; Fernandez-Ruiz, Daniel; Sebina, Ismail; Montandon, Ruddy; Soon, Megan S. F.; Fogg, Lily G.; Nair, Arya Sheela; Liligeto, Urijah; Stubbington, Michael J. T.; Ly, Lam-Ha; Bagger, Frederik Otzen; Zwiessele, Max; Lawrence, Neil D.; Souza-Fonseca-Guimaraes, Fernando; Bunn, Patrick T.; Engwerda, Christian R.; Heath, William R.; Billker, Oliver; Stegle, Oliver; Haque, Ashraful; Teichmann, Sarah A.

2017-01-01

Differentiation of naïve CD4+ T cells into functionally distinct T helper subsets is crucial for the orchestration of immune responses. Due to extensive heterogeneity and multiple overlapping transcriptional programs in differentiating T cell populations, this process has remained a challenge for systematic dissection in vivo. By using single-cell transcriptomics and computational analysis using a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of Th1 and Tfh cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous TCR sequences, we first demonstrated that Th1/Tfh bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with Th1 or Tfh fates, and demonstrated a T-cell intrinsic role for Galectin-1 in supporting a Th1 differentiation. We also revealed the close molecular relationship between Th1 and IL-10-producing Tr1 cells in this infection. Th1 and Tfh fates emerged from a highly proliferative precursor that upregulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell in driving Th1/Tfh fates. In particular, we found that precursor Th cells were coached towards a Th1 but not a Tfh fate by inflammatory monocytes. Thus, by integrating genomic and computational approaches, our study has provided two unique resources, a database www.PlasmoTH.org, which facilitates discovery of novel factors controlling Th1/Tfh fate commitment, and more generally, GPfates, a modelling framework for characterizing cell differentiation towards multiple fates. PMID:28345074

Differential muscle regulatory factor gene expression between larval and adult myogenesis in the frog Xenopus laevis: adult myogenic cell-specific myf5 upregulation and its relation to the notochord suppression of adult muscle differentiation.

PubMed

Yamane, Hitomi; Nishikawa, Akio

2013-08-01

During Xenopus laevis metamorphosis, larval-to-adult muscle conversion depends on the differential responses of adult and larval myogenic cells to thyroid hormone. Essential differences in cell growth, differentiation, and hormone-dependent life-or-death fate have been reported between cultured larval (tail) and adult (hindlimb) myogenic cells. A previous study revealed that tail notochord cells suppress terminal differentiation in adult (but not larval) myogenic cells. However, little is known about the differences in expression patterns of myogenic regulatory factors (MRF) and the satellite cell marker Pax7 between adult and larval myogenic cells. In the present study, we compared mRNA expression of these factors between the two types. At first, reverse transcription polymerase chain reaction analysis of hindlimb buds showed sequential upregulation of myf5, myogenin, myod, and mrf4 during stages 50-54, when limb buds elongate and muscles begin to form. By contrast, in the tail, there was no such increase during the same period. Secondary, these results were duplicated in vitro: adult myogenic cells upregulated myf5, myod, and pax7 in the early culture period, followed by myogenin upregulation and myotube differentiation, while larval myogenic cells did not upregulate these genes and precociously started myotube differentiation. Thirdly, myf5 upregulation and early-phase proliferation in adult myogenic cells were potently inhibited by the presence of notochord cells, suggesting that notochord cells suppress adult myogenesis through inhibiting the transition from Myf5(-) stem cells to Myf5(+) committed myoblasts. All of the data presented here suggest that myf5 upregulation can be a good criterion for the activation of adult myogenesis during X. laevis metamorphosis.

The potential of 3-dimensional construct engineered from poly(lactic-co-glycolic acid)/fibrin hybrid scaffold seeded with bone marrow mesenchymal stem cells for in vitro cartilage tissue engineering.

PubMed

Abdul Rahman, Rozlin; Mohamad Sukri, Norhamiza; Md Nazir, Noorhidayah; Ahmad Radzi, Muhammad Aa'zamuddin; Zulkifly, Ahmad Hafiz; Che Ahmad, Aminudin; Hashi, Abdurezak Abdulahi; Abdul Rahman, Suzanah; Sha'ban, Munirah

2015-08-01

Articular cartilage is well known for its simple uniqueness of avascular and aneural structure that has limited capacity to heal itself when injured. The use of three dimensional construct in tissue engineering holds great potential in regenerating cartilage defects. This study evaluated the in vitro cartilaginous tissue formation using rabbit's bone marrow mesenchymal stem cells (BMSCs)-seeded onto poly(lactic-co-glycolic acid) PLGA/fibrin and PLGA scaffolds. The in vitro cartilaginous engineered constructs were evaluated by gross inspection, histology, cell proliferation, gene expression and sulphated glycosaminoglycan (sGAG) production at week 1, 2 and 3. After 3 weeks of culture, the PLGA/fibrin construct demonstrated gross features similar to the native tissue with smooth, firm and glistening appearance, superior histoarchitectural and better cartilaginous extracellular matrix compound in concert with the positive glycosaminoglycan accumulation on Alcian blue. Significantly higher cell proliferation in PLGA/fibrin construct was noted at day-7, day-14 and day-21 (p<0.05 respectively). Both constructs expressed the accumulation of collagen type II, collagen type IX, aggrecan and sox9, showed down-regulation of collagen type I as well as produced relative sGAG content with PLGA/fibrin construct exhibited better gene expression in all profiles and showed significantly higher relative sGAG content at each time point (p<0.05). This study suggested that with optimum in vitro manipulation, PLGA/fibrin when seeded with pluripotent non-committed BMSCs has the capability to differentiate into chondrogenic lineage and may serve as a prospective construct to be developed as functional tissue engineered cartilage. Copyright © 2015 Elsevier Ltd. All rights reserved.

Enhancing solubility of deoxyxylulose phosphate pathway enzymes for microbial isoprenoid production

PubMed Central

2012-01-01

Background Recombinant proteins are routinely overexpressed in metabolic engineering. It is well known that some over-expressed heterologous recombinant enzymes are insoluble with little or no enzymatic activity. This study examined the solubility of over-expressed homologous enzymes of the deoxyxylulose phosphate pathway (DXP) and the impact of inclusion body formation on metabolic engineering of microbes. Results Four enzymes of this pathway (DXS, ISPG, ISPH and ISPA), but not all, were highly insoluble, regardless of the expression systems used. Insoluble dxs (the committed enzyme of DXP pathway) was found to be inactive. Expressions of fusion tags did not significantly improve the solubility of dxs. However, hypertonic media containing sorbitol, an osmolyte, successfully doubled the solubility of dxs, with the concomitant improvement in microbial production of the metabolite, DXP. Similarly, sorbitol significantly improved the production of soluble and functional ERG12, the committed enzyme in the mevalonate pathway. Conclusion This study demonstrated the unanticipated findings that some over-expressed homologous enzymes of the DXP pathway were highly insoluble, forming inclusion bodies, which affected metabolite formation. Sorbitol was found to increase both the solubility and function of some of these over-expressed enzymes, a strategy to increase the production of secondary metabolites. PMID:23148661

Enforcement of γδ-lineage commitment by the pre-T-cell receptor in precursors with weak γδ-TCR signals.

PubMed

Zarin, Payam; Wong, Gladys W; Mohtashami, Mahmood; Wiest, David L; Zúñiga-Pflücker, Juan Carlos

2014-04-15

Developing thymocytes bifurcate from a bipotent precursor into αβ- or γδ-lineage T cells. Considering this common origin and the fact that the T-cell receptor (TCR) β-, γ-, and δ-chains simultaneously rearrange at the double negative (DN) stage of development, the possibility exists that a given DN cell can express and transmit signals through both the pre-TCR and γδ-TCR. Here, we tested this scenario by defining the differentiation outcomes and criteria for lineage choice when both TCR-β and γδ-TCR are simultaneously expressed in Rag2(-/-) DN cells via retroviral transduction. Our results showed that Rag2(-/-) DN cells expressing both TCRs developed along the γδ-lineage, down-regulated CD24 expression, and up-regulated CD73 expression, showed a γδ-biased gene-expression profile, and produced IFN-γ in response to stimulation. However, in the absence of Inhibitor of DNA-binding 3 expression and strong γδ-TCR ligand, γδ-expressing cells showed a lower propensity to differentiate along the γδ-lineage. Importantly, differentiation along the γδ-lineage was restored by pre-TCR coexpression, which induced greater down-regulation of CD24, higher levels of CD73, Nr4a2, and Rgs1, and recovery of functional competence to produce IFN-γ. These results confirm a requirement for a strong γδ-TCR ligand engagement to promote maturation along the γδ T-cell lineage, whereas additional signals from the pre-TCR can serve to enforce a γδ-lineage choice in the case of weaker γδ-TCR signals. Taken together, these findings further cement the view that the cumulative signal strength sensed by developing DN cells serves to dictate its lineage choice.

GEM-TREND: a web tool for gene expression data mining toward relevant network discovery

PubMed Central

Feng, Chunlai; Araki, Michihiro; Kunimoto, Ryo; Tamon, Akiko; Makiguchi, Hiroki; Niijima, Satoshi; Tsujimoto, Gozoh; Okuno, Yasushi

2009-01-01

Background DNA microarray technology provides us with a first step toward the goal of uncovering gene functions on a genomic scale. In recent years, vast amounts of gene expression data have been collected, much of which are available in public databases, such as the Gene Expression Omnibus (GEO). To date, most researchers have been manually retrieving data from databases through web browsers using accession numbers (IDs) or keywords, but gene-expression patterns are not considered when retrieving such data. The Connectivity Map was recently introduced to compare gene expression data by introducing gene-expression signatures (represented by a set of genes with up- or down-regulated labels according to their biological states) and is available as a web tool for detecting similar gene-expression signatures from a limited data set (approximately 7,000 expression profiles representing 1,309 compounds). In order to support researchers to utilize the public gene expression data more effectively, we developed a web tool for finding similar gene expression data and generating its co-expression networks from a publicly available database. Results GEM-TREND, a web tool for searching gene expression data, allows users to search data from GEO using gene-expression signatures or gene expression ratio data as a query and retrieve gene expression data by comparing gene-expression pattern between the query and GEO gene expression data. The comparison methods are based on the nonparametric, rank-based pattern matching approach of Lamb et al. (Science 2006) with the additional calculation of statistical significance. The web tool was tested using gene expression ratio data randomly extracted from the GEO and with in-house microarray data, respectively. The results validated the ability of GEM-TREND to retrieve gene expression entries biologically related to a query from GEO. For further analysis, a network visualization interface is also provided, whereby genes and gene annotations are dynamically linked to external data repositories. Conclusion GEM-TREND was developed to retrieve gene expression data by comparing query gene-expression pattern with those of GEO gene expression data. It could be a very useful resource for finding similar gene expression profiles and constructing its gene co-expression networks from a publicly available database. GEM-TREND was designed to be user-friendly and is expected to support knowledge discovery. GEM-TREND is freely available at . PMID:19728865

GEM-TREND: a web tool for gene expression data mining toward relevant network discovery.

PubMed

Feng, Chunlai; Araki, Michihiro; Kunimoto, Ryo; Tamon, Akiko; Makiguchi, Hiroki; Niijima, Satoshi; Tsujimoto, Gozoh; Okuno, Yasushi

2009-09-03

DNA microarray technology provides us with a first step toward the goal of uncovering gene functions on a genomic scale. In recent years, vast amounts of gene expression data have been collected, much of which are available in public databases, such as the Gene Expression Omnibus (GEO). To date, most researchers have been manually retrieving data from databases through web browsers using accession numbers (IDs) or keywords, but gene-expression patterns are not considered when retrieving such data. The Connectivity Map was recently introduced to compare gene expression data by introducing gene-expression signatures (represented by a set of genes with up- or down-regulated labels according to their biological states) and is available as a web tool for detecting similar gene-expression signatures from a limited data set (approximately 7,000 expression profiles representing 1,309 compounds). In order to support researchers to utilize the public gene expression data more effectively, we developed a web tool for finding similar gene expression data and generating its co-expression networks from a publicly available database. GEM-TREND, a web tool for searching gene expression data, allows users to search data from GEO using gene-expression signatures or gene expression ratio data as a query and retrieve gene expression data by comparing gene-expression pattern between the query and GEO gene expression data. The comparison methods are based on the nonparametric, rank-based pattern matching approach of Lamb et al. (Science 2006) with the additional calculation of statistical significance. The web tool was tested using gene expression ratio data randomly extracted from the GEO and with in-house microarray data, respectively. The results validated the ability of GEM-TREND to retrieve gene expression entries biologically related to a query from GEO. For further analysis, a network visualization interface is also provided, whereby genes and gene annotations are dynamically linked to external data repositories. GEM-TREND was developed to retrieve gene expression data by comparing query gene-expression pattern with those of GEO gene expression data. It could be a very useful resource for finding similar gene expression profiles and constructing its gene co-expression networks from a publicly available database. GEM-TREND was designed to be user-friendly and is expected to support knowledge discovery. GEM-TREND is freely available at http://cgs.pharm.kyoto-u.ac.jp/services/network.

GUN4-Porphyrin Complexes Bind the ChlH/GUN5 Subunit of Mg-Chelatase and Promote Chlorophyll Biosynthesis in Arabidopsis[W

PubMed Central

Adhikari, Neil D.; Froehlich, John E.; Strand, Deserah D.; Buck, Stephanie M.; Kramer, David M.; Larkin, Robert M.

2011-01-01

The GENOMES UNCOUPLED4 (GUN4) protein stimulates chlorophyll biosynthesis by activating Mg-chelatase, the enzyme that commits protoporphyrin IX to chlorophyll biosynthesis. This stimulation depends on GUN4 binding the ChlH subunit of Mg-chelatase and the porphyrin substrate and product of Mg-chelatase. After binding porphyrins, GUN4 associates more stably with chloroplast membranes and was proposed to promote interactions between ChlH and chloroplast membranes—the site of Mg-chelatase activity. GUN4 was also proposed to attenuate the production of reactive oxygen species (ROS) by binding and shielding light-exposed porphyrins from collisions with O2. To test these proposals, we first engineered Arabidopsis thaliana plants that express only porphyrin binding–deficient forms of GUN4. Using these transgenic plants and particular mutants, we found that the porphyrin binding activity of GUN4 and Mg-chelatase contribute to the accumulation of chlorophyll, GUN4, and Mg-chelatase subunits. Also, we found that the porphyrin binding activity of GUN4 and Mg-chelatase affect the associations of GUN4 and ChlH with chloroplast membranes and have various effects on the expression of ROS-inducible genes. Based on our findings, we conclude that ChlH and GUN4 use distinct mechanisms to associate with chloroplast membranes and that mutant alleles of GUN4 and Mg-chelatase genes cause sensitivity to intense light by a mechanism that is potentially complex. PMID:21467578

Understanding the role of growth factors in modulating stem cell tenogenesis.

PubMed

Gonçalves, Ana I; Rodrigues, Márcia T; Lee, Sang-Jin; Atala, Anthony; Yoo, James J; Reis, Rui L; Gomes, Manuela E

2013-01-01

Current treatments for tendon injuries often fail to fully restore joint biomechanics leading to the recurrence of symptoms, and thus resulting in a significant health problem with a relevant social impact worldwide. Cell-based approaches involving the use of stem cells might enable tailoring a successful tendon regeneration outcome. As growth factors (GFs) powerfully regulate the cell biological response, their exogenous addition can further stimulate stem cells into the tenogenic lineage, which might eventually depend on stem cells source. In the present study we investigate the tenogenic differentiation potential of human- amniotic fluid stem cells (hAFSCs) and adipose-derived stem cells (hASCs) with several GFs associated to tendon development and healing; namely, EGF, bFGF, PDGF-BB and TGF-β1. Stem cells response to biochemical stimuli was studied by screening of tendon-related genes (collagen type I, III, decorin, tenascin C and scleraxis) and proteins found in tendon extracellular matrix (ECM) (Collagen I, III, and Tenascin C). Despite the fact that GFs did not seem to influence the synthesis of tendon ECM proteins, EGF and bFGF influenced the expression of tendon-related genes in hAFSCs, while EGF and PDGF-BB stimulated the genetic expression in hASCs. Overall results on cellular alignment morphology, immunolocalization and PCR analysis indicated that both stem cell source can be biochemically induced towards tenogenic commitment, validating the potential of hASCs and hAFSCs for tendon regeneration strategies.

Enhanced neuro-therapeutic potential of Wharton's Jelly-derived mesenchymal stem cells in comparison with bone marrow mesenchymal stem cells culture.

PubMed

Drela, Katarzyna; Lech, Wioletta; Figiel-Dabrowska, Anna; Zychowicz, Marzena; Mikula, Michał; Sarnowska, Anna; Domanska-Janik, Krystyna

2016-04-01

Substantial inconsistencies in mesenchymal stem (stromal) cell (MSC) therapy reported in early translational and clinical studies may indicate need for selection of the proper cell population for any particular therapeutic purpose. In the present study we have examined stromal stem cells derived either from umbilical cord Wharton's Jelly (WJ-MSC) or bone marrow (BM-MSC) of adult, healthy donors. The cells characterized in accordance with the International Society for Cellular Therapy (ISCT) indications as well as other phenotypic and functional parameters have been compared under strictly controlled culture conditions. WJ-MSC, in comparison with BM-MSC, exhibited a higher proliferation rate, a greater expansion capability being additionally stimulated under low-oxygen atmosphere, enhanced neurotrophic factors gene expression and spontaneous tendency toward a neural lineage differentiation commitment confirmed by protein and gene marker induction. Our data suggest that WJ-MSC may represent an example of immature-type "pre-MSC," where a substantial cellular component is embryonic-like, pluripotent derivatives with the default neural-like differentiation. These cells may contribute in different extents to nearly all classical MSC populations adversely correlated with the age of cell donors. Our data suggest that neuro-epithelial markers, like nestin, stage specific embryonic antigens-4 or α-smooth muscle actin expressions, may serve as useful indicators of MSC culture neuro-regeneration-associated potency. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells

PubMed Central

Sviderskaya, Elena V.; Easty, David J.; Lawrence, Mark A.; Sánchez, Daniel P.; Negulyaev, Yuri A.; Patel, Ricken H.; Anand, Praveen; Korchev, Yuri E.; Bennett, Dorothy C.

2009-01-01

Stem cells, that is, cells that can both reproduce themselves and differentiate into functional cell types, attract much interest as potential aids to healing and disease therapy. Embryonic neural crest is pluripotent and generates the peripheral nervous system, melanocytes, and some connective tissues. Neural-crest-related stem cells have been reported previously in postnatal skin: committed melanocytic stem cells in the hair follicle, and pluripotent cell types from the hair follicle and papilla that can produce various sets of lineages. Here we describe novel pluripotent neural crest-like stem cells from neonatal mouse epidermis, with different potencies, isolated as 3 independent immortal lines. Using alternative regulatory factors, they could be converted to large numbers of either Schwann precursor cells, pigmented melanocytes, chondrocytes, or functional sensory neurons showing voltage-gated sodium channels. Some of the neurons displayed abundant active TRPV1 and TRPA1 receptors. Such functional neurons have previously been obtained in culture only with difficulty, by explantation. The system was also used to generate comparative gene expression data for the stem cells, melanocytes, and melanoblasts that sufficiently explain the lack of pigment in melanoblasts and provide a rationale for some genes expressed apparently ectopically in melanomas, such as ephrin receptors.—Sviderskaya, E. V., Easty, D. J., Lawrence, M. A., Sánchez, D. P., Negulyaev, Y. A., Patel, R. H., Anand, P., Korchev, Y. E., Bennett, D. C. Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells. PMID:19447881

Neighboring Genes Show Correlated Evolution in Gene Expression

PubMed Central

Ghanbarian, Avazeh T.; Hurst, Laurence D.

2015-01-01

When considering the evolution of a gene’s expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. PMID:25743543

Smad1/5/8 are myogenic regulators of murine and human mesoangioblasts

PubMed Central

Costamagna, Domiziana; Quattrocelli, Mattia; van Tienen, Florence; Umans, Lieve; de Coo, Irineus F. M.; Zwijsen, An; Huylebroeck, Danny; Sampaolesi, Maurilio

2016-01-01

Mesoangioblasts (MABs) are vessel-associated stem cells that express pericyte marker genes and participate in skeletal muscle regeneration. Molecular circuits that regulate the myogenic commitment of MABs are still poorly characterized. The critical role of bone morphogenetic protein (BMP) signalling during proliferation and differentiation of adult myogenic precursors, such as satellite cells, has recently been established. We evaluated whether BMP signalling impacts on the myogenic potential of embryonic and adult MABs both in vitro and in vivo. Addition of BMP inhibited MAB myogenic differentiation, whereas interference with the interactions between BMPs and receptor complexes induced differentiation. Similarly, siRNA-mediated knockdown of Smad8 in Smad1/5-null MABs or inhibition of SMAD1/5/8 phosphorylation with Dorsomorphin (DM) also improved myogenic differentiation, demonstrating a novel role of SMAD8. Moreover, using a transgenic mouse model of Smad8 deletion, we demonstrated that the absence of SMAD8 protein improved MAB myogenic differentiation. Furthermore, once injected into α-Sarcoglycan (Sgca)-null muscles, DM-treated MABs were more efficacious to restore α-sarcoglycan (αSG) protein levels and re-establish functional muscle properties. Similarly, in acute muscle damage, DM-treated MABs displayed a better myogenic potential compared with BMP-treated and untreated cells. Finally, SMADs also control the myogenic commitment of human MABs (hMABs). BMP signalling antagonists are therefore novel candidates to improve the therapeutic effects of hMABs. PMID:26450990

Impact on community organisations that partnered with the Act-Belong-Commit mental health promotion campaign.

PubMed

Jalleh, Geoffrey; Anwar-McHenry, Julia; Donovan, Robert J; Laws, Amberlee

2013-04-01

A primary aim of the pilot phase of the Act-Belong-Commit mental health promotion campaign was to form partnerships with community organisations. As a component of the broader campaign strategy, collaborating organisations promoted their activities under the Act-Belong-Commit banner in exchange for resources, promotional opportunities and capacity building in event management and funding. The impact of the Act-Belong-Commit campaign on the capacity and activities of collaborating organisations during the pilot phase was evaluated using self-completed mail surveys in 2006 and 2008. Collaboration with the campaign had a positive impact on community organisations' capacity, including staff expertise, media publicity and funding applications. Collaborating organisations had strong positive perceptions of Act-Belong-Commit officers and all expressed a willingness to collaborate in future events and activities. The partnership model used during the pilot phase of the Act-Belong-Commit campaign was successful in creating mutually beneficial exchanges with collaborating organisations. So what? Community partnerships are necessary for the effective delivery of mental health promotion campaigns at a local level. Successful partnerships involve the provision of real and valuable benefits to collaborating organisations in return for their cooperation in promoting health messages.

Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

PubMed Central

Oliveira, L M A; Falomir-Lockhart, L J; Botelho, M G; Lin, K-H; Wales, P; Koch, J C; Gerhardt, E; Taschenberger, H; Outeiro, T F; Lingor, P; Schüle, B; Arndt-Jovin, D J; Jovin, T M

2015-01-01

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson's disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson's disease progression, particularly in the context of bioenergetic dysfunction. PMID:26610207

Identification of Human HK Genes and Gene Expression Regulation Study in Cancer from Transcriptomics Data Analysis

PubMed Central

Zhang, Zhang; Liu, Jingxing; Wu, Jiayan; Yu, Jun

2013-01-01

The regulation of gene expression is essential for eukaryotes, as it drives the processes of cellular differentiation and morphogenesis, leading to the creation of different cell types in multicellular organisms. RNA-Sequencing (RNA-Seq) provides researchers with a powerful toolbox for characterization and quantification of transcriptome. Many different human tissue/cell transcriptome datasets coming from RNA-Seq technology are available on public data resource. The fundamental issue here is how to develop an effective analysis method to estimate expression pattern similarities between different tumor tissues and their corresponding normal tissues. We define the gene expression pattern from three directions: 1) expression breadth, which reflects gene expression on/off status, and mainly concerns ubiquitously expressed genes; 2) low/high or constant/variable expression genes, based on gene expression level and variation; and 3) the regulation of gene expression at the gene structure level. The cluster analysis indicates that gene expression pattern is higher related to physiological condition rather than tissue spatial distance. Two sets of human housekeeping (HK) genes are defined according to cell/tissue types, respectively. To characterize the gene expression pattern in gene expression level and variation, we firstly apply improved K-means algorithm and a gene expression variance model. We find that cancer-associated HK genes (a HK gene is specific in cancer group, while not in normal group) are expressed higher and more variable in cancer condition than in normal condition. Cancer-associated HK genes prefer to AT-rich genes, and they are enriched in cell cycle regulation related functions and constitute some cancer signatures. The expression of large genes is also avoided in cancer group. These studies will help us understand which cell type-specific patterns of gene expression differ among different cell types, and particularly for cancer. PMID:23382867

Neighboring Genes Show Correlated Evolution in Gene Expression.

PubMed

Ghanbarian, Avazeh T; Hurst, Laurence D

2015-07-01

When considering the evolution of a gene's expression profile, we commonly assume that this is unaffected by its genomic neighborhood. This is, however, in contrast to what we know about the lack of autonomy between neighboring genes in gene expression profiles in extant taxa. Indeed, in all eukaryotic genomes genes of similar expression-profile tend to cluster, reflecting chromatin level dynamics. Does it follow that if a gene increases expression in a particular lineage then the genomic neighbors will also increase in their expression or is gene expression evolution autonomous? To address this here we consider evolution of human gene expression since the human-chimp common ancestor, allowing for both variation in estimation of current expression level and error in Bayesian estimation of the ancestral state. We find that in all tissues and both sexes, the change in gene expression of a focal gene on average predicts the change in gene expression of neighbors. The effect is highly pronounced in the immediate vicinity (<100 kb) but extends much further. Sex-specific expression change is also genomically clustered. As genes increasing their expression in humans tend to avoid nuclear lamina domains and be enriched for the gene activator 5-hydroxymethylcytosine, we conclude that, most probably owing to chromatin level control of gene expression, a change in gene expression of one gene likely affects the expression evolution of neighbors, what we term expression piggybacking, an analog of hitchhiking. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The Human Cell Atlas.

PubMed

Regev, Aviv; Teichmann, Sarah A; Lander, Eric S; Amit, Ido; Benoist, Christophe; Birney, Ewan; Bodenmiller, Bernd; Campbell, Peter; Carninci, Piero; Clatworthy, Menna; Clevers, Hans; Deplancke, Bart; Dunham, Ian; Eberwine, James; Eils, Roland; Enard, Wolfgang; Farmer, Andrew; Fugger, Lars; Göttgens, Berthold; Hacohen, Nir; Haniffa, Muzlifah; Hemberg, Martin; Kim, Seung; Klenerman, Paul; Kriegstein, Arnold; Lein, Ed; Linnarsson, Sten; Lundberg, Emma; Lundeberg, Joakim; Majumder, Partha; Marioni, John C; Merad, Miriam; Mhlanga, Musa; Nawijn, Martijn; Netea, Mihai; Nolan, Garry; Pe'er, Dana; Phillipakis, Anthony; Ponting, Chris P; Quake, Stephen; Reik, Wolf; Rozenblatt-Rosen, Orit; Sanes, Joshua; Satija, Rahul; Schumacher, Ton N; Shalek, Alex; Shapiro, Ehud; Sharma, Padmanee; Shin, Jay W; Stegle, Oliver; Stratton, Michael; Stubbington, Michael J T; Theis, Fabian J; Uhlen, Matthias; van Oudenaarden, Alexander; Wagner, Allon; Watt, Fiona; Weissman, Jonathan; Wold, Barbara; Xavier, Ramnik; Yosef, Nir

2017-12-05

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

The Human Cell Atlas

PubMed Central

Amit, Ido; Benoist, Christophe; Birney, Ewan; Bodenmiller, Bernd; Campbell, Peter; Carninci, Piero; Clatworthy, Menna; Clevers, Hans; Deplancke, Bart; Dunham, Ian; Eberwine, James; Eils, Roland; Enard, Wolfgang; Farmer, Andrew; Fugger, Lars; Göttgens, Berthold; Hacohen, Nir; Haniffa, Muzlifah; Hemberg, Martin; Kim, Seung; Klenerman, Paul; Kriegstein, Arnold; Lein, Ed; Linnarsson, Sten; Lundberg, Emma; Lundeberg, Joakim; Majumder, Partha; Marioni, John C; Merad, Miriam; Mhlanga, Musa; Nawijn, Martijn; Netea, Mihai; Nolan, Garry; Pe'er, Dana; Phillipakis, Anthony; Ponting, Chris P; Quake, Stephen; Reik, Wolf; Rozenblatt-Rosen, Orit; Sanes, Joshua; Satija, Rahul; Schumacher, Ton N; Shalek, Alex; Shapiro, Ehud; Sharma, Padmanee; Shin, Jay W; Stegle, Oliver; Stratton, Michael; Stubbington, Michael J T; Theis, Fabian J; Uhlen, Matthias; van Oudenaarden, Alexander; Wagner, Allon; Watt, Fiona; Weissman, Jonathan; Wold, Barbara; Xavier, Ramnik; Yosef, Nir

2017-01-01

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community. PMID:29206104

Growth rate regulation of Escherichia coli acetyl coenzyme A carboxylase, which catalyzes the first committed step of lipid biosynthesis.

PubMed Central

Li, S J; Cronan, J E

1993-01-01

Acetyl coenzyme A (CoA) carboxylase catalyzes the synthesis of malonyl-CoA, the first intermediate of fatty acid synthesis. The Escherichia coli enzyme is encoded by four subunits located at three different positions on the E. coli chromosome. The accBC genes lie in a small operon at min 72, whereas accA and accD are located at min 4.3 and 50, respectively. We examined the expression of the genes that encode the E. coli acetyl-CoA carboxylase subunits (accA, accBC, and accD) under a variety of growth conditions by quantitative Northern (RNA) blot analysis. We found a direct correlation between the levels of transcription of the acc genes and the rate of cellular growth. Consistent results were also obtained upon nutritional upshift and downshift experiments and upon dilution of stationary-phase cultures into fresh media. We also determined the 5' end of the accA and accD mRNAs by primer extension and did transcriptional fusion analysis of the previously reported accBC promoter. Several interesting features were found in the promoter regions of these genes, including a bent DNA sequence and an open reading frame within the unusually long leader mRNA of the accBC operon, potential stem-loop structures in the accA and accD mRNA leader regions, and a stretch of GC-rich sequences followed by AT-rich sequences common to all three promoters. In addition, both accA and accD are located in complex gene clusters. For example, the accA promoter was localized within the upstream polC gene (which encodes the DNA polymerase III catalytic subunit), suggesting that additional regulatory mechanisms exist. Images PMID:7678242

Love, Hate and Murder: Commitment Devices in Violent Relationships

PubMed Central

Aizer, Anna; Bó, Pedro Dal

2009-01-01

Many violent relationships are characterized by a high degree of cyclicality: women who are the victims of domestic violence often leave and return multiple times. To explain this we develop a model of time inconsistent preferences in the context of domestic violence. This time inconsistency generates a demand for commitment. We present supporting evidence that women in violent relationships display time inconsistent preferences by examining their demand for commitment devices. We find that no-drop policies – which compel the prosecutor to continue with prosecution even if the victim expresses a desire to drop the charges – result in an increase in reporting. No-drop policies also result in a decrease in the number of men murdered by intimates suggesting that some women in violent relationships move away from an extreme type of commitment device when a less costly one is offered. PMID:24244055

Dual Character Concepts in Social Cognition: Commitments and the Normative Dimension of Conceptual Representation.

PubMed

Del Pinal, Guillermo; Reuter, Kevin

2017-04-01

The concepts expressed by social role terms such as artist and scientist are unique in that they seem to allow two independent criteria for categorization, one of which is inherently normative (Knobe, Prasada, & Newman, 2013). This study presents and tests an account of the content and structure of the normative dimension of these "dual character concepts." Experiment 1 suggests that the normative dimension of a social role concept represents the commitment to fulfill the idealized basic function associated with the role. Background information can affect which basic function is associated with each social role. However, Experiment 2 indicates that the normative dimension always represents the relevant commitment as an end in itself. We argue that social role concepts represent the commitments to basic functions because that information is crucial to predict the future social roles and role-dependent behavior of others. Copyright © 2016 Cognitive Science Society, Inc.

Application of community phylogenetic approaches to understand gene expression: differential exploration of venom gene space in predatory marine gastropods.

PubMed

Chang, Dan; Duda, Thomas F

2014-06-05

Predatory marine gastropods of the genus Conus exhibit substantial variation in venom composition both within and among species. Apart from mechanisms associated with extensive turnover of gene families and rapid evolution of genes that encode venom components ('conotoxins'), the evolution of distinct conotoxin expression patterns is an additional source of variation that may drive interspecific differences in the utilization of species' 'venom gene space'. To determine the evolution of expression patterns of venom genes of Conus species, we evaluated the expression of A-superfamily conotoxin genes of a set of closely related Conus species by comparing recovered transcripts of A-superfamily genes that were previously identified from the genomes of these species. We modified community phylogenetics approaches to incorporate phylogenetic history and disparity of genes and their expression profiles to determine patterns of venom gene space utilization. Less than half of the A-superfamily gene repertoire of these species is expressed, and only a few orthologous genes are coexpressed among species. Species exhibit substantially distinct expression strategies, with some expressing sets of closely related loci ('under-dispersed' expression of available genes) while others express sets of more disparate genes ('over-dispersed' expression). In addition, expressed genes show higher dN/dS values than either unexpressed or ancestral genes; this implies that expression exposes genes to selection and facilitates rapid evolution of these genes. Few recent lineage-specific gene duplicates are expressed simultaneously, suggesting that expression divergence among redundant gene copies may be established shortly after gene duplication. Our study demonstrates that venom gene space is explored differentially by Conus species, a process that effectively permits the independent and rapid evolution of venoms in these species.

Opposing actions of Arx and Pax4 in endocrine pancreas development

PubMed Central

Collombat, Patrick; Mansouri, Ahmed; Hecksher-Sørensen, Jacob; Serup, Palle; Krull, Jens; Gradwohl, Gerard; Gruss, Peter

2003-01-01

Genes encoding homeodomain-containing proteins potentially involved in endocrine pancreas development were isolated by combined in silico and nested-PCR approaches. One such transcription factor, Arx, exhibits Ngn3-dependent expression throughout endocrine pancreas development in α, β-precursor, and δ cells. We have used gene targeting in mouse embryonic stem cells to generate Arx loss-of-function mice. Arx-deficient animals are born at the expected Mendelian frequency, but develop early-onset hypoglycemia, dehydration, and weakness, and die 2 d after birth. Immunohistological analysis of pancreas from Arx mutants reveals an early-onset loss of mature endocrine α cells with a concomitant increase in β-and δ-cell numbers, whereas islet morphology remains intact. Our study indicates a requirement of Arx for α-cell fate acquisition and a repressive action on β-and δ-cell destiny, which is exactly the opposite of the action of Pax4 in endocrine commitment. Using multiplex reverse transcriptase PCR (RT-PCR), we demonstrate an accumulation of Pax4 and Arx transcripts in Arx and Pax4 mutant mice, respectively. We propose that the antagonistic functions of Arx and Pax4 for proper islet cell specification are related to the pancreatic levels of the respective transcripts. PMID:14561778

Opposing actions of Arx and Pax4 in endocrine pancreas development.

PubMed

Collombat, Patrick; Mansouri, Ahmed; Hecksher-Sorensen, Jacob; Serup, Palle; Krull, Jens; Gradwohl, Gerard; Gruss, Peter

2003-10-15

Genes encoding homeodomain-containing proteins potentially involved in endocrine pancreas development were isolated by combined in silico and nested-PCR approaches. One such transcription factor, Arx, exhibits Ngn3-dependent expression throughout endocrine pancreas development in alpha, beta-precursor, and delta cells. We have used gene targeting in mouse embryonic stem cells to generate Arx loss-of-function mice. Arx-deficient animals are born at the expected Mendelian frequency, but develop early-onset hypoglycemia, dehydration, and weakness, and die 2 d after birth. Immunohistological analysis of pancreas from Arx mutants reveals an early-onset loss of mature endocrine alpha cells with a concomitant increase in beta-and delta-cell numbers, whereas islet morphology remains intact. Our study indicates a requirement of Arx for alpha-cell fate acquisition and a repressive action on beta-and delta-cell destiny, which is exactly the opposite of the action of Pax4 in endocrine commitment. Using multiplex reverse transcriptase PCR (RT-PCR), we demonstrate an accumulation of Pax4 and Arx transcripts in Arx and Pax4 mutant mice, respectively. We propose that the antagonistic functions of Arx and Pax4 for proper islet cell specification are related to the pancreatic levels of the respective transcripts.

Bradykinin regulates osteoblast differentiation by Akt/ERK/NFκB signaling axis.

PubMed

Srivastava, Swati; Sharma, Kirti; Kumar, Narender; Roy, Partha

2014-12-01

Bradykinin (BK), a well known mediator of pain and inflammation, is also known to be involved in the process of bone resorption. The present study therefore evaluated the role of BK in osteoblast lineage commitment. Our data showed that BK inhibits the migration of bone marrow mesenchymal stem cells, but does not affect their viability. Moreover, BK also inhibits osteoblastic differentiation by significantly downregulating the levels of mRNAs for osteopontin, runX2, col24, osterix, osteocalcin genes and bone mineralization (P < 0.05). Further, BK was found to elicit the BK receptors (BDKR1 and BDKR2) mediated activation of ERK1/2 and Akt pathways, which finally led to the activation of NFκB. BK also promoted the osteoclast differentiation of bone marrow derived preosteoclast cells by upregulating the expression of c-fos, NFATC1, TRAP, clcn7, cathK, and OSCAR genes and increasing TRAP activity through NFκB pathway. In conclusion, our data suggest that BK decreases the differentiation of osteoblasts with concomitant increase in osteoclast formation and thus provides new insight into the mechanism of action of BK in modulating bone resorption. © 2014 Wiley Periodicals, Inc.

Genomics and drug profiling of fatal TCF3-HLF-positive acute lymphoblastic leukemia identifies recurrent mutation patterns and therapeutic options

PubMed Central

Bornhauser, Beat; Gombert, Michael; Kratsch, Christina; Stütz, Adrian M.; Sultan, Marc; Tchinda, Joelle; Worth, Catherine L.; Amstislavskiy, Vyacheslav; Badarinarayan, Nandini; Baruchel, André; Bartram, Thies; Basso, Giuseppe; Canpolat, Cengiz; Cario, Gunnar; Cavé, Hélène; Dakaj, Dardane; Delorenzi, Mauro; Dobay, Maria Pamela; Eckert, Cornelia; Ellinghaus, Eva; Eugster, Sabrina; Frismantas, Viktoras; Ginzel, Sebastian; Haas, Oskar A.; Heidenreich, Olaf; Hemmrich-Stanisak, Georg; Hezaveh, Kebria; Höll, Jessica I.; Hornhardt, Sabine; Husemann, Peter; Kachroo, Priyadarshini; Kratz, Christian P.; te Kronnie, Geertruy; Marovca, Blerim; Niggli, Felix; McHardy, Alice C.; Moorman, Anthony V.; Panzer-Grümayer, Renate; Petersen, Britt S.; Raeder, Benjamin; Ralser, Meryem; Rosenstiel, Philip; Schäfer, Daniel; Schrappe, Martin; Schreiber, Stefan; Schütte, Moritz; Stade, Björn; Thiele, Ralf; von der Weid, Nicolas; Vora, Ajay; Zaliova, Marketa; Zhang, Langhui; Zichner, Thomas; Zimmermann, Martin; Lehrach, Hans; Borkhardt, Arndt; Bourquin, Jean-Pierre; Franke, Andre; Korbel, Jan O.; Stanulla, Martin; Yaspo, Marie-Laure

2015-01-01

TCF3-HLF-fusion positive acute lymphoblastic leukemia (ALL) is currently incurable. Employing an integrated approach, we uncovered distinct mutation, gene expression, and drug response profiles in TCF3-HLF-positive and treatment-responsive TCF3-PBX1-positive ALL. Recurrent intragenic deletions of PAX5 or VPREB1 were identified in constellation with TCF3-HLF. Moreover somatic mutations in the non-translocated allele of TCF3 and a reduction of PAX5 gene dosage in TCF3-HLF ALL suggest cooperation within a restricted genetic context. The enrichment for stem cell and myeloid features in the TCF3-HLF signature may reflect reprogramming by TCF3-HLF of a lymphoid-committed cell of origin towards a hybrid, drug-resistant hematopoietic state. Drug response profiling of matched patient-derived xenografts revealed a distinct profile for TCF3-HLF ALL with resistance to conventional chemotherapeutics, but sensitivity towards glucocorticoids, anthracyclines and agents in clinical development. Striking on-target sensitivity was achieved with the BCL2-specific inhibitor venetoclax (ABT-199). This integrated approach thus provides alternative treatment options for this deadly disease. PMID:26214592

The paradox of Foxd3: how does it function in pluripotency and differentiation of embryonic stem cells?

PubMed

Plank-Bazinet, Jennifer L; Mundell, Nathan A

2016-01-01

Uncommitted cells of the early mammalian embryo transition through distinct stages of pluripotency, including establishment of ground state "naïve" pluripotency in the early epiblast, transition to a post-implantation "primed" state, and subsequent lineage commitment of the gastrulating epiblast. Previous transcriptional profiling of in vitro models to recapitulate early to late epiblast transition and differentiation suggest that distinct gene regulatory networks are likely to function in each of these states. While the mechanisms underlying transition between pluripotent states are poorly understood, the forkhead family transcription factor Foxd3 has emerged as a key regulatory factor. Foxd3 is required to maintain pluripotent cells of the murine epiblast and for survival, self-renewal and pluripotency of embryonic stem cells (ESCs). Two recent, simultaneous studies have shed light on how Foxd3 regulates gene expression in early cell fate transitions of progenitor cells. While the two publications shared some common findings, they also presented some conflicting results and suggest different models for the mechanisms underlying Foxd3 function. Here, we discuss the key similarities and differences between the publications, highlight data from the literature relevant to their findings, and hypothesize a potential mechanism of Foxd3 action.

Convergent microRNA actions coordinate neocortical development.

PubMed

Barca-Mayo, Olga; De Pietri Tonelli, Davide

2014-08-01

Neocortical development is a complex process that, at the cellular level, involves tight control of self-renewal, cell fate commitment, survival, differentiation and delamination/migration. These processes require, at the molecular level, the precise regulation of intrinsic signaling pathways and extrinsic factors with coordinated action in a spatially and temporally specific manner. Transcriptional regulation plays an important role during corticogenesis; however, microRNAs (miRNAs) are emerging as important post-transcriptional regulators of various aspects of central nervous system development. miRNAs are a class of small, single-stranded noncoding RNA molecules that control the expression of the majority of protein coding genes (i.e., targets). How do different miRNAs achieve precise control of gene networks during neocortical development? Here, we critically review all the miRNA-target interactions validated in vivo, with relevance to the generation and migration of pyramidal-projection glutamatergic neurons, and for the initial formation of cortical layers in the embryonic development of rodent neocortex. In particular, we focus on convergent miRNA actions, which are still a poorly understood layer of complexity in miRNA signaling, but potentially one of the keys to disclosing how miRNAs achieve the precise coordination of complex biological processes such as neocortical development.

Social Support, Religious Endorsement, and Career Commitment: A Study on Saudi Nurses

PubMed Central

Azim, Mohammad T.

2018-01-01

The present study investigates the effect of perceived social support (PSS) and perceived religious endorsement (PRE) on career commitment (CC) of Saudi nurses. The investigation also extends to the moderating role of different demographic and organizational factors in the extent of PSS, and career commitment these nurses report. Data required for meeting these study objectives were collected from male and female Saudi nurses through a structured questionnaire. Multiple regressions using Partial Least Squares based Structural Equation Model, Smart-PLS version 3.0, and independent sample t-test using SPSS version 22.0, were used to analyze data. The study findings reveal that both perceived social support and perceived religious endorsement are important antecedents of career commitment of Saudi nurses. However, private-sector nurses are found to exhibit a significantly higher level of career commitment compared to their public-sector counterparts. Nurses with greater educational attainment perceive higher level of social support and express greater career commitment than their less educated peers. These findings suggest that nursing as a profession should be more openly discussed in both secular and religious contexts, to ensure an adequate level of respect and compassion on behalf of the public. In particular, endorsement from the individual nurses’ social networks is vital in maintaining their wellbeing and career commitment. Given the religious influence in all aspects of life in the Saudi society, the current practice of gender-based segregation in Saudi hospitals and clinics seems to be meaningful for sustaining the career commitment of the nurses. PMID:29320417

Effects of a signature on rates of change: a randomized controlled trial involving continuing education and the commitment-to-change model.

PubMed

Mazmanian, P E; Johnson, R E; Zhang, A; Boothby, J; Yeatts, E J

2001-06-01

Physicians frequently are asked to sign commitments to change practice, based upon their involvement in continuing medical education (CME) activities. Although use of the commitment-to-change model is increasingly widespread in CME, the effect of signing such commitments on rates of change is not well understood. Immediately after a CME session, 110 physicians were asked to specify a change they intended to make in practice and to designate a level of commitment to change. To determine the effects of a signature on rates of change, physicians were randomly assigned to control (signature) and experimental (non-signature) groups. Follow-up surveys were conducted at two and three months to determine rates of change. In all, 88 physicians completed the first questionnaire, and 64 of them completed the follow-up. Consistent with prior studies involving the commitment-to-change model, those expressing an intention to change were significantly more likely to change on follow-up (p =.035). There was no significant difference between signature and non-signature groups (p =.99), regardless of age or gender. Signatures appear unimportant to assuring compliance with commitments to change used in CME conferences. A physician's behavior can be expected to change if the specified change is consistent with the physician's beliefs and sense of what is important. The relative influences of components of the commitment-to-change model require further study to determine more clearly their roles in causation and measurement.

Social Support, Religious Endorsement, and Career Commitment: A Study on Saudi Nurses.

PubMed

Azim, Mohammad T; Islam, Mazharul M

2018-01-10

The present study investigates the effect of perceived social support (PSS) and perceived religious endorsement (PRE) on career commitment (CC) of Saudi nurses. The investigation also extends to the moderating role of different demographic and organizational factors in the extent of PSS, and career commitment these nurses report. Data required for meeting these study objectives were collected from male and female Saudi nurses through a structured questionnaire. Multiple regressions using Partial Least Squares based Structural Equation Model, Smart-PLS version 3.0, and independent sample t -test using SPSS version 22.0, were used to analyze data. The study findings reveal that both perceived social support and perceived religious endorsement are important antecedents of career commitment of Saudi nurses. However, private-sector nurses are found to exhibit a significantly higher level of career commitment compared to their public-sector counterparts. Nurses with greater educational attainment perceive higher level of social support and express greater career commitment than their less educated peers. These findings suggest that nursing as a profession should be more openly discussed in both secular and religious contexts, to ensure an adequate level of respect and compassion on behalf of the public. In particular, endorsement from the individual nurses' social networks is vital in maintaining their wellbeing and career commitment. Given the religious influence in all aspects of life in the Saudi society, the current practice of gender-based segregation in Saudi hospitals and clinics seems to be meaningful for sustaining the career commitment of the nurses.

Initiation of olfactory placode development and neurogenesis is blocked in mice lacking both Six1 and Six4.

PubMed

Chen, Binglai; Kim, Eun-Hee; Xu, Pin-Xian

2009-02-01

Mouse olfactory epithelium (OE) originates from ectodermally derived placode, the olfactory placode that arises at the anterior end of the neural plate. Tissue grafting and recombination experiments suggest that the placode is derived from a common preplacodal domain around the neural plate and its development is directed by signals arising from the underlying mesoderm and adjacent neuroectoderm. In mice, loss of Six1 affects OE morphogenesis but not placode formation. We show here that embryos lacking both Six1 and Six4 failed to form the olfactory placode but the preplacodal region appeared to be specified as judged by the expression of Eya2, which marks the common preplacodal domain, suggesting a synergistic requirement of Six1 and Six4 in patterning the preplacodal ectoderm to a morphologic placode. Our results show that Six1 and Six4 are coexpressed in the preplacodal ectoderm from E8.0. In the olfactory pit, Six4 expression was observed in the peripheral precursors that overlap with Mash1-expressing cells, the early committed neuronal lineage. In contrast, Six1 is highly distributed in the peripheral regions where stem cells reside at E10.5 and it overlaps with Sox2 expression. Both genes are expressed in the basal and apical neuronal progenitors in the OE. Analyses of Six1;Six4 double mutant embryos demonstrated that the slightly thickened epithelium observed in the mutant was not induced for neuronal development. In contrast, in Six1(-/-) embryos, all neuronal lineage markers were initially expressed but the pattern of their expression was altered. Although very few, the pioneer neurons were initially present in the Six1 mutant OE. However, neurogenesis ceased by E12.5 due to markedly increased cell apoptosis and reduced proliferation, thus defining the cellular defects occurring in Six1(-/-) OE that have not been previously observed. Our findings demonstrate that Six1/4 function at the top of early events controlling olfactory placode formation and neuronal development. Our analyses show that the threshold of Six1/4 may be crucial for the expression of olfactory specific genes and that Six1 and Six4 may act synergistically to mediate olfactory placode specification and patterning through Fgf and Bmp signaling pathways.

In situ hybridization analysis of anterior pituitary hormone gene expression during fetal mouse development.

PubMed

Japón, M A; Rubinstein, M; Low, M J

1994-08-01

We used 35S-labeled oligonucleotides and cRNAs (riboprobes) to detect the temporal order and spatial pattern of anterior pituitary hormone gene expression in (B6CBF1 x B6CBF1)F2 fetal mice from embryonic Day 9.5 (E9.5) to postnatal Day 1 (P1). Pro-opiomelanocortin (POMC) mRNA was expressed in the basal diencephalon on Day E10.5, in the ventromedial zone of the pars distalis on Day E12.5, and in the pars intermedia on Day E14.5. The common alpha-glycoprotein subunit (alpha-GSU) mRNA first appeared in the anterior wall of Rathke's pouch on Day E11.5 and extended to the pars tuberalis and ventromedial zone of the pars distalis on Day E12.5. Thyroid-stimulating hormone-beta (TSH beta) subunit mRNA was expressed initially in both the pas tuberalis and ventromedial pars distalis on Day E14.5, with an identical spatial distribution to alpha-GSU at the time. In contrast, luteinizing hormone-beta (LH beta) subunit and follicle-stimulating hormone beta (FSH beta) subunit mRNAs were detected initially only in the ventromedial pars distalis on Days E16.5 and E17.5, respectively, in an identical distribution to each other. POMC-, alpha-GSU-, TSH beta, LH beta-, and FSH beta-positive cells within the pars distalis all increased in number and autoradiographic signal with differing degrees of spatial expansion posteriorly, laterally, and dorsally up to Day P1. POMC expression was typically the most intense and extended circumferentially to include the entire lateral and dorsal surfaces of the pars distalis. The expression of both growth hormone (GH) and prolactin (PRL) started coincidentally on Day E15.5. However PRL cells localized in the ventromedial area similarly to POMC and the glycoprotein hormone subunits, whereas GH cells were found initially in a more lateral and central distribution within the lobes of the pars distalis. Somatotrophs increased dramatically in number and autoradiographic signal, extending throughout the pars distalis except for the most peripheral layer of cells on Day E17.5. Mammotrophs also increased in number but less abundantly than somatotrophs, and PRL expression remained more confined to central-medial and ventrolateral areas of the pars distalis up to Day P1. These data demonstrate distinctive patterns of expression for each of the major anterior pituitary hormone genes during development of the mouse pituitary gland and suggest that different groups of committed cells are the immediate precursors to the terminally differentiated hormone-secreting cell types.

FARO server: Meta-analysis of gene expression by matching gene expression signatures to a compendium of public gene expression data.

PubMed

Manijak, Mieszko P; Nielsen, Henrik B

2011-06-11

Although, systematic analysis of gene annotation is a powerful tool for interpreting gene expression data, it sometimes is blurred by incomplete gene annotation, missing expression response of key genes and secondary gene expression responses. These shortcomings may be partially circumvented by instead matching gene expression signatures to signatures of other experiments. To facilitate this we present the Functional Association Response by Overlap (FARO) server, that match input signatures to a compendium of 242 gene expression signatures, extracted from more than 1700 Arabidopsis microarray experiments. Hereby we present a publicly available tool for robust characterization of Arabidopsis gene expression experiments which can point to similar experimental factors in other experiments. The server is available at http://www.cbs.dtu.dk/services/faro/.

Digital gene expression analysis of the zebra finch genome

PubMed Central

2010-01-01

Background In order to understand patterns of adaptation and molecular evolution it is important to quantify both variation in gene expression and nucleotide sequence divergence. Gene expression profiling in non-model organisms has recently been facilitated by the advent of massively parallel sequencing technology. Here we investigate tissue specific gene expression patterns in the zebra finch (Taeniopygia guttata) with special emphasis on the genes of the major histocompatibility complex (MHC). Results Almost 2 million 454-sequencing reads from cDNA of six different tissues were assembled and analysed. A total of 11,793 zebra finch transcripts were represented in this EST data, indicating a transcriptome coverage of about 65%. There was a positive correlation between the tissue specificity of gene expression and non-synonymous to synonymous nucleotide substitution ratio of genes, suggesting that genes with a specialised function are evolving at a higher rate (or with less constraint) than genes with a more general function. In line with this, there was also a negative correlation between overall expression levels and expression specificity of contigs. We found evidence for expression of 10 different genes related to the MHC. MHC genes showed relatively tissue specific expression levels and were in general primarily expressed in spleen. Several MHC genes, including MHC class I also showed expression in brain. Furthermore, for all genes with highest levels of expression in spleen there was an overrepresentation of several gene ontology terms related to immune function. Conclusions Our study highlights the usefulness of next-generation sequence data for quantifying gene expression in the genome as a whole as well as in specific candidate genes. Overall, the data show predicted patterns of gene expression profiles and molecular evolution in the zebra finch genome. Expression of MHC genes in particular, corresponds well with expression patterns in other vertebrates. PMID:20359325

A WRKY transcription factor participates in dehydration tolerance in Boea hygrometrica by binding to the W-box elements of the galactinol synthase (BhGolS1) promoter.

PubMed

Wang, Zhi; Zhu, Yan; Wang, Lili; Liu, Xia; Liu, Yongxiu; Phillips, Jonathan; Deng, Xin

2009-11-01

Accumulation of compatible osmolytes, such as soluble sugars, in plants is an important osmoprotective mechanism. Sugars play a role in osmotic adjustment and are associated with stabilization of proteins and cell structures, reactive oxygen species scavenging, signaling functions or induction of adaptive pathways. Galactinol is the galactosyl donor for the synthesis of raffinose family oligosaccharides (RFOs) and its synthesis by galactinol synthase (GolS) is the first committed step of the RFOs biosynthetic pathway. GolS genes are induced by a variety of stresses in both stress-sensitive and tolerant-plant species; however, the mechanism of transcriptional regulation is not fully established. In this paper, we characterized a GolS gene (BhGolS1) that was dehydration and ABA-inducible in the resurrection plant Boea hygrometrica and conferred dehydration tolerance in a transgenic tobacco system. Four W-box cis-elements were identified in the BhGolS1 promoter and shown to be bound by an early dehydration and ABA-inducible WRKY gene (BhWRKY1). These data suggest a mechanism where BhWRKY1 is likely to function in an ABA-dependent signal pathway to regulate BhGolS1 expression, which leads to the accumulation of RFOs in desiccation-tolerant B. hygrometrica leaves.

Correction of X-linked immunodeficient mice by competitive reconstitution with limiting numbers of normal bone marrow cells.

PubMed

Rohrer, J; Conley, M E

1999-11-15

Gene therapy for inherited disorders is more likely to succeed if gene-corrected cells have a proliferative or survival advantage compared with mutant cells. We used a competitive reconstitution model to evaluate the strength of the selective advantage that Btk normal cells have in Btk-deficient xid mice. Whereas 2,500 normal bone marrow cells when mixed with 497,500 xid cells restored serum IgM and IgG3 levels to near normal concentrations in 3 of 5 lethally irradiated mice, 25,000 normal cells mixed with 475,000 xid cells reliably restored serum IgM and IgG3 concentrations and the thymus-independent antibody response in all transplanted mice. Reconstitution was not dependent on lethal irradiation, because sublethally irradiated mice all had elevated serum IgM and IgG3 by 30 weeks postreconstitution when receiving 25,000 normal cells. Furthermore, the xid defect was corrected with as few as 10% of the splenic B cells expressing a normal Btk. When normal donor cells were sorted into B220(+)/CD19(+) committed B cells and B220(-)/CD19(-) cell populations, only the B220(-)/CD19(-) cells provided long-term B-cell reconstitution in sublethally irradiated mice. These findings suggest that even inefficient gene therapy may provide clinical benefit for patients with XLA.

Radio electric conveyed fields directly reprogram human dermal skin fibroblasts toward cardiac, neuronal, and skeletal muscle-like lineages.

PubMed

Maioli, Margherita; Rinaldi, Salvatore; Santaniello, Sara; Castagna, Alessandro; Pigliaru, Gianfranco; Gualini, Sara; Cavallini, Claudia; Fontani, Vania; Ventura, Carlo

2013-01-01

Somatic cells can be directly reprogrammed to alternative differentiated fates without first becoming stem/progenitor cells. Nevertheless, the initial need for viral-mediated gene delivery renders this strategy unsafe in humans. Here, we provide evidence that exposure of human skin fibroblasts to a Radio Electric Asymmetric Conveyer (REAC), an innovative device delivering radio electric conveyed fields at a radiofrequency of 2.4 GHz, afforded remarkable commitment toward cardiac, neuronal, and skeletal muscle lineages. REAC induced the transcription of tissue-restricted genes, including Mef2c, Tbx5, GATA4, Nkx2.5, and prodynorphin for cardiac reprogramming, as well as myoD, and neurogenin 1 for skeletal myogenesis and neurogenesis, respectively. Conversely, REAC treatment elicited a biphasic effect on a number of stemness-related genes, leading to early transcriptional increase of Oct4, Sox2, cMyc, Nanog, and Klf4 within 6-20 h, followed by a downregulation at later times. The REAC action bypassed a persistent reprogramming toward an induced pluripotent stem cell-like state and involved the transcriptional induction of the NADPH oxidase subunit Nox4. Our results show for the first time the feasibility of using a physical stimulus to afford the expression of pluripotentiality in human adult somatic cells up to the attainment of three major target lineages for regenerative medicine.

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data.

PubMed

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R; Del Río-Navarro, Blanca E; Mendoza-Vargas, Alfredo; Sánchez, Filiberto; Ochoa-Leyva, Adrian

2017-01-01

In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6-10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments.

IL-4/IL-13 Signaling Inhibits the Potential of Early Thymic Progenitors To Commit to the T Cell Lineage.

PubMed

Barik, Subhasis; Miller, Mindy M; Cattin-Roy, Alexis N; Ukah, Tobechukwu K; Chen, Weirong; Zaghouani, Habib

2017-10-15

Early thymic progenitors (ETPs) are endowed with diverse potencies and can give rise to myeloid and lymphoid lineage progenitors. How the thymic environment guides ETP commitment and maturation toward a specific lineage remains obscure. We have previously shown that ETPs expressing the heteroreceptor (HR) comprising IL-4Rα and IL-13Rα1 give rise to myeloid cells but not T cells. In this article, we show that signaling through the HR inhibits ETP maturation to the T cell lineage but enacts commitment toward the myeloid cells. Indeed, HR + ETPs, but not HR - ETPs, exhibit activated STAT6 transcription factor, which parallels with downregulation of Notch1, a critical factor for T cell development. Meanwhile, the myeloid-specific transcription factor C/EBPα, usually under the control of Notch1, is upregulated. Furthermore, in vivo inhibition of STAT6 phosphorylation restores Notch1 expression in HR + ETPs, which regain T lineage potential. In addition, upon stimulation with IL-4 or IL-13, HR - ETPs expressing virally transduced HR also exhibit STAT6 phosphorylation and downregulation of Notch1, leading to inhibition of lymphoid, but not myeloid, lineage potential. These observations indicate that environmental cytokines play a role in conditioning ETP lineage choice, which would impact T cell development. Copyright © 2017 by The American Association of Immunologists, Inc.

Differential gene expression analysis in glioblastoma cells and normal human brain cells based on GEO database.

PubMed

Wang, Anping; Zhang, Guibin

2017-11-01

The differentially expressed genes between glioblastoma (GBM) cells and normal human brain cells were investigated to performed pathway analysis and protein interaction network analysis for the differentially expressed genes. GSE12657 and GSE42656 gene chips, which contain gene expression profile of GBM were obtained from Gene Expression Omniub (GEO) database of National Center for Biotechnology Information (NCBI). The 'limma' data packet in 'R' software was used to analyze the differentially expressed genes in the two gene chips, and gene integration was performed using 'RobustRankAggreg' package. Finally, pheatmap software was used for heatmap analysis and Cytoscape, DAVID, STRING and KOBAS were used for protein-protein interaction, Gene Ontology (GO) and KEGG analyses. As results: i) 702 differentially expressed genes were identified in GSE12657, among those genes, 548 were significantly upregulated and 154 were significantly downregulated (p<0.01, fold-change >1), and 1,854 differentially expressed genes were identified in GSE42656, among the genes, 1,068 were significantly upregulated and 786 were significantly downregulated (p<0.01, fold-change >1). A total of 167 differentially expressed genes including 100 upregulated genes and 67 downregulated genes were identified after gene integration, and the genes showed significantly different expression levels in GBM compared with normal human brain cells (p<0.05). ii) Interactions between the protein products of 101 differentially expressed genes were identified using STRING and expression network was established. A key gene, called CALM3, was identified by Cytoscape software. iii) GO enrichment analysis showed that differentially expressed genes were mainly enriched in 'neurotransmitter:sodium symporter activity' and 'neurotransmitter transporter activity', which can affect the activity of neurotransmitter transportation. KEGG pathway analysis showed that the differentially expressed genes were mainly enriched in 'protein processing in endoplasmic reticulum', which can affect protein processing in endoplasmic reticulum. The results showed that: i) 167 differentially expressed genes were identified from two gene chips after integration; and ii) protein interaction network was established, and GO and KEGG pathway analyses were successfully performed to identify and annotate the key gene, which provide new insights for the studies on GBN at gene level.

Discovery and validation of a glioblastoma co-expressed gene module

PubMed Central

Dunwoodie, Leland J.; Poehlman, William L.; Ficklin, Stephen P.; Feltus, Frank Alexander

2018-01-01

Tumors exhibit complex patterns of aberrant gene expression. Using a knowledge-independent, noise-reducing gene co-expression network construction software called KINC, we created multiple RNAseq-based gene co-expression networks relevant to brain and glioblastoma biology. In this report, we describe the discovery and validation of a glioblastoma-specific gene module that contains 22 co-expressed genes. The genes are upregulated in glioblastoma relative to normal brain and lower grade glioma samples; they are also hypo-methylated in glioblastoma relative to lower grade glioma tumors. Among the proneural, neural, mesenchymal, and classical glioblastoma subtypes, these genes are most-highly expressed in the mesenchymal subtype. Furthermore, high expression of these genes is associated with decreased survival across each glioblastoma subtype. These genes are of interest to glioblastoma biology and our gene interaction discovery and validation workflow can be used to discover and validate co-expressed gene modules derived from any co-expression network. PMID:29541392

Discovery and validation of a glioblastoma co-expressed gene module.

PubMed

Dunwoodie, Leland J; Poehlman, William L; Ficklin, Stephen P; Feltus, Frank Alexander

2018-02-16

Tumors exhibit complex patterns of aberrant gene expression. Using a knowledge-independent, noise-reducing gene co-expression network construction software called KINC, we created multiple RNAseq-based gene co-expression networks relevant to brain and glioblastoma biology. In this report, we describe the discovery and validation of a glioblastoma-specific gene module that contains 22 co-expressed genes. The genes are upregulated in glioblastoma relative to normal brain and lower grade glioma samples; they are also hypo-methylated in glioblastoma relative to lower grade glioma tumors. Among the proneural, neural, mesenchymal, and classical glioblastoma subtypes, these genes are most-highly expressed in the mesenchymal subtype. Furthermore, high expression of these genes is associated with decreased survival across each glioblastoma subtype. These genes are of interest to glioblastoma biology and our gene interaction discovery and validation workflow can be used to discover and validate co-expressed gene modules derived from any co-expression network.

Hyper-reactive cloned mice generated by direct nuclear transfer of antigen-specific CD4+ T cells.

PubMed

Kaminuma, Osamu; Katayama, Kazufumi; Inoue, Kimiko; Saeki, Mayumi; Nishimura, Tomoe; Kitamura, Noriko; Shimo, Yusuke; Tofukuji, Soichi; Ishida, Satoru; Ogonuki, Narumi; Kamimura, Satoshi; Oikawa, Mami; Katoh, Shigeki; Mori, Akio; Shichijo, Michitaka; Hiroi, Takachika; Ogura, Atsuo

2017-06-01

T-cell receptor (TCR)-transgenic mice have been employed for evaluating antigen-response mechanisms, but their non-endogenous TCR might induce immune response differently than the physiologically expressed TCR Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen-specific CD4 + T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre-rearranged TCRα (rTα) and TCRβ (rTβ) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic-type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTβ is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter-regulated antigen-specific TCR are a unique animal model with allergic predisposition for investigating CD4 + T-cell-mediated pathogenesis and cellular commitment in immune diseases. © 2017 The Authors.

Gender-Specific Gene Expression in Post-Mortem Human Brain: Localization to Sex Chromosomes

PubMed Central

Vawter, Marquis P; Evans, Simon; Choudary, Prabhakara; Tomita, Hiroaki; Meador-Woodruff, Jim; Molnar, Margherita; Li, Jun; Lopez, Juan F; Myers, Rick; Cox, David; Watson, Stanley J; Akil, Huda; Jones, Edward G; Bunney, William E

2011-01-01

Gender differences in brain development and in the prevalence of neuropsychiatric disorders such as depression have been reported. Gender differences in human brain might be related to patterns of gene expression. Microarray technology is one useful method for investigation of gene expression in brain. We investigated gene expression, cell types, and regional expression patterns of differentially expressed sex chromosome genes in brain. We profiled gene expression in male and female dorsolateral prefrontal cortex, anterior cingulate cortex, and cerebellum using the Affymetrix oligonucleotide microarray platform. Differentially expressed genes between males and females on the Y chromosome (DBY, SMCY, UTY, RPS4Y, and USP9Y) and X chromosome (XIST) were confirmed using real-time PCR measurements. In situ hybridization confirmed the differential expression of gender-specific genes and neuronal expression of XIST, RPS4Y, SMCY, and UTY in three brain regions examined. The XIST gene, which silences gene expression on regions of the X chromosome, is expressed in a subset of neurons. Since a subset of neurons express gender-specific genes, neural subpopulations may exhibit a subtle sexual dimorphism at the level of differences in gene regulation and function. The distinctive pattern of neuronal expression of XIST, RPS4Y, SMCY, and UTY and other sex chromosome genes in neuronal subpopulations may possibly contribute to gender differences in prevalence noted for some neuropsychiatric disorders. Studies of the protein expression of these sex- chromosome-linked genes in brain tissue are required to address the functional consequences of the observed gene expression differences. PMID:14583743

An in silico investigation into the causes of telomere length heterogeneity and its implications for the Hayflick limit.

PubMed

Golubev, A; Khrustalev, S; Butov, A

2003-11-21

In telomerase-negative cell populations the mean telomere length (TL) decreases with increasing population doubling number (PD). A critically small TL is believed to stop cell proliferation at a cell-, age- and species-specific PD thus defining the Hayflick limit. However, positively skewed TL distributions are broad compared to differences between initial and final mean TL and strongly overlap at middle and late PD, which is inconsistent with a limiting role of TL. We used computer-assisted modelling to define what set of premises may account for the above. Our model incorporates the following concepts. DNA end replication problem: telomeres loose 1 shortening unit (SU) upon each cell division. Free radical-caused TL decrease: telomeres experience random events resulting in the loss of a random SU number within a remaining TL. Stochasticity of gene expression and cell differentiation: cells experience random events inducing mitoses or committing cells to proliferation arrest, the latter option requiring a specified number of mitoses to be passed. Cells whose TL reaches 1SU cannot divide. The proliferation kinetics of such virtual cells conforms to the transition probability model of cell cycle. When no committing events occur and at realistic SU estimates of the initial TL, maximal PD values far exceed the Hayflick limit observed in normal cells and are consistent with the crisis stage entered by transformed cells that have surpassed the Hayflick limit. At intermediate PD, symmetrical TL distributions are yielded. Upon introduction of committing events making the ratio of the rates of proliferating and committing events (P/C) range from 1.10 to 1.25, TL distributions at intermediate PD become positively skewed, and virtual cell clones show bimodal size distributions. At P/C as high as 1.25 the majority of virtual cells at maximal PD contain telomeres with TL>1SU. A 10% increase in P/C within the 1.10-1.25 range produces a two-fold increase in the maximal PD, which can reach values of up to 25 observed in rodent and some human cells. Increasing the number of committed mitoses from 0 to 10 can increases PD to about 50 observed in human fibroblasts. Introduction of the random TL breakage makes the shapes of TL distributions quite dissimilar from those observed in real cells. Telomere length decrease is a correlate of cell proliferation that cannot alone account for the Hayflick limit, which primarily depends on parameters of cell population kinetics. Free radical damage influences the Hayflick limit not through TL but rather by affecting the ratio of the rates of events that commit cells to mitoses or to proliferation arrest.

Redeployment of germ layers related TFs shows regionalized expression during two non-embryonic developments.

PubMed

Ricci, Lorenzo; Cabrera, Fabien; Lotito, Sonia; Tiozzo, Stefano

2016-08-01

In all non-vertebrate metazoan phyla, species that evolved non-embryonic developmental pathways as means of propagation or regeneration can be found. In this context, new bodies arise through asexual reproduction processes (such as budding) or whole body regeneration, that lack the familiar temporal and spatial cues classically associated with embryogenesis, like maternal determinants, or gastrulation. The molecular mechanisms underlying those non-embryonic developments (i.e., regeneration and asexual reproduction), and their relationship to those deployed during embryogenesis are poorly understood. We have addressed this question in the colonial ascidian Botryllus schlosseri, which undergoes an asexual reproductive process via palleal budding (PB), as well as a whole body regeneration by vascular budding (VB). We identified early regenerative structures during VB and then followed the fate of differentiating tissues during both non-embryonic developments (PB and VB) by monitoring the expression of genes known to play key functions in germ layer specification with well conserved expression patterns in solitary ascidian embryogenesis. The expression patterns of FoxA1, GATAa, GATAb, Otx, Bra, Gsc and Tbx2/3 were analysed during both PB and VB. We found that the majority of these transcription factors were expressed during both non-embryonic developmental processes, revealing a regionalization of the palleal and vascular buds. Knockdown of GATAa by siRNA in palleal buds confirmed that preventing the correct development of one of these regions blocks further tissue specification. Our results indicate that during both normal and injury-induced budding, a similar alternative developmental program operates via early commitment of epithelial regions. Copyright © 2016. Published by Elsevier Inc.

Gene expression variability in human hepatic drug metabolizing enzymes and transporters.

PubMed

Yang, Lun; Price, Elvin T; Chang, Ching-Wei; Li, Yan; Huang, Ying; Guo, Li-Wu; Guo, Yongli; Kaput, Jim; Shi, Leming; Ning, Baitang

2013-01-01

Interindividual variability in the expression of drug-metabolizing enzymes and transporters (DMETs) in human liver may contribute to interindividual differences in drug efficacy and adverse reactions. Published studies that analyzed variability in the expression of DMET genes were limited by sample sizes and the number of genes profiled. We systematically analyzed the expression of 374 DMETs from a microarray data set consisting of gene expression profiles derived from 427 human liver samples. The standard deviation of interindividual expression for DMET genes was much higher than that for non-DMET genes. The 20 DMET genes with the largest variability in the expression provided examples of the interindividual variation. Gene expression data were also analyzed using network analysis methods, which delineates the similarities of biological functionalities and regulation mechanisms for these highly variable DMET genes. Expression variability of human hepatic DMET genes may affect drug-gene interactions and disease susceptibility, with concomitant clinical implications.

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data

PubMed Central

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R.; del Río-Navarro, Blanca E.; Mendoza-Vargas, Alfredo; Sánchez, Filiberto

2017-01-01

Background In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. Methods We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6–10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). Results From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Discussion Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments. PMID:29230367

Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum

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

Pasquo, Alessandra; Bonamore, Alessandra; Franceschini, Stefano

The cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from T. flavum, a protein which catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids, are reported. Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 Å resolution using a synchrotron-radiation source. The crystals belong to themore » trigonal space group P3{sub 1}21, with unit-cell parameters a = b = 86.31, c = 118.36 Å. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 Å resolution. The model is under construction.« less

Accelerated Evolution of Developmentally Biased Genes in the Tetraphenic Ant Cardiocondyla obscurior.

PubMed

Schrader, Lukas; Helanterä, Heikki; Oettler, Jan

2017-03-01

Plastic gene expression underlies phenotypic plasticity and plastically expressed genes evolve under different selection regimes compared with ubiquitously expressed genes. Social insects are well-suited models to elucidate the evolutionary dynamics of plastic genes for their genetically and environmentally induced discrete polymorphisms. Here, we study the evolution of plastically expressed genes in the ant Cardiocondyla obscurior-a species that produces two discrete male morphs in addition to the typical female polymorphism of workers and queens. Based on individual-level gene expression data from 28 early third instar larvae, we test whether the same evolutionary dynamics that pertain to plastically expressed genes in adults also pertain to genes with plastic expression during development. In order to quantify plasticity of gene expression over multiple contrasts, we develop a novel geometric measure. For genes expressed during development, we show that plasticity of expression is positively correlated with evolutionary rates. We furthermore find a strong correlation between expression plasticity and expression variation within morphs, suggesting a close link between active and passive plasticity of gene expression. Our results support the notion of relaxed selection and neutral processes as important drivers in the evolution of adaptive plasticity. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Analysis of multiplex gene expression maps obtained by voxelation.

PubMed

An, Li; Xie, Hongbo; Chin, Mark H; Obradovic, Zoran; Smith, Desmond J; Megalooikonomou, Vasileios

2009-04-29

Gene expression signatures in the mammalian brain hold the key to understanding neural development and neurological disease. Researchers have previously used voxelation in combination with microarrays for acquisition of genome-wide atlases of expression patterns in the mouse brain. On the other hand, some work has been performed on studying gene functions, without taking into account the location information of a gene's expression in a mouse brain. In this paper, we present an approach for identifying the relation between gene expression maps obtained by voxelation and gene functions. To analyze the dataset, we chose typical genes as queries and aimed at discovering similar gene groups. Gene similarity was determined by using the wavelet features extracted from the left and right hemispheres averaged gene expression maps, and by the Euclidean distance between each pair of feature vectors. We also performed a multiple clustering approach on the gene expression maps, combined with hierarchical clustering. Among each group of similar genes and clusters, the gene function similarity was measured by calculating the average gene function distances in the gene ontology structure. By applying our methodology to find similar genes to certain target genes we were able to improve our understanding of gene expression patterns and gene functions. By applying the clustering analysis method, we obtained significant clusters, which have both very similar gene expression maps and very similar gene functions respectively to their corresponding gene ontologies. The cellular component ontology resulted in prominent clusters expressed in cortex and corpus callosum. The molecular function ontology gave prominent clusters in cortex, corpus callosum and hypothalamus. The biological process ontology resulted in clusters in cortex, hypothalamus and choroid plexus. Clusters from all three ontologies combined were most prominently expressed in cortex and corpus callosum. The experimental results confirm the hypothesis that genes with similar gene expression maps might have similar gene functions. The voxelation data takes into account the location information of gene expression level in mouse brain, which is novel in related research. The proposed approach can potentially be used to predict gene functions and provide helpful suggestions to biologists.

Co-expression network analysis of duplicate genes in maize (Zea mays L.) reveals no subgenome bias.

PubMed

Li, Lin; Briskine, Roman; Schaefer, Robert; Schnable, Patrick S; Myers, Chad L; Flagel, Lex E; Springer, Nathan M; Muehlbauer, Gary J

2016-11-04

Gene duplication is prevalent in many species and can result in coding and regulatory divergence. Gene duplications can be classified as whole genome duplication (WGD), tandem and inserted (non-syntenic). In maize, WGD resulted in the subgenomes maize1 and maize2, of which maize1 is considered the dominant subgenome. However, the landscape of co-expression network divergence of duplicate genes in maize is still largely uncharacterized. To address the consequence of gene duplication on co-expression network divergence, we developed a gene co-expression network from RNA-seq data derived from 64 different tissues/stages of the maize reference inbred-B73. WGD, tandem and inserted gene duplications exhibited distinct regulatory divergence. Inserted duplicate genes were more likely to be singletons in the co-expression networks, while WGD duplicate genes were likely to be co-expressed with other genes. Tandem duplicate genes were enriched in the co-expression pattern where co-expressed genes were nearly identical for the duplicates in the network. Older gene duplications exhibit more extensive co-expression variation than younger duplications. Overall, non-syntenic genes primarily from inserted duplications show more co-expression divergence. Also, such enlarged co-expression divergence is significantly related to duplication age. Moreover, subgenome dominance was not observed in the co-expression networks - maize1 and maize2 exhibit similar levels of intra subgenome correlations. Intriguingly, the level of inter subgenome co-expression was similar to the level of intra subgenome correlations, and genes from specific subgenomes were not likely to be the enriched in co-expression network modules and the hub genes were not predominantly from any specific subgenomes in maize. Our work provides a comprehensive analysis of maize co-expression network divergence for three different types of gene duplications and identifies potential relationships between duplication types, duplication ages and co-expression consequences.

Tissue Non-Specific Genes and Pathways Associated with Diabetes: An Expression Meta-Analysis.

PubMed

Mei, Hao; Li, Lianna; Liu, Shijian; Jiang, Fan; Griswold, Michael; Mosley, Thomas

2017-01-21

We performed expression studies to identify tissue non-specific genes and pathways of diabetes by meta-analysis. We searched curated datasets of the Gene Expression Omnibus (GEO) database and identified 13 and five expression studies of diabetes and insulin responses at various tissues, respectively. We tested differential gene expression by empirical Bayes-based linear method and investigated gene set expression association by knowledge-based enrichment analysis. Meta-analysis by different methods was applied to identify tissue non-specific genes and gene sets. We also proposed pathway mapping analysis to infer functions of the identified gene sets, and correlation and independent analysis to evaluate expression association profile of genes and gene sets between studies and tissues. Our analysis showed that PGRMC1 and HADH genes were significant over diabetes studies, while IRS1 and MPST genes were significant over insulin response studies, and joint analysis showed that HADH and MPST genes were significant over all combined data sets. The pathway analysis identified six significant gene sets over all studies. The KEGG pathway mapping indicated that the significant gene sets are related to diabetes pathogenesis. The results also presented that 12.8% and 59.0% pairwise studies had significantly correlated expression association for genes and gene sets, respectively; moreover, 12.8% pairwise studies had independent expression association for genes, but no studies were observed significantly different for expression association of gene sets. Our analysis indicated that there are both tissue specific and non-specific genes and pathways associated with diabetes pathogenesis. Compared to the gene expression, pathway association tends to be tissue non-specific, and a common pathway influencing diabetes development is activated through different genes at different tissues.

Phosphorylation of serine 248 of C/EBPα is dispensable for myelopoiesis but its disruption leads to a low penetrant myeloid disorder with long latency.

PubMed

Hasemann, Marie S; Schuster, Mikkel B; Frank, Anne-Katrine; Theilgaard-Mönch, Kim; Pedersen, Thomas Å; Nerlov, Claus; Porse, Bo T

2012-01-01

Transcription factors play a key role in lineage commitment and differentiation of stem cells into distinct mature cells. In hematopoiesis, they regulate lineage-specific gene expression in a stage-specific manner through various physical and functional interactions with regulatory proteins that are simultanously recruited and activated to ensure timely gene expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is such a factor and is essential for the development of granulocytic/monocytic cells. The activity of C/EBPα is regulated on several levels including gene expression, alternative translation, protein interactions and posttranslational modifications, such as phosphorylation. In particular, the phosphorylation of serine 248 of the transactivation domain has been shown to be of crucial importance for granulocytic differentiation of 32Dcl3 cells in vitro. Here, we use mouse genetics to investigate the significance of C/EBPα serine 248 in vivo through the construction and analysis of Cebpa(S248A/S248A) knock-in mice. Surprisingly, 8-week old Cebpa(S248A/S248A) mice display normal steady-state hematopoiesis including unaltered development of mature myeloid cells. However, over time some of the animals develop a hematopoietic disorder with accumulation of multipotent, megakaryocytic and erythroid progenitor cells and a mild impairment of differentiation along the granulocytic-monocytic lineage. Furthermore, BM cells from Cebpa(S248A/S248A) animals display a competitive advantage compared to wild type cells in a transplantation assay. Taken together, our data shows that the substitution of C/EBPα serine 248 to alanine favors the selection of the megakaryocytic/erythroid lineage over the monocytic/granulocytic compartment in old mice and suggests that S248 phosphorylation may be required to maintain proper hematopoietic homeostasis in response to changes in the wiring of cellular signalling networks. More broadly, the marked differences between the phenotype of the S248A variant in vivo and in vitro highlight the need to exert caution when extending in vitro phenotypes to the more appropriate in vivo context.

Phosphorylation of Serine 248 of C/EBPα Is Dispensable for Myelopoiesis but Its Disruption Leads to a Low Penetrant Myeloid Disorder with Long Latency

PubMed Central

Frank, Anne-Katrine; Theilgaard-Mönch, Kim; Pedersen, Thomas Å.; Nerlov, Claus; Porse, Bo T.

2012-01-01

Background Transcription factors play a key role in lineage commitment and differentiation of stem cells into distinct mature cells. In hematopoiesis, they regulate lineage-specific gene expression in a stage-specific manner through various physical and functional interactions with regulatory proteins that are simultanously recruited and activated to ensure timely gene expression. The transcription factor CCAAT/enhancer binding protein α (C/EBPα) is such a factor and is essential for the development of granulocytic/monocytic cells. The activity of C/EBPα is regulated on several levels including gene expression, alternative translation, protein interactions and posttranslational modifications, such as phosphorylation. In particular, the phosphorylation of serine 248 of the transactivation domain has been shown to be of crucial importance for granulocytic differentiation of 32Dcl3 cells in vitro. Methodology/Principal Findings Here, we use mouse genetics to investigate the significance of C/EBPα serine 248 in vivo through the construction and analysis of Cebpa S248A/S248A knock-in mice. Surprisingly, 8-week old Cebpa S248A/S248A mice display normal steady-state hematopoiesis including unaltered development of mature myeloid cells. However, over time some of the animals develop a hematopoietic disorder with accumulation of multipotent, megakaryocytic and erythroid progenitor cells and a mild impairment of differentiation along the granulocytic-monocytic lineage. Furthermore, BM cells from Cebpa S248A/S248A animals display a competitive advantage compared to wild type cells in a transplantation assay. Conclusions/Significance Taken together, our data shows that the substitution of C/EBPα serine 248 to alanine favors the selection of the megakaryocytic/erythroid lineage over the monocytic/granulocytic compartment in old mice and suggests that S248 phosphorylation may be required to maintain proper hematopoietic homeostasis in response to changes in the wiring of cellular signalling networks. More broadly, the marked differences between the phenotype of the S248A variant in vivo and in vitro highlight the need to exert caution when extending in vitro phenotypes to the more appropriate in vivo context. PMID:22715416

Transcriptional profiling defines dynamics of parasite tissue sequestration during malaria infection.

PubMed

Pelle, Karell G; Oh, Keunyoung; Buchholz, Kathrin; Narasimhan, Vagheesh; Joice, Regina; Milner, Danny A; Brancucci, Nicolas Mb; Ma, Siyuan; Voss, Till S; Ketman, Ken; Seydel, Karl B; Taylor, Terrie E; Barteneva, Natasha S; Huttenhower, Curtis; Marti, Matthias

2015-01-01

During intra-erythrocytic development, late asexually replicating Plasmodium falciparum parasites sequester from peripheral circulation. This facilitates chronic infection and is linked to severe disease and organ-specific pathology including cerebral and placental malaria. Immature gametocytes - sexual stage precursor cells - likewise disappear from circulation. Recent work has demonstrated that these sexual stage parasites are located in the hematopoietic system of the bone marrow before mature gametocytes are released into the bloodstream to facilitate mosquito transmission. However, as sequestration occurs only in vivo and not during in vitro culture, the mechanisms by which it is regulated and enacted (particularly by the gametocyte stage) remain poorly understood. We generated the most comprehensive P. falciparum functional gene network to date by integrating global transcriptional data from a large set of asexual and sexual in vitro samples, patient-derived in vivo samples, and a new set of in vitro samples profiling sexual commitment. We defined more than 250 functional modules (clusters) of genes that are co-expressed primarily during the intra-erythrocytic parasite cycle, including 35 during sexual commitment and gametocyte development. Comparing the in vivo and in vitro datasets allowed us, for the first time, to map the time point of asexual parasite sequestration in patients to 22 hours post-invasion, confirming previous in vitro observations on the dynamics of host cell modification and cytoadherence. Moreover, we were able to define the properties of gametocyte sequestration, demonstrating the presence of two circulating gametocyte populations: gametocyte rings between 0 and approximately 30 hours post-invasion and mature gametocytes after around 7 days post-invasion. This study provides a bioinformatics resource for the functional elucidation of parasite life cycle dynamics and specifically demonstrates the presence of the gametocyte ring stages in circulation, adding significantly to our understanding of the dynamics of gametocyte sequestration in vivo.

Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood–derived hematopoietic progenitor cells

PubMed Central

Martin, Colin H.; Woll, Petter S.; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos

2008-01-01

Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34+ cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34+ cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34+ cells also did not produce B cells in vitro. In contrast, CD34+ cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor–induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs. PMID:18621931

Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood-derived hematopoietic progenitor cells.

PubMed

Martin, Colin H; Woll, Petter S; Ni, Zhenya; Zúñiga-Pflücker, Juan Carlos; Kaufman, Dan S

2008-10-01

Hematopoietic progenitor cells derived from human embryonic stem cells (hESCs) develop into diverse mature hematopoietic lineages, including lymphocytes. Whereas functional natural killer (NK) cells can be efficiently generated in vitro from hESC-derived CD34(+) cells, studies of T- and B-cell development from hESCs have been much more limited. Here, we demonstrate that despite expressing functional Notch-1, CD34(+) cells from hESCs did not derive T cells when cocultured with OP9 cells expressing Delta-like 1, or in fetal thymus organ culture. hESC-derived CD34(+) cells also did not produce B cells in vitro. In contrast, CD34(+) cells isolated from UCB routinely generated T and B cells when cultured in the same conditions. Notably, both undifferentiated hESCs, and sorted hESC-derived populations with hematopoietic developmental potential exhibited constitutive expression of ID family genes and of transcriptional targets of stem cell factor-induced signaling. These pathways both inhibit T-cell development and promote NK-cell development. Together, these results demonstrate fundamental differences between hESC-derived hematopoietic progenitors and analogous primary human cells. Therefore, hESCs can be more readily supported to differentiate into certain cell types than others, findings that have important implications for derivation of defined lineage-committed populations from hESCs.

Interleukin-6 inhibits early differentiation of ATDC5 chondrogenic progenitor cells.

PubMed

Nakajima, Shoko; Naruto, Takuya; Miyamae, Takako; Imagawa, Tomoyuki; Mori, Masaaki; Nishimaki, Shigeru; Yokota, Shumpei

2009-08-01

Interleukin (IL)-6 is a causative agent of systemic juvenile idiopathic arthritis (sJIA), a chronic inflammatory disease complicated with severe growth impairment. Recent trials of anti-IL-6 receptor monoclonal antibody, tocilizumab, indicated that tocilizumab blocks IL-6/IL-6 receptor-mediated inflammation, and induces catch-up growth in children with sJIA. This study evaluates the effects of IL-6 on chondrogenesis by ATDC5 cells, a clonal murine chondrogenic cell line that provides an excellent model for studying endochondral ossification at growth plate. ATDC5 cells were examined for the expression of IL-6 receptor and gp130 by fluorescence-activated cell sorting analysis. Recombinant murine IL-6 was added to ATDC5 cultures to observe cell differentiation, using a quantitative RT-PCR for the chondrogenic differentiation markers type II collagen, aggrecan, and type X collagen. To block IL-6, the anti-mouse IL-6 receptor monoclonal antibody MR16-1 was added. As a result, the cells expressed IL-6 receptor and gp130. The expression of chondrogenic differentiation marker gene was reduced by IL-6, but this was abrogated by MR16-1. We conclude that IL-6 inhibits early chondrogenesis of ATDC5 cells suggesting that IL-6 may affect committed stem cells at a cellular level during chondrogenic differentiation of growth plate chondrocytes, and that IL-6 may be a cellular-level factor in growth impairment in sJIA.

Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiae.

PubMed

Solomon, Kevin V; Ovadia, Elisa; Yu, Fujio; Mizunashi, Wataru; O'Malley, Michelle A

2016-12-01

Bio-based isobutantol is a sustainable 'drop in' substitute for petroleum-based fuels. However, well-studied production routes, such as the Ehrlich pathway, have yet to be commercialized despite more than a century of research. The more versatile bacterial valine catabolism may be a competitive alternate route producing not only an isobutanol precursor but several carboxylic acids with applications as biomonomers, and building blocks for other advanced biofuels. Here, we transfer the first two committed steps of the pathway from pathogenic Pseudomonas aeruginosa PAO1 to yeast to evaluate their activity in a safer model organism. Genes encoding the heteroligomeric branched chain keto-acid dehydrogenase (BCKAD; bkdA1, bkdA2, bkdB, lpdV ), and the homooligomeric acyl-CoA dehydrogenase (ACD; acd1 ) were tagged with fluorescence epitopes and targeted for expression in either the mitochondria or cytoplasm of S. cerevisiae . We verified the localization of our constructs with confocal fluorescence microscopy before measuring the activity of tag-free constructs. Despite reduced heterologous expression of mitochondria-targeted enzymes, their specific activities were significantly improved with total enzyme activities up to 138% greater than those of enzymes expressed in the cytoplasm. In total, our results demonstrate that the choice of protein localization in yeast has significant impact on heterologous activity, and suggests a new path forward for isobutanol production.

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome.

PubMed

Hurst, Laurence D; Ghanbarian, Avazeh T; Forrest, Alistair R R; Huminiecki, Lukasz

2015-12-01

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X's gene content, gene expression, and evolution.

The Constrained Maximal Expression Level Owing to Haploidy Shapes Gene Content on the Mammalian X Chromosome

PubMed Central

Hurst, Laurence D.; Ghanbarian, Avazeh T.; Forrest, Alistair R. R.; Huminiecki, Lukasz

2015-01-01

X chromosomes are unusual in many regards, not least of which is their nonrandom gene content. The causes of this bias are commonly discussed in the context of sexual antagonism and the avoidance of activity in the male germline. Here, we examine the notion that, at least in some taxa, functionally biased gene content may more profoundly be shaped by limits imposed on gene expression owing to haploid expression of the X chromosome. Notably, if the X, as in primates, is transcribed at rates comparable to the ancestral rate (per promoter) prior to the X chromosome formation, then the X is not a tolerable environment for genes with very high maximal net levels of expression, owing to transcriptional traffic jams. We test this hypothesis using The Encyclopedia of DNA Elements (ENCODE) and data from the Functional Annotation of the Mammalian Genome (FANTOM5) project. As predicted, the maximal expression of human X-linked genes is much lower than that of genes on autosomes: on average, maximal expression is three times lower on the X chromosome than on autosomes. Similarly, autosome-to-X retroposition events are associated with lower maximal expression of retrogenes on the X than seen for X-to-autosome retrogenes on autosomes. Also as expected, X-linked genes have a lesser degree of increase in gene expression than autosomal ones (compared to the human/Chimpanzee common ancestor) if highly expressed, but not if lowly expressed. The traffic jam model also explains the known lower breadth of expression for genes on the X (and the Z of birds), as genes with broad expression are, on average, those with high maximal expression. As then further predicted, highly expressed tissue-specific genes are also rare on the X and broadly expressed genes on the X tend to be lowly expressed, both indicating that the trend is shaped by the maximal expression level not the breadth of expression per se. Importantly, a limit to the maximal expression level explains biased tissue of expression profiles of X-linked genes. Tissues whose tissue-specific genes are very highly expressed (e.g., secretory tissues, tissues abundant in structural proteins) are also tissues in which gene expression is relatively rare on the X chromosome. These trends cannot be fully accounted for in terms of alternative models of biased expression. In conclusion, the notion that it is hard for genes on the Therian X to be highly expressed, owing to transcriptional traffic jams, provides a simple yet robustly supported rationale of many peculiar features of X’s gene content, gene expression, and evolution. PMID:26685068

Electrospun biomimetic scaffold of hydroxyapatite/chitosan supports enhanced osteogenic differentiation of mMSCs

NASA Astrophysics Data System (ADS)

Peng, Hongju; Yin, Zi; Liu, Huanhuan; Chen, Xiao; Feng, Bei; Yuan, Huihua; Su, Bo; Ouyang, Hongwei; Zhang, Yanzhong

2012-12-01

Engaging functional biomaterial scaffolds to regulate stem cell differentiation has drawn a great deal of attention in the tissue engineering and regenerative medicine community. In this study, biomimetic composite nanofibrous scaffolds of hydroxyapatite/chitosan (HAp/CTS) were prepared to investigate their capacity for inducing murine mesenchymal stem cells (mMSCs) to differentiate into the osteogenic lineage, in the absence and presence of an osteogenic supplementation (i.e., ascorbic acid, β-glycerol phosphate, and dexamethasone), respectively. Using electrospun chitosan (CTS) nanofibrous scaffolds as the control, cell morphology, growth, specific osteogenic genes expression, and quantified proteins secretion on the HAp/CTS scaffolds were sequentially examined and assessed. It appeared that the HAp/CTS scaffolds supported better attachment and proliferation of the mMSCs. Most noteworthy was that in the absence of the osteogenic supplementation, expression of osteogenic genes including collagen I (Col I), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OCN) were significantly upregulated in mMSCs cultured on the HAp/CTS nanofibrous scaffolds. Also increased secretion of the osteogenesis protein markers of alkaline phosphatase and collagen confirmed that the HAp/CTS nanofibrous scaffold markedly promoted the osteogenic commitment in the mMSCs. Moreover, the presence of osteogenic supplementation proved an enhanced efficacy of mMSC osteogenesis on the HAp/CTS nanofibrous scaffolds. Collectively, this study demonstrated that the biomimetic nanofibrous HAp/CTS scaffolds could support and enhance the adhesion, proliferation, and particularly osteogenic differentiation of the mMSCs. It also substantiated the potential of using biomimetic nanofibrous scaffolds of HAp/CTS for functional bone repair and regeneration applications.

Validating internal controls for quantitative plant gene expression studies.

PubMed

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-08-18

Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments.

General statistics of stochastic process of gene expression in eukaryotic cells.

PubMed Central

Kuznetsov, V A; Knott, G D; Bonner, R F

2002-01-01

Thousands of genes are expressed at such very low levels (< or =1 copy per cell) that global gene expression analysis of rarer transcripts remains problematic. Ambiguity in identification of rarer transcripts creates considerable uncertainty in fundamental questions such as the total number of genes expressed in an organism and the biological significance of rarer transcripts. Knowing the distribution of the true number of genes expressed at each level and the corresponding gene expression level probability function (GELPF) could help resolve these uncertainties. We found that all observed large-scale gene expression data sets in yeast, mouse, and human cells follow a Pareto-like distribution model skewed by many low-abundance transcripts. A novel stochastic model of the gene expression process predicts the universality of the GELPF both across different cell types within a multicellular organism and across different organisms. This model allows us to predict the frequency distribution of all gene expression levels within a single cell and to estimate the number of expressed genes in a single cell and in a population of cells. A random "basal" transcription mechanism for protein-coding genes in all or almost all eukaryotic cell types is predicted. This fundamental mechanism might enhance the expression of rarely expressed genes and, thus, provide a basic level of phenotypic diversity, adaptability, and random monoallelic expression in cell populations. PMID:12136033

The changes of gene expression profiling between segmental vitiligo, generalized vitiligo and healthy individual.

PubMed

Wang, Ping; Li, Yong; Nie, Huiqiong; Zhang, Xiaoyan; Shao, Qiongyan; Hou, Xiuli; Xu, Wen; Hong, Weisong; Xu, Aie

2016-10-01

Vitiligo is a common acquired depigmentation skin disease characterized by loss or dysfunction of melanocytes within the skin lesion, but its pathologenesis is far from lucid. The gene expression profiling of segmental vitiligo (SV) and generalized vitiligo (GV) need further investigation. To better understanding the common and distinct factors, especially in the view of gene expression profile, which were involved in the diseases development and maintenance of segmental vitiligo (SV) and generalized vitiligo (GV). Peripheral bloods were collected from SV, GV and healthy individual (HI), followed by leukocytes separation and total RNA extraction. The high-throughput whole genome expression microarrays were used to assay the gene expression profiles between HI, SV and GV. Bioinformatics tools were employed to annotated the biological function of differently expressed genes. Quantitative PCR assay was used to validate the gene expression of array. Compared to HI, 239 over-expressed genes and 175 down-expressed genes detected in SV, 688 over-expressed genes and 560 down-expressed genes were found in GV, following the criteria of log2 (fold change)≥0.585 and P value<0.05. In these differently expressed genes, 60 over-expressed genes and 60 down-expressed genes had similar tendency in SV and GV. Compared to SV, 223 genes were up regulated and 129 genes were down regulated in GV. In the SV with HI as control, the differently expressed genes were mainly involved in the adaptive immune response, cytokine-cytokine receptor interaction, chemokine signaling, focal adhesion and sphingolipid metabolism. The differently expressed genes between GV and HI were mainly involved in the innate immune, autophagy, apoptosis, melanocyte biology, ubiquitin mediated proteolysis and tyrosine metabolism, which was different from SV. While the differently expressed genes between SV and GV were mainly involved in the metabolism pathway of purine, pyrimidine, glycolysis and sphingolipid. Above results suggested that they not only shared part bio-process and signal pathway, but more important, they utilized different biological mechanism in their pathogenesis and maintenance. Our results provide a comprehensive view on the gene expression profiling change between SV and GV especially in the side of leukocytes, and may facilitate the future study on their molecular mechanism and theraputic targets. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Identification of reference genes in human myelomonocytic cells for gene expression studies in altered gravity.

PubMed

Thiel, Cora S; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Unverdorben, Felix; Buttron, Isabell; Lauber, Beatrice; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E; Ullrich, Oliver

2015-01-01

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes ("housekeeping genes") are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity.

Adaptation of Musca domestica L. Field Population to Laboratory Breeding Causes Transcriptional Alterations

PubMed Central

Højland, Dorte H.; Jensen, Karl-Martin Vagn; Kristensen, Michael

2014-01-01

Background The housefly, Musca domestica, has developed resistance to most insecticides applied for its control. Expression of genes coding for detoxification enzymes play a role in the response of the housefly when encountered by a xenobiotic. The highest level of constitutive gene expression of nine P450 genes was previously found in a newly-collected susceptible field population in comparison to three insecticide-resistant laboratory strains and a laboratory reference strain. Results We compared gene expression of five P450s by qPCR as well as global gene expression by RNAseq in the newly-acquired field population (845b) in generation F1, F13 and F29 to test how gene expression changes following laboratory adaption. Four (CYP6A1, CYP6A36, CYP6D3, CYP6G4) of five investigated P450 genes adapted to breeding by decreasing expression. CYP6D1 showed higher female expression in F29 than in F1. For males, about half of the genes accessed in the global gene expression were up-regulated in F13 and F29 in comparison with the F1 population. In females, 60% of the genes were up-regulated in F13 in comparison with F1, while 33% were up-regulated in F29. Forty potential P450 genes were identified. In most cases, P450 gene expression was decreased in F13 flies in comparison with F1. Gene expression then increased from F13 to F29 in males and decreased further in females. Conclusion The global gene expression changes massively during adaptation to laboratory breeding. In general, global expression decreased as a result of laboratory adaption in males, while female expression was not unidirectional. Expression of P450 genes was in general down-regulated as a result of laboratory adaption. Expression of hexamerin, coding for a storage protein was increased, while gene expression of genes coding for amylases decreased. This suggests a major impact of the surrounding environment on gene response to xenobiotics and genetic composition of housefly strains. PMID:24489682

A consumer satisfaction survey of civilly committed sex offenders in Illinois.

PubMed

Levenson, Jill S; Prescott, David S; Jumper, Shan

2014-04-01

The purpose of this study was to obtain feedback from civilly committed sex offenders (N = 113) about the components of treatment that they believed to be most important and helpful in preventing reoffense. Participants were also asked to rate their satisfaction with the treatment process and therapists. Victim empathy and accountability were rated as the most important elements of treatment, along with skills for preventing relapse and methods for controlling sexual arousal. There was a fairly robust correlation between client perceptions of importance and satisfaction on most treatment components. Some clients expressed concerns about respect, confidentiality, and judgmental attitudes of some therapists. Because civilly committed sex offenders are considered to be among the most likely to reoffend, strategies are discussed for engagement of this population in a meaningful process of change.

An RNA-Seq based gene expression atlas of the common bean.

PubMed

O'Rourke, Jamie A; Iniguez, Luis P; Fu, Fengli; Bucciarelli, Bruna; Miller, Susan S; Jackson, Scott A; McClean, Philip E; Li, Jun; Dai, Xinbin; Zhao, Patrick X; Hernandez, Georgina; Vance, Carroll P

2014-10-06

Common bean (Phaseolus vulgaris) is grown throughout the world and comprises roughly 50% of the grain legumes consumed worldwide. Despite this, genetic resources for common beans have been lacking. Next generation sequencing, has facilitated our investigation of the gene expression profiles associated with biologically important traits in common bean. An increased understanding of gene expression in common bean will improve our understanding of gene expression patterns in other legume species. Combining recently developed genomic resources for Phaseolus vulgaris, including predicted gene calls, with RNA-Seq technology, we measured the gene expression patterns from 24 samples collected from seven tissues at developmentally important stages and from three nitrogen treatments. Gene expression patterns throughout the plant were analyzed to better understand changes due to nodulation, seed development, and nitrogen utilization. We have identified 11,010 genes differentially expressed with a fold change ≥ 2 and a P-value < 0.05 between different tissues at the same time point, 15,752 genes differentially expressed within a tissue due to changes in development, and 2,315 genes expressed only in a single tissue. These analyses identified 2,970 genes with expression patterns that appear to be directly dependent on the source of available nitrogen. Finally, we have assembled this data in a publicly available database, The Phaseolus vulgaris Gene Expression Atlas (Pv GEA), http://plantgrn.noble.org/PvGEA/ . Using the website, researchers can query gene expression profiles of their gene of interest, search for genes expressed in different tissues, or download the dataset in a tabular form. These data provide the basis for a gene expression atlas, which will facilitate functional genomic studies in common bean. Analysis of this dataset has identified genes important in regulating seed composition and has increased our understanding of nodulation and impact of the nitrogen source on assimilation and distribution throughout the plant.

Analysis of bHLH coding genes using gene co-expression network approach.

PubMed

Srivastava, Swati; Sanchita; Singh, Garima; Singh, Noopur; Srivastava, Gaurava; Sharma, Ashok

2016-07-01

Network analysis provides a powerful framework for the interpretation of data. It uses novel reference network-based metrices for module evolution. These could be used to identify module of highly connected genes showing variation in co-expression network. In this study, a co-expression network-based approach was used for analyzing the genes from microarray data. Our approach consists of a simple but robust rank-based network construction. The publicly available gene expression data of Solanum tuberosum under cold and heat stresses were considered to create and analyze a gene co-expression network. The analysis provide highly co-expressed module of bHLH coding genes based on correlation values. Our approach was to analyze the variation of genes expression, according to the time period of stress through co-expression network approach. As the result, the seed genes were identified showing multiple connections with other genes in the same cluster. Seed genes were found to be vary in different time periods of stress. These analyzed seed genes may be utilized further as marker genes for developing the stress tolerant plant species.

The metabotropic P2Y4 receptor participates in the commitment to differentiation and cell death of human neuroblastoma SH-SY5Y cells.

PubMed

Cavaliere, Fabio; Nestola, Valeria; Amadio, Susanna; D'Ambrosi, Nadia; Angelini, Daniela F; Sancesario, Giuseppe; Bernardi, Giorgio; Volonté, Cinzia

2005-02-01

Extracellular nucleotides exert a variety of biological actions through different subtypes of P2 receptors. Here we characterized in the human neuroblastoma SH-SY5Y cells the simultaneous presence of various P2 receptors, belonging to the P2X ionotropic and P2Y metabotropic families. Western blot analysis detected the P2X1,2,4,5,6,7 and P2Y1,2,4,6, but not the P2X3 and P2Y12 receptors. We then investigated which biological effects were mediated by the P2Y4 subtype and its physiological pyrimidine agonist UTP. We found that neuronal differentiation of the SH-SY5Y cells with dibutiryl-cAMP increased the expression of the P2Y4 protein and that UTP itself was able to positively interfere with neuritogenesis. Moreover, transient transfection and activation of P2Y4 also facilitated neuritogenesis in SH-SY5Y cells, as detected by morphological phase contrast analysis and confocal examination of neurofilament proteins NFL. This was concurrent with increased transcription of immediate-early genes linked to differentiation such as cdk-5 and NeuroD6, and activity of AP-1 transcription family members such as c-fos, fos-B, and jun-D. Nevertheless, a prolonged activation of the P2Y4 receptor by UTP also induced cell death, both in naive, differentiated, and P2Y4-transfected SH-SY5Y cells, as measured by direct count of intact nuclei and cytofluorimetric analysis of damaged DNA. Taken together, our data indicate that the high expression and activation of the P2Y4 receptor participates in the neuronal differentiation and commitment to death of SH-SY5Y cells.

Low oxygen tension enhances endothelial fate of human pluripotent stem cells.

PubMed

Kusuma, Sravanti; Peijnenburg, Elizabeth; Patel, Parth; Gerecht, Sharon

2014-04-01

A critical regulator of the developing or regenerating vasculature is low oxygen tension. Precise elucidation of the role of low oxygen environments on endothelial commitment from human pluripotent stem cells necessitates controlled in vitro differentiation environments. We used a feeder-free, 2-dimensional differentiation system in which we could monitor accurately dissolved oxygen levels during human pluripotent stem cell differentiation toward early vascular cells (EVCs). We found that oxygen uptake rate of differentiating human pluripotent stem cells is lower in 5% O2 compared with atmospheric conditions. EVCs differentiated in 5% O2 had an increased vascular endothelial cadherin expression with clusters of vascular endothelial cadherin+ cells surrounded by platelet-derived growth factor β+ cells. When we assessed the temporal effects of low oxygen differentiation environments, we determined that low oxygen environments during the early stages of EVC differentiation enhance endothelial lineage commitment. EVCs differentiated in 5% O2 exhibited an increased expression of vascular endothelial cadherin and CD31 along with their localization to the membrane, enhanced lectin binding and acetylated low-density lipoprotein uptake, rapid cord-like structure formation, and increased expression of arterial endothelial cell markers. Inhibition of reactive oxygen species generation during the early stages of differentiation abrogated the endothelial inductive effects of the low oxygen environments. Low oxygen tension during early stages of EVC derivation induces endothelial commitment and maturation through the accumulation of reactive oxygen species, highlighting the importance of regulating oxygen tensions during human pluripotent stem cell-vascular differentiation.

Validating internal controls for quantitative plant gene expression studies

PubMed Central

Brunner, Amy M; Yakovlev, Igor A; Strauss, Steven H

2004-01-01

Background Real-time reverse transcription PCR (RT-PCR) has greatly improved the ease and sensitivity of quantitative gene expression studies. However, accurate measurement of gene expression with this method relies on the choice of a valid reference for data normalization. Studies rarely verify that gene expression levels for reference genes are adequately consistent among the samples used, nor compare alternative genes to assess which are most reliable for the experimental conditions analyzed. Results Using real-time RT-PCR to study the expression of 10 poplar (genus Populus) housekeeping genes, we demonstrate a simple method for determining the degree of stability of gene expression over a set of experimental conditions. Based on a traditional method for analyzing the stability of varieties in plant breeding, it defines measures of gene expression stability from analysis of variance (ANOVA) and linear regression. We found that the potential internal control genes differed widely in their expression stability over the different tissues, developmental stages and environmental conditions studied. Conclusion Our results support that quantitative comparisons of candidate reference genes are an important part of real-time RT-PCR studies that seek to precisely evaluate variation in gene expression. The method we demonstrated facilitates statistical and graphical evaluation of gene expression stability. Selection of the best reference gene for a given set of experimental conditions should enable detection of biologically significant changes in gene expression that are too small to be revealed by less precise methods, or when highly variable reference genes are unknowingly used in real-time RT-PCR experiments. PMID:15317655

Characterization of basal gene expression trends over a diurnal cycle in Xiphophorus maculatus skin, brain and liver.

PubMed

Lu, Yuan; Reyes, Jose; Walter, Sean; Gonzalez, Trevor; Medrano, Geraldo; Boswell, Mikki; Boswell, William; Savage, Markita; Walter, Ronald

2018-06-01

Evolutionarily conserved diurnal circadian mechanisms maintain oscillating patterns of gene expression based on the day-night cycle. Xiphophorus fish have been used to evaluate transcriptional responses after exposure to various light sources and it was determined that each source incites distinct genetic responses in skin tissue. However, basal expression levels of genes that show oscillating expression patterns in day-night cycle, may affect the outcomes of such experiments, since basal gene expression levels at each point in the circadian path may influence the profile of identified light responsive genes. Lack of knowledge regarding diurnal fluctuations in basal gene expression patterns may confound the understanding of genetic responses to external stimuli (e.g., light) since the dynamic nature of gene expression implies animals subjected to stimuli at different times may be at very different stages within the continuum of genetic homeostasis. We assessed basal gene expression changes over a 24-hour period in 200 select Xiphophorus gene targets known to transcriptionally respond to various types of light exposure. We identified 22 genes in skin, 36 genes in brain and 28 genes in liver that exhibit basal oscillation of expression patterns. These genes, including known circadian regulators, produced the expected expression patterns over a 24-hour cycle when compared to circadian regulatory genes identified in other species, especially human and other vertebrate animal models. Our results suggest the regulatory network governing diurnal oscillating gene expression is similar between Xiphophorus and other vertebrates for the three Xiphophorus organs tested. In addition, we were able to categorize light responsive gene sets in Xiphophorus that do, and do not, exhibit circadian based oscillating expression patterns. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of Reference Genes in Human Myelomonocytic Cells for Gene Expression Studies in Altered Gravity

PubMed Central

Thiel, Cora S.; Hauschild, Swantje; Tauber, Svantje; Paulsen, Katrin; Raig, Christiane; Raem, Arnold; Biskup, Josefine; Gutewort, Annett; Hürlimann, Eva; Philpot, Claudia; Lier, Hartwin; Engelmann, Frank; Layer, Liliana E.

2015-01-01

Gene expression studies are indispensable for investigation and elucidation of molecular mechanisms. For the process of normalization, reference genes (“housekeeping genes”) are essential to verify gene expression analysis. Thus, it is assumed that these reference genes demonstrate similar expression levels over all experimental conditions. However, common recommendations about reference genes were established during 1 g conditions and therefore their applicability in studies with altered gravity has not been demonstrated yet. The microarray technology is frequently used to generate expression profiles under defined conditions and to determine the relative difference in expression levels between two or more different states. In our study, we searched for potential reference genes with stable expression during different gravitational conditions (microgravity, normogravity, and hypergravity) which are additionally not altered in different hardware systems. We were able to identify eight genes (ALB, B4GALT6, GAPDH, HMBS, YWHAZ, ABCA5, ABCA9, and ABCC1) which demonstrated no altered gene expression levels in all tested conditions and therefore represent good candidates for the standardization of gene expression studies in altered gravity. PMID:25654098

Life-cycle and growth-phase-dependent regulation of the ubiquitin genes of Trypanosoma cruzi.

PubMed

Manning-Cela, Rebeca; Jaishankar, Sobha; Swindle, John

2006-07-01

Trypanosoma cruzi, the causative agent of Chagas disease, exhibits a complex life cycle that is accompanied by the stage-specific gene expression. At the molecular level, very little is known about gene regulation in trypanosomes. Complex gene organizations coupled with polycistronic transcription units make the analysis of regulated gene expression difficult in trypanosomes. The ubiquitin genes of T. cruzi are a good example of this complexity. They are organized as a single cluster containing five ubiquitin fusion (FUS) and five polyubiquitin (PUB) genes that are polycistronically transcribed but expressed differently in response to developmental and environmental changes. Gene replacements were used to study FUS and PUB gene expression at different stages of growth and at different points in the life cycle of T. cruzi. Based on the levels of reporter gene expression, it was determined that FUS1 expression was downregulated as the parasites approached stationary phase, whereas PUB12.5 polyubiquitin gene expression increased. Conversely, FUS1 expression increases when epimastigotes and amastigotes differentiate into trypomastigotes, whereas the expression of PUB12.5 decreases when epimastigotes differentiate into amastigotes and trypomastigotes. Although the level of CAT activity in logarithmic growing epimastigotes is six- to seven-fold higher when the gene was expressed from the FUS1 locus than when expressed from the PUB12.5 locus, the rate of transcription from the two loci was the same implying that post-transcriptional mechanisms play a dominant role in the regulation of gene expression.

The three keys to quality customer service: opening the doors to exceptional performance.

PubMed

Myers, Pennie; Nance, Don W

2002-01-01

Excellence in customer service requires three things. The first is a commitment to a set of principles. These principles reflect beliefs and assumptions that people are valuable and deserve to be treated with dignity and respect. This commitment is expressed to customers by an adherence to six customer-friendly processes and by personnel who possess a high level of skill in the behaviors that make up customer service. When these three requirements are met, laboratorians not only maintain a competitive edge but also fulfill their mission as health-care professionals.

Gene expression changes with age in skin, adipose tissue, blood and brain.

PubMed

Glass, Daniel; Viñuela, Ana; Davies, Matthew N; Ramasamy, Adaikalavan; Parts, Leopold; Knowles, David; Brown, Andrew A; Hedman, Asa K; Small, Kerrin S; Buil, Alfonso; Grundberg, Elin; Nica, Alexandra C; Di Meglio, Paola; Nestle, Frank O; Ryten, Mina; Durbin, Richard; McCarthy, Mark I; Deloukas, Panagiotis; Dermitzakis, Emmanouil T; Weale, Michael E; Bataille, Veronique; Spector, Tim D

2013-07-26

Previous studies have demonstrated that gene expression levels change with age. These changes are hypothesized to influence the aging rate of an individual. We analyzed gene expression changes with age in abdominal skin, subcutaneous adipose tissue and lymphoblastoid cell lines in 856 female twins in the age range of 39-85 years. Additionally, we investigated genotypic variants involved in genotype-by-age interactions to understand how the genomic regulation of gene expression alters with age. Using a linear mixed model, differential expression with age was identified in 1,672 genes in skin and 188 genes in adipose tissue. Only two genes expressed in lymphoblastoid cell lines showed significant changes with age. Genes significantly regulated by age were compared with expression profiles in 10 brain regions from 100 postmortem brains aged 16 to 83 years. We identified only one age-related gene common to the three tissues. There were 12 genes that showed differential expression with age in both skin and brain tissue and three common to adipose and brain tissues. Skin showed the most age-related gene expression changes of all the tissues investigated, with many of the genes being previously implicated in fatty acid metabolism, mitochondrial activity, cancer and splicing. A significant proportion of age-related changes in gene expression appear to be tissue-specific with only a few genes sharing an age effect in expression across tissues. More research is needed to improve our understanding of the genetic influences on aging and the relationship with age-related diseases.

Transgelin is a TGFβ-inducible gene that regulates osteoblastic and adipogenic differentiation of human skeletal stem cells through actin cytoskeleston organization

PubMed Central

Elsafadi, M; Manikandan, M; Dawud, R A; Alajez, N M; Hamam, R; Alfayez, M; Kassem, M; Aldahmash, A; Mahmood, A

2016-01-01

Regenerative medicine is a novel approach for treating conditions in which enhanced bone regeneration is required. We identified transgelin (TAGLN), a transforming growth factor beta (TGFβ)-inducible gene, as an upregulated gene during in vitro osteoblastic and adipocytic differentiation of human bone marrow-derived stromal (skeletal) stem cells (hMSC). siRNA-mediated gene silencing of TAGLN impaired lineage differentiation into osteoblasts and adipocytes but enhanced cell proliferation. Additional functional studies revealed that TAGLN deficiency impaired hMSC cell motility and in vitro transwell cell migration. On the other hand, TAGLN overexpression reduced hMSC cell proliferation, but enhanced cell migration, osteoblastic and adipocytic differentiation, and in vivo bone formation. In addition, deficiency or overexpression of TAGLN in hMSC was associated with significant changes in cellular and nuclear morphology and cytoplasmic organelle composition as demonstrated by high content imaging and transmission electron microscopy that revealed pronounced alterations in the distribution of the actin filament and changes in cytoskeletal organization. Molecular signature of TAGLN-deficient hMSC showed that several genes and genetic pathways associated with cell differentiation, including regulation of actin cytoskeleton and focal adhesion pathways, were downregulated. Our data demonstrate that TAGLN has a role in generating committed progenitor cells from undifferentiated hMSC by regulating cytoskeleton organization. Targeting TAGLN is a plausible approach to enrich for committed hMSC cells needed for regenerative medicine application. PMID:27490926

Differential expression profiles and pathways of genes in sugarcane leaf at elongation stage in response to drought stress

PubMed Central

Li, Changning; Nong, Qian; Solanki, Manoj Kumar; Liang, Qiang; Xie, Jinlan; Liu, Xiaoyan; Li, Yijie; Wang, Weizan; Yang, Litao; Li, Yangrui

2016-01-01

Water stress causes considerable yield losses in sugarcane. To investigate differentially expressed genes under water stress, a pot experiment was performed with the sugarcane variety GT21 at three water-deficit levels (mild, moderate, and severe) during the elongation stage and gene expression was analyzed using microarray technology. Physiological parameters of sugarcane showed significant alterations in response to drought stress. Based on the expression profile of 15,593 sugarcane genes, 1,501 (9.6%) genes were differentially expressed under different water-level treatments; 821 genes were upregulated and 680 genes were downregulated. A gene similarity analysis showed that approximately 62.6% of the differentially expressed genes shared homology with functional proteins. In a Gene Ontology (GO) analysis, 901 differentially expressed genes were assigned to 36 GO categories. Moreover, 325 differentially expressed genes were classified into 101 pathway categories involved in various processes, such as the biosynthesis of secondary metabolites, ribosomes, carbon metabolism, etc. In addition, some unannotated genes were detected; these may provide a basis for studies of water-deficit tolerance. The reliability of the observed expression patterns was confirmed by RT-PCR. The results of this study may help identify useful genes for improving drought tolerance in sugarcane. PMID:27170459

Nuclear translocation of PKCα isoenzyme is involved in neurogenic commitment of human neural crest-derived periodontal ligament stem cells.

PubMed

Trubiani, Oriana; Guarnieri, Simone; Diomede, Francesca; Mariggiò, Maria A; Merciaro, Ilaria; Morabito, Caterina; Cavalcanti, Marcos F X B; Cocco, Lucio; Ramazzotti, Giulia

2016-11-01

Stem cells isolated from human adult tissue niche represent a promising source for neural differentiation. Human Periodontal Ligament Stem Cells (hPDLSCs) originating from the neural crest are particularly suitable for induction of neural commitment. In this study, under xeno-free culture conditions, in undifferentiated hPDLSCs and in hPDLSCs induced to neuronal differentiation by basic Fibroblast Growth Factor, the level of some neural markers have been analyzed. The hPDLSCs spontaneously express Nestin, a neural progenitor marker. In these cells, the neurogenic process induced to rearrange the cytoskeleton, form neurospheres and express higher levels of Nestin and Tyrosine Hydroxylase, indicating neural induction. Protein Kinase C (PKC) is highly expressed in neural tissue and has a key role in neuronal functions. In particular the Ca(2+) and diacylglycerol-dependent activation of PKCα isozyme is involved in the regulation of neuronal differentiation. Another main component of the pathways controlling neuronal differentiation is the Growth Associated Protein-43 (GAP-43), whose activity is strictly regulated by PKC. The aim of this study is to investigate the role of PKCα/GAP-43 nuclear signal transduction pathway during neuronal commitment of hPDLSCs. During hPDLSCs neurogenic commitment the levels of p-PKC and p-GAP-43 increased both in cytoplasmic and nuclear compartment. PKCα nuclear translocation induced GAP-43 movement to the cytoplasm, where it is known to regulate growth cone dynamics and neuronal differentiation. Moreover, the degree of cytosolic Ca(2+) mobilization appeared to be more pronounced in differentiated hPDLSCs than in undifferentiated cells. This study provides evidences of a new PKCα/GAP-43 nuclear signalling pathway that controls neuronal differentiation in hPDLSCs, leading the way to a potential use of these cells in cell-based therapy in neurodegenerative diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Chemical and genetic blockade of HDACs enhances osteogenic differentiation of human adipose tissue-derived stem cells by oppositely affecting osteogenic and adipogenic transcription factors

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

Maroni, Paola; Brini, Anna Teresa; Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Universita degli Studi di Milano, Milano

2012-11-16

Highlights: Black-Right-Pointing-Pointer Acetylation affected hASCs osteodifferentiation through Runx2-PPAR{gamma}. Black-Right-Pointing-Pointer HDACs knocking-down favoured the commitment effect of osteogenic medium. Black-Right-Pointing-Pointer HDACs silencing early activated Runx2 and ALP. Black-Right-Pointing-Pointer PPAR{gamma} reduction and calcium/collagen deposition occurred later. Black-Right-Pointing-Pointer Runx2/PPAR{gamma} target genes were modulated in line with HDACs role in osteo-commitment. -- Abstract: The human adipose-tissue derived stem/stromal cells (hASCs) are an interesting source for bone-tissue engineering applications. Our aim was to clarify in hASCs the role of acetylation in the control of Runt-related transcription factor 2 (Runx2) and Peroxisome proliferator activated receptor (PPAR) {gamma}. These key osteogenic and adipogenic transcription factors are oppositelymore » involved in osteo-differentiation. The hASCs, committed or not towards bone lineage with osteoinductive medium, were exposed to HDACs chemical blockade with Trichostatin A (TSA) or were genetically silenced for HDACs. Alkaline phosphatase (ALP) and collagen/calcium deposition, considered as early and late osteogenic markers, were evaluated concomitantly as index of osteo-differentiation. TSA pretreatment, useful experimental protocol to analyse pan-HDAC-chemical inhibition, and switch to osteogenic medium induced early-osteoblast maturation gene Runx2, while transiently decreased PPAR{gamma} and scarcely affected late-differentiation markers. Time-dependent effects were observed after knocking-down of HDAC1 and 3: Runx2 and ALP underwent early activation, followed by late-osteogenic markers increase and by PPAR{gamma}/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPAR{gamma} target genes, respectively. Noteworthy, HDACs knocking-down favoured the commitment effect of osteogenic medium. Our results reveal a role for HDACs in orchestrating osteo-differentiation of hASCs at transcriptional level, and might provide new insights into the modulation of hASCs-based regenerative therapy.« less

GSEH: A Novel Approach to Select Prostate Cancer-Associated Genes Using Gene Expression Heterogeneity.

PubMed

Kim, Hyunjin; Choi, Sang-Min; Park, Sanghyun

2018-01-01

When a gene shows varying levels of expression among normal people but similar levels in disease patients or shows similar levels of expression among normal people but different levels in disease patients, we can assume that the gene is associated with the disease. By utilizing this gene expression heterogeneity, we can obtain additional information that abets discovery of disease-associated genes. In this study, we used collaborative filtering to calculate the degree of gene expression heterogeneity between classes and then scored the genes on the basis of the degree of gene expression heterogeneity to find "differentially predicted" genes. Through the proposed method, we discovered more prostate cancer-associated genes than 10 comparable methods. The genes prioritized by the proposed method are potentially significant to biological processes of a disease and can provide insight into them.

The Relationship between Religiosity and Attitudes of Nurses Aides toward Sexual Expression by Older Adults in Nursing Homes.

ERIC Educational Resources Information Center

Stevenson, Robert T.; Courtenay, Bradley C.

Systematic research on attitudes of nursing home staff toward the sexual expression of older residents is sparse and of recent origin. In order to determine the relationship between the degree of religiosity (religious commitment) of nursing home aides and their degree of tolerance concerning sexuality and aging, female nursing assistants (N=101)…

Circular RNA and gene expression profiles in gastric cancer based on microarray chip technology.

PubMed

Sui, Weiguo; Shi, Zhoufang; Xue, Wen; Ou, Minglin; Zhu, Ying; Chen, Jiejing; Lin, Hua; Liu, Fuhua; Dai, Yong

2017-03-01

The aim of the present study was to screen gastric cancer (GC) tissue and adjacent tissue for differences in mRNA and circular (circRNA) expression, to analyze the differences in circRNA and mRNA expression, and to investigate the circRNA expression in gastric carcinoma and its mechanism. circRNA and mRNA differential expression profiles generated using Agilent microarray technology were analyzed in the GC tissues and adjacent tissues. qRT-PCR was used to verify the differential expression of circRNAs and mRNAs according to the interactions between circRNAs and miRNAs as well as the possible existence of miRNA and mRNA interactions. We found that: i) the circRNA expression profile revealed 1,285 significant differences in circRNA expression, with circRNA expression downregulated in 594 samples and upregulated in 691 samples via interactions with miRNAs. The qRT-PCR validation experiments showed that hsa_circRNA_400071, hsa_circRNA_000543 and hsa_circRNA_001959 expression was consistent with the microarray analysis results. ii) 29,112 genes were found in the GC tissues and adjacent tissues, including 5,460 differentially expressed genes. Among them, 2,390 differentially expressed genes were upregulated and 3,070 genes were downregulated. Gene Ontology (GO) analysis of the differentially expressed genes revealed these genes involved in biological process classification, cellular component classification and molecular function classification. Pathway analysis of the differentially expressed genes identified 83 significantly enriched genes, including 28 upregulated genes and 55 downregulated genes. iii) 69 differentially expressed circRNAs were found that might adsorb specific miRNAs to regulate the expression of their target gene mRNAs. The conclusions are: i) differentially expressed circRNAs had corresponding miRNA binding sites. These circRNAs regulated the expression of target genes through interactions with miRNAs and might become new molecular biomarkers for GC in the future. ii) Differentially expressed genes may be involved in the occurrence of GC via a variety of mechanisms. iii) CD44, CXXC5, MYH9, MALAT1 and other genes may have important implications for the occurrence and development of GC through the regulation, interaction, and mutual influence of circRNA-miRNA-mRNA via different mechanisms.

A P-Norm Robust Feature Extraction Method for Identifying Differentially Expressed Genes

PubMed Central

Liu, Jian; Liu, Jin-Xing; Gao, Ying-Lian; Kong, Xiang-Zhen; Wang, Xue-Song; Wang, Dong

2015-01-01

In current molecular biology, it becomes more and more important to identify differentially expressed genes closely correlated with a key biological process from gene expression data. In this paper, based on the Schatten p-norm and Lp-norm, a novel p-norm robust feature extraction method is proposed to identify the differentially expressed genes. In our method, the Schatten p-norm is used as the regularization function to obtain a low-rank matrix and the Lp-norm is taken as the error function to improve the robustness to outliers in the gene expression data. The results on simulation data show that our method can obtain higher identification accuracies than the competitive methods. Numerous experiments on real gene expression data sets demonstrate that our method can identify more differentially expressed genes than the others. Moreover, we confirmed that the identified genes are closely correlated with the corresponding gene expression data. PMID:26201006

A P-Norm Robust Feature Extraction Method for Identifying Differentially Expressed Genes.

PubMed

Liu, Jian; Liu, Jin-Xing; Gao, Ying-Lian; Kong, Xiang-Zhen; Wang, Xue-Song; Wang, Dong

2015-01-01

In current molecular biology, it becomes more and more important to identify differentially expressed genes closely correlated with a key biological process from gene expression data. In this paper, based on the Schatten p-norm and Lp-norm, a novel p-norm robust feature extraction method is proposed to identify the differentially expressed genes. In our method, the Schatten p-norm is used as the regularization function to obtain a low-rank matrix and the Lp-norm is taken as the error function to improve the robustness to outliers in the gene expression data. The results on simulation data show that our method can obtain higher identification accuracies than the competitive methods. Numerous experiments on real gene expression data sets demonstrate that our method can identify more differentially expressed genes than the others. Moreover, we confirmed that the identified genes are closely correlated with the corresponding gene expression data.

Preferences, constraints or schemas of devotion? Exploring Norwegian mothers' withdrawals from high-commitment careers.

PubMed

Halrynjo, Sigtona; Lyng, Selma Therese

2009-06-01

Despite decades of focus on gender equality and work-family balance, parenthood still affects mothers' and fathers' careers differently. Drawing on in-depth interviews with Norwegian mothers who are relinquishing high-commitment careers of law and consultancy, this paper questions the adequacy of established explanations emphasizing constraints vs. individual preferences. Our sample of female professionals living in a well-developed welfare state is particularly apt to explore the processes and mechanisms upholding the statistically gendered pattern of women reducing their work commitment after childbirth. These doubly privileged mothers might be considered to have the best odds for combining career and work commitment with motherhood. Thus, we argue that the approach emphasizing practical constraints does not sufficiently account for the withdrawal from high-commitment careers among these female professionals. Nevertheless, we are not content with the claim of Preference Theory that this shift in commitment is merely a matter of 'not-so-dedicated' women discovering their 'genuine' preferences. Rather, in order to understand why and how this shift occurs, we explore the culturally constructed rationalities and schemas of both work and family devotions. We specifically examine the circumstances, mechanisms and steps in a seemingly individual process of making the shift in commitment from a promising career to a family-friendly job. Moreover, the analysis demonstrates how generous parental leave arrangements designed to enhance gender equality and work-family balance by simply reducing practical constraints may have limited--or even counterproductive--impact within high-commitment occupations where the 'irreplaceability' of workers is taken for granted. Our findings indicate that unless the culturally (re)produced discourses, demands and expectations of both work and family are exposed and challenged, even intentionally gender neutral work-family policies will continue to facilitate mothers' career withdrawals, expressed as modified individual preferences.

Genetic and epigenetic architecture of sex-biased expression in the jewel wasps Nasonia vitripennis and giraulti

PubMed Central

Wang, Xu; Werren, John H.; Clark, Andrew G.

2015-01-01

There is extraordinary diversity in sexual dimorphism (SD) among animals, but little is known about its epigenetic basis. To study the epigenetic architecture of SD in a haplodiploid system, we performed RNA-seq and whole-genome bisulfite sequencing of adult females and males from two closely related parasitoid wasps, Nasonia vitripennis and Nasonia giraulti. More than 75% of expressed genes displayed significantly sex-biased expression. As a consequence, expression profiles are more similar between species within each sex than between sexes within each species. Furthermore, extremely male- and female-biased genes are enriched for totally different functional categories: male-biased genes for key enzymes in sex-pheromone synthesis and female-biased genes for genes involved in epigenetic regulation of gene expression. Remarkably, just 70 highly expressed, extremely male-biased genes account for 10% of all transcripts in adult males. Unlike expression profiles, DNA methylomes are highly similar between sexes within species, with no consistent sex differences in methylation found. Therefore, methylation changes cannot explain the extensive level of sex-biased gene expression observed. Female-biased genes have smaller sequence divergence between species, higher conservation to other hymenopterans, and a broader expression range across development. Overall, female-biased genes have been recruited from genes with more conserved and broadly expressing “house-keeping” functions, whereas male-biased genes are more recently evolved and are predominately testis specific. In summary, Nasonia accomplish a striking degree of sex-biased expression without sex chromosomes or epigenetic differences in methylation. We propose that methylation provides a general signal for constitutive gene expression, whereas other sex-specific signals cause sex-biased gene expression. PMID:26100871

Extent and character of circadian gene expression in Drosophila melanogaster: identification of twenty oscillating mRNAs in the fly head.

PubMed

Van Gelder, R N; Bae, H; Palazzolo, M J; Krasnow, M A

1995-12-01

Although mRNAs expressed with a circadian rhythm have been isolated from many species, the extent and character of circadianly regulated gene expression is unknown for any animal. In Drosophila melanogaster, only the period (per) gene, an essential component of the circadian pacemaker, is known to show rhythmic mRNA expression. Recent work suggests that the encoded Per protein controls its own transcription by an autoregulatory feedback loop. Per might also control the rhythmic expression of other genes to generate circadian behavior and physiology. The goals of this work were to evaluate the extent and character of circadian control of gene expression in Drosophila, and to identify genes dependent on per for circadian expression. A large collection of anonymous, independent cDNA clones was used to screen for transcripts that are rhythmically expressed in the fly head. 20 of the 261 clones tested detected mRNAs with a greater than two-fold daily change in abundance. Three mRNAs were maximally expressed in the morning, whereas 17 mRNAs were most abundant in the evening--when per mRNA is also maximally expressed (but when the flies are inactive). Further analysis of the three 'morning' cDNAs showed that each has a unique dependence on the presence of a light-dark cycle, on timed feeding, and on the function of the per gene for its oscillation. These dependencies were different from those determined for per and for a novel 'evening' gene. Sequence analysis indicated that all but one of the 20 cDNAs identified previously uncloned genes. Diurnal control of gene expression is a significant but limited phenomenon in the fly head, which involves many uncharacterized genes. Diurnal control is mediated by multiple endogenous and exogenous mechanisms, even at the level of individual genes. A subset of circadianly expressed genes are predominantly or exclusively dependent on per for their rhythmic expression. The per gene can therefore influence the expression of genes other than itself, but for many rhythmically expressed genes, per functions in conjunction with external inputs to control their daily expression patterns.

Polycistronic gene expression in Aspergillus niger.

PubMed

Schuetze, Tabea; Meyer, Vera

2017-09-25

Genome mining approaches predict dozens of biosynthetic gene clusters in each of the filamentous fungal genomes sequenced so far. However, the majority of these gene clusters still remain cryptic because they are not expressed in their natural host. Simultaneous expression of all genes belonging to a biosynthetic pathway in a heterologous host is one approach to activate biosynthetic gene clusters and to screen the metabolites produced for bioactivities. Polycistronic expression of all pathway genes under control of a single and tunable promoter would be the method of choice, as this does not only simplify cloning procedures, but also offers control on timing and strength of expression. However, polycistronic gene expression is a feature not commonly found in eukaryotic host systems, such as Aspergillus niger. In this study, we tested the suitability of the viral P2A peptide for co-expression of three genes in A. niger. Two genes descend from Fusarium oxysporum and are essential to produce the secondary metabolite enniatin (esyn1, ekivR). The third gene (luc) encodes the reporter luciferase which was included to study position effects. Expression of the polycistronic gene cassette was put under control of the Tet-On system to ensure tunable gene expression in A. niger. In total, three polycistronic expression cassettes which differed in the position of luc were constructed and targeted to the pyrG locus in A. niger. This allowed direct comparison of the luciferase activity based on the position of the luciferase gene. Doxycycline-mediated induction of the Tet-On expression cassettes resulted in the production of one long polycistronic mRNA as proven by Northern analyses, and ensured comparable production of enniatin in all three strains. Notably, gene position within the polycistronic expression cassette matters, as, luciferase activity was lowest at position one and had a comparable activity at positions two and three. The P2A peptide can be used to express at least three genes polycistronically in A. niger. This approach can now be applied to heterologously express entire secondary metabolite gene clusters polycistronically or to co-express any genes of interest in equimolar amounts.

Intracellular high cholesterol content disorders the clock genes, apoptosis-related genes and fibrinolytic-related genes rhythmic expressions in human plaque-derived vascular smooth muscle cells.

PubMed

Lin, Changpo; Tang, Xiao; Xu, Lirong; Qian, Ruizhe; Shi, Zhenyu; Wang, Lixin; Cai, Tingting; Yan, Dong; Fu, Weiguo; Guo, Daqiao

2017-07-10

The clock genes are involved in regulating cardiovascular functions, and their expression disorders would lead to circadian rhythm disruptions of clock-controlled genes (CCGs), resulting in atherosclerotic plaque formation and rupture. Our previous study revealed the rhythmic expression of clock genes were attenuated in human plaque-derived vascular smooth muscle cells (PVSMCs), but failed to detect the downstream CCGs expressions and the underlying molecular mechanism. In this study, we examined the difference of CCGs rhythmic expression between human normal carotid VSMCs (NVSMCs) and PVSMCs. Furthermore, we compared the cholesterol and triglycerides levels between two groups and the link to clock genes and CCGs expressions. Seven health donors' normal carotids and 19 carotid plaques yielded viable cultured NVSMCs and PVSMCs. The expression levels of target genes were measured by quantitative real-time PCR and Western-blot. The intracellular cholesterol and triglycerides levels were measured by kits. The circadian expressions of apoptosis-related genes and fibrinolytic-related genes were disordered. Besides, the cholesterol levels were significant higher in PVSMCs. After treated with cholesterol or oxidized low density lipoprotein (ox-LDL), the expressions of clock genes were inhibited; and the rhythmic expressions of clock genes, apoptosis-related genes and fibrinolytic-related genes were disturbed in NVSMCs, which were similar to PVSMCs. The results suggested that intracellular high cholesterol content of PVSMCs would lead to the disorders of clock genes and CCGs rhythmic expressions. And further studies should be conducted to demonstrate the specific molecular mechanisms involved.

Double-filter identification of vascular-expressed genes using Arabidopsis plants with vascular hypertrophy and hypotrophy.

PubMed

Ckurshumova, Wenzislava; Scarpella, Enrico; Goldstein, Rochelle S; Berleth, Thomas

2011-08-01

Genes expressed in vascular tissues have been identified by several strategies, usually with a focus on mature vascular cells. In this study, we explored the possibility of using two opposite types of altered tissue compositions in combination with a double-filter selection to identify genes with a high probability of vascular expression in early organ primordia. Specifically, we generated full-transcriptome microarray profiles of plants with (a) genetically strongly reduced and (b) pharmacologically vastly increased vascular tissues and identified a reproducible cohort of 158 transcripts that fulfilled the dual requirement of being underrepresented in (a) and overrepresented in (b). In order to assess the predictive value of our identification scheme for vascular gene expression, we determined the expression patterns of genes in two unbiased subsamples. First, we assessed the expression patterns of all twenty annotated transcription factor genes from the cohort of 158 genes and found that seventeen of the twenty genes were preferentially expressed in leaf vascular cells. Remarkably, fifteen of these seventeen vascular genes were clearly expressed already very early in leaf vein development. Twelve genes with published leaf expression patterns served as a second subsample to monitor the representation of vascular genes in our cohort. Of those twelve genes, eleven were preferentially expressed in leaf vascular tissues. Based on these results we propose that our compendium of 158 genes represents a sample that is highly enriched for genes expressed in vascular tissues and that our approach is particularly suited to detect genes expressed in vascular cell lineages at early stages of their inception. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Increased gene expression noise in human cancers is correlated with low p53 and immune activities as well as late stage cancer.

PubMed

Han, Rongfei; Huang, Guanqun; Wang, Yejun; Xu, Yafei; Hu, Yueming; Jiang, Wenqi; Wang, Tianfu; Xiao, Tian; Zheng, Duo

2016-11-01

Gene expression in metazoans is delicately organized. As genetic information transmits from DNA to RNA and protein, expression noise is inevitably generated. Recent studies begin to unveil the mechanisms of gene expression noise control, but the changes of gene expression precision in pathologic conditions like cancers are unknown. Here we analyzed the transcriptomic data of human breast, liver, lung and colon cancers, and found that the expression noise of more than 74.9% genes was increased in cancer tissues as compared to adjacent normal tissues. This suggested that gene expression precision controlling collapsed during cancer development. A set of 269 genes with noise increased more than 2-fold were identified across different cancer types. These genes were involved in cell adhesion, catalytic and metabolic functions, implying the vulnerability of deregulation of these processes in cancers. We also observed a tendency of increased expression noise in patients with low p53 and immune activity in breast, liver and lung caners but not in colon cancers, which indicated the contributions of p53 signaling and host immune surveillance to gene expression noise in cancers. Moreover, more than 53.7% genes had increased noise in patients with late stage than early stage cancers, suggesting that gene expression precision was associated with cancer outcome. Together, these results provided genomic scale explorations of gene expression noise control in human cancers.

[Differential expression genes of bone tissues surrounding implants in diabetic rats by gene chip].

PubMed

Wang, Xin-xin; Ma, Yue; Li, Qing; Jiang, Bao-qi; Lan, Jing

2012-10-01

To compare mRNA expression profiles of bone tissues surrounding implants between normal rats and rats with diabetes using microarray technology. Six Wistar rats were randomly selected and divided into normal model group and diabetic group. Diabetic model condition was established by injecting Streptozotocin into peritoneal space. Titanium implants were implanted into the epiphyseal end of the rats' tibia. Bone tissues surrounding implant were harvested and sampled after 3 months to perform comprehensive RNA gene expression profiling, including 17983 for genome-wide association study.GO analysis was used to compare different gene expression and real-time PCR was used to confirm the results on core samples. The results indicated that there were 1084 differential gene expression. In the diabetic model, there were 352 enhanced expression genes, 732 suppressed expression genes. GO analysis involved 1154 different functional type. Osteoblast related gene expressions in bone tissue samples of diabetic rats were decreased, and lipid metabolism pathway related gene expression was increased.

Gene Architectures that Minimize Cost of Gene Expression.

PubMed

Frumkin, Idan; Schirman, Dvir; Rotman, Aviv; Li, Fangfei; Zahavi, Liron; Mordret, Ernest; Asraf, Omer; Wu, Song; Levy, Sasha F; Pilpel, Yitzhak

2017-01-05

Gene expression burdens cells by consuming resources and energy. While numerous studies have investigated regulation of expression level, little is known about gene design elements that govern expression costs. Here, we ask how cells minimize production costs while maintaining a given protein expression level and whether there are gene architectures that optimize this process. We measured fitness of ∼14,000 E. coli strains, each expressing a reporter gene with a unique 5' architecture. By comparing cost-effective and ineffective architectures, we found that cost per protein molecule could be minimized by lowering transcription levels, regulating translation speeds, and utilizing amino acids that are cheap to synthesize and that are less hydrophobic. We then examined natural E. coli genes and found that highly expressed genes have evolved more forcefully to minimize costs associated with their expression. Our study thus elucidates gene design elements that improve the economy of protein expression in natural and heterologous systems. Copyright © 2017 Elsevier Inc. All rights reserved.

Transcriptome sequencing of Eucalyptus camaldulensis seedlings subjected to water stress reveals functional single nucleotide polymorphisms and genes under selection

PubMed Central

2012-01-01

Background Water stress limits plant survival and production in many parts of the world. Identification of genes and alleles responding to water stress conditions is important in breeding plants better adapted to drought. Currently there are no studies examining the transcriptome wide gene and allelic expression patterns under water stress conditions. We used RNA sequencing (RNA-seq) to identify the candidate genes and alleles and to explore the evolutionary signatures of selection. Results We studied the effect of water stress on gene expression in Eucalyptus camaldulensis seedlings derived from three natural populations. We used reference-guided transcriptome mapping to study gene expression. Several genes showed differential expression between control and stress conditions. Gene ontology (GO) enrichment tests revealed up-regulation of 140 stress-related gene categories and down-regulation of 35 metabolic and cell wall organisation gene categories. More than 190,000 single nucleotide polymorphisms (SNPs) were detected and 2737 of these showed differential allelic expression. Allelic expression of 52% of these variants was correlated with differential gene expression. Signatures of selection patterns were studied by estimating the proportion of nonsynonymous to synonymous substitution rates (Ka/Ks). The average Ka/Ks ratio among the 13,719 genes was 0.39 indicating that most of the genes are under purifying selection. Among the positively selected genes (Ka/Ks > 1.5) apoptosis and cell death categories were enriched. Of the 287 positively selected genes, ninety genes showed differential expression and 27 SNPs from 17 positively selected genes showed differential allelic expression between treatments. Conclusions Correlation of allelic expression of several SNPs with total gene expression indicates that these variants may be the cis-acting variants or in linkage disequilibrium with such variants. Enrichment of apoptosis and cell death gene categories among the positively selected genes reveals the past selection pressures experienced by the populations used in this study. PMID:22853646

Functional clustering of time series gene expression data by Granger causality

PubMed Central

2012-01-01

Background A common approach for time series gene expression data analysis includes the clustering of genes with similar expression patterns throughout time. Clustered gene expression profiles point to the joint contribution of groups of genes to a particular cellular process. However, since genes belong to intricate networks, other features, besides comparable expression patterns, should provide additional information for the identification of functionally similar genes. Results In this study we perform gene clustering through the identification of Granger causality between and within sets of time series gene expression data. Granger causality is based on the idea that the cause of an event cannot come after its consequence. Conclusions This kind of analysis can be used as a complementary approach for functional clustering, wherein genes would be clustered not solely based on their expression similarity but on their topological proximity built according to the intensity of Granger causality among them. PMID:23107425

Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

PubMed Central

Richter, Karin; Wirta, Valtteri; Dahl, Lina; Bruce, Sara; Lundeberg, Joakim; Carlsson, Leif; Williams, Cecilia

2006-01-01

Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin. PMID:16600034

Integrated analysis of gene expression and methylation profiles of 48 candidate genes in breast cancer patients.

PubMed

Li, Zibo; Heng, Jianfu; Yan, Jinhua; Guo, Xinwu; Tang, Lili; Chen, Ming; Peng, Limin; Wu, Yepeng; Wang, Shouman; Xiao, Zhi; Deng, Zhongping; Dai, Lizhong; Wang, Jun

2016-11-01

Gene-specific methylation and expression have shown biological and clinical importance for breast cancer diagnosis and prognosis. Integrated analysis of gene methylation and gene expression may identify genes associated with biology mechanism and clinical outcome of breast cancer and aid in clinical management. Using high-throughput microfluidic quantitative PCR, we analyzed the expression profiles of 48 candidate genes in 96 Chinese breast cancer patients and investigated their correlation with gene methylation and associations with breast cancer clinical parameters. Breast cancer-specific gene expression alternation was found in 25 genes with significant expression difference between paired tumor and normal tissues. A total of 9 genes (CCND2, EGFR, GSTP1, PGR, PTGS2, RECK, SOX17, TNFRSF10D, and WIF1) showed significant negative correlation between methylation and gene expression, which were validated in the TCGA database. Total 23 genes (ACADL, APC, BRCA2, CADM1, CAV1, CCND2, CST6, EGFR, ESR2, GSTP1, ICAM5, NPY, PGR, PTGS2, RECK, RUNX3, SFRP1, SOX17, SYK, TGFBR2, TNFRSF10D, WIF1, and WRN) annotated with potential TFBSs in the promoter regions showed negative correlation between methylation and expression. In logistics regression analysis, 31 of the 48 genes showed improved performance in disease prediction with combination of methylation and expression coefficient. Our results demonstrated the complex correlation and the possible regulatory mechanisms between DNA methylation and gene expression. Integration analysis of methylation and expression of candidate genes could improve performance in breast cancer prediction. These findings would contribute to molecular characterization and identification of biomarkers for potential clinical applications.

Genome-wide profiling identifies a subset of methamphetamine (METH)-induced genes associated with METH-induced increased H4K5Ac binding in the rat striatum

PubMed Central

2013-01-01

Background METH is an illicit drug of abuse that influences gene expression in the rat striatum. Histone modifications regulate gene transcription. Methods We therefore used microarray analysis and genome-scale approaches to examine potential relationships between the effects of METH on gene expression and on DNA binding of histone H4 acetylated at lysine 4 (H4K5Ac) in the rat dorsal striatum of METH-naïve and METH-pretreated rats. Results Acute and chronic METH administration caused differential changes in striatal gene expression. METH also increased H4K5Ac binding around the transcriptional start sites (TSSs) of genes in the rat striatum. In order to relate gene expression to histone acetylation, we binned genes of similar expression into groups of 100 genes and proceeded to relate gene expression to H4K5Ac binding. We found a positive correlation between gene expression and H4K5Ac binding in the striatum of control rats. Similar correlations were observed in METH-treated rats. Genes that showed acute METH-induced increased expression in saline-pretreated rats also showed METH-induced increased H4K5Ac binding. The acute METH injection caused similar increases in H4K5Ac binding in METH-pretreated rats, without affecting gene expression to the same degree. Finally, genes that showed METH-induced decreased expression exhibited either decreases or no changes in H4K5Ac binding. Conclusion Acute METH injections caused increased gene expression of genes that showed increased H4K5Ac binding near their transcription start sites. PMID:23937714

Genome-wide profiling identifies a subset of methamphetamine (METH)-induced genes associated with METH-induced increased H4K5Ac binding in the rat striatum.

PubMed

Cadet, Jean Lud; Jayanthi, Subramaniam; McCoy, Michael T; Ladenheim, Bruce; Saint-Preux, Fabienne; Lehrmann, Elin; De, Supriyo; Becker, Kevin G; Brannock, Christie

2013-08-12

METH is an illicit drug of abuse that influences gene expression in the rat striatum. Histone modifications regulate gene transcription. We therefore used microarray analysis and genome-scale approaches to examine potential relationships between the effects of METH on gene expression and on DNA binding of histone H4 acetylated at lysine 4 (H4K5Ac) in the rat dorsal striatum of METH-naïve and METH-pretreated rats. Acute and chronic METH administration caused differential changes in striatal gene expression. METH also increased H4K5Ac binding around the transcriptional start sites (TSSs) of genes in the rat striatum. In order to relate gene expression to histone acetylation, we binned genes of similar expression into groups of 100 genes and proceeded to relate gene expression to H4K5Ac binding. We found a positive correlation between gene expression and H4K5Ac binding in the striatum of control rats. Similar correlations were observed in METH-treated rats. Genes that showed acute METH-induced increased expression in saline-pretreated rats also showed METH-induced increased H4K5Ac binding. The acute METH injection caused similar increases in H4K5Ac binding in METH-pretreated rats, without affecting gene expression to the same degree. Finally, genes that showed METH-induced decreased expression exhibited either decreases or no changes in H4K5Ac binding. Acute METH injections caused increased gene expression of genes that showed increased H4K5Ac binding near their transcription start sites.

President Kennedy's Speech at Rice University

NASA Technical Reports Server (NTRS)

1988-01-01

This video tape presents unedited film footage of President John F. Kennedy's speech at Rice University, Houston, Texas, September 12, 1962. The speech expresses the commitment of the United States to landing an astronaut on the Moon.

Evaluating the consistency of gene sets used in the analysis of bacterial gene expression data.

PubMed

Tintle, Nathan L; Sitarik, Alexandra; Boerema, Benjamin; Young, Kylie; Best, Aaron A; Dejongh, Matthew

2012-08-08

Statistical analyses of whole genome expression data require functional information about genes in order to yield meaningful biological conclusions. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) are common sources of functionally grouped gene sets. For bacteria, the SEED and MicrobesOnline provide alternative, complementary sources of gene sets. To date, no comprehensive evaluation of the data obtained from these resources has been performed. We define a series of gene set consistency metrics directly related to the most common classes of statistical analyses for gene expression data, and then perform a comprehensive analysis of 3581 Affymetrix® gene expression arrays across 17 diverse bacteria. We find that gene sets obtained from GO and KEGG demonstrate lower consistency than those obtained from the SEED and MicrobesOnline, regardless of gene set size. Despite the widespread use of GO and KEGG gene sets in bacterial gene expression data analysis, the SEED and MicrobesOnline provide more consistent sets for a wide variety of statistical analyses. Increased use of the SEED and MicrobesOnline gene sets in the analysis of bacterial gene expression data may improve statistical power and utility of expression data.

Validation of housekeeping genes as an internal control for gene expression studies in Giardia lamblia using quantitative real-time PCR.

PubMed

Marcial-Quino, Jaime; Fierro, Francisco; De la Mora-De la Mora, Ignacio; Enríquez-Flores, Sergio; Gómez-Manzo, Saúl; Vanoye-Carlo, America; Garcia-Torres, Itzhel; Sierra-Palacios, Edgar; Reyes-Vivas, Horacio

2016-04-25

The analysis of transcript levels of specific genes is important for understanding transcriptional regulation and for the characterization of gene function. Real-time quantitative reverse transcriptase PCR (RT-qPCR) has become a powerful tool to quantify gene expression. The objective of this study was to identify reliable housekeeping genes in Giardia lamblia. Twelve genes were selected for this purpose, and their expression was analyzed in the wild type WB strain and in two strains with resistance to nitazoxanide (NTZ) and metronidazole (MTZ), respectively. RefFinder software analysis showed that the expression of the genes is different in the three strains. The integrated data from the four analyses showed that the NADH oxidase (NADH) and aldolase (ALD) genes were the most steadily expressed genes, whereas the glyceraldehyde-3-phosphate dehydrogenase gene was the most unstable. Additionally, the relative expression of seven genes were quantified in the NTZ- and MTZ-resistant strains by RT-qPCR, using the aldolase gene as the internal control, and the results showed a consistent differential pattern of expression in both strains. The housekeeping genes found in this work will facilitate the analysis of mRNA expression levels of other genes of interest in G. lamblia. Copyright © 2016 Elsevier B.V. All rights reserved.

Targeting gene expression selectively in cancer cells by using the progression-elevated gene-3 promoter.

PubMed

Su, Zhao-Zhong; Sarkar, Devanand; Emdad, Luni; Duigou, Gregory J; Young, Charles S H; Ware, Joy; Randolph, Aaron; Valerie, Kristoffer; Fisher, Paul B

2005-01-25

One impediment to effective cancer-specific gene therapy is the rarity of regulatory sequences targeting gene expression selectively in tumor cells. Although many tissue-specific promoters are recognized, few cancer-selective gene promoters are available. Progression-elevated gene-3 (PEG-3) is a rodent gene identified by subtraction hybridization that displays elevated expression as a function of transformation by diversely acting oncogenes, DNA damage, and cancer cell progression. The promoter of PEG-3, PEG-Prom, displays robust expression in a broad spectrum of human cancer cell lines with marginal expression in normal cellular counterparts. Whereas GFP expression, when under the control of a CMV promoter, is detected in both normal and cancer cells, when GFP is expressed under the control of the PEG-Prom, cancer-selective expression is evident. Mutational analysis identifies the AP-1 and PEA-3 transcription factors as primary mediators of selective, cancer-specific expression of the PEG-Prom. Synthesis of apoptosis-inducing genes, under the control of the CMV promoter, inhibits the growth of both normal and cancer cells, whereas PEG-Prom-mediated expression of these genes kills only cancer cells and spares normal cells. The efficacy of the PEG-Prom as part of a cancer gene therapeutic regimen is further documented by in vivo experiments in which PEG-Prom-controlled expression of an apoptosis-inducing gene completely inhibited prostate cancer xenograft growth in nude mice. These compelling observations indicate that the PEG-Prom, with its cancer-specific expression, provides a means of selectively delivering genes to cancer cells, thereby providing a crucial component in developing effective cancer gene therapies.

Evaluation of Allelic Expression of Imprinted Genes in Adult Human Blood

PubMed Central

Frost, Jennifer M.; Monk, Dave; Stojilkovic-Mikic, Taita; Woodfine, Kathryn; Chitty, Lyn S.; Murrell, Adele; Stanier, Philip; Moore, Gudrun E.

2010-01-01

Background Imprinted genes are expressed from only one allele in a parent-of-origin dependent manner. Loss of imprinted (LOI) expression can result in a variety of human disorders and is frequently reported in cancer. Biallelic expression of imprinted genes in adult blood has been suggested as a useful biomarker and is currently being investigated in colorectal cancer. In general, the expression profiles of imprinted genes are well characterised during human and mouse fetal development, but not in human adults. Methodology/Principal Findings We investigated quantitative expression of 36 imprinted genes in adult human peripheral blood leukocytes obtained from healthy individuals. Allelic expression was also investigated in B and T lymphocytes and myeloid cells. We found that 21 genes were essentially undetectable in adult blood. Only six genes were demonstrably monoallelic, and most importantly, we found that nine genes were either biallelic or showed variable expression in different individuals. Separated leukocyte populations showed the same expression patterns as whole blood. Differential methylation at each of the imprinting control loci analysed was maintained, including regions that contained biallelically expressed genes. This suggests in some cases methylation has become uncoupled from its role in regulating gene expression. Conclusions/Significance We conclude that only a limited set of imprinted genes, including IGF2 and SNRPN, may be useful for LOI cancer biomarker studies. In addition, blood is not a good tissue to use for the discovery of new imprinted genes. Finally, lymphocyte DNA methylation status in the adult may not always be a reliable indicator of monoallelic gene expression. PMID:21042416

Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.).

PubMed

Wang, Min; Wang, Qinglian; Zhang, Baohong

2013-11-01

Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions. © 2013.

ΔNp63 is an ectodermal gatekeeper of epidermal morphogenesis

PubMed Central

Shalom-Feuerstein, R; Lena, A M; Zhou, H; De La Forest Divonne, S; Van Bokhoven, H; Candi, E; Melino, G; Aberdam, D

2011-01-01

p63, a member of p53 family, has a significant role in the development and maintenance of stratified epithelia. However, a persistent dispute remained over the last decade concerning the interpretation of the severe failure of p63-null embryos to develop stratified epithelia. In this study, by investigating both p63-deficient strains, we demonstrated that p63-deficient epithelia failed to develop beyond ectodermal stage as they remained a monolayer of non-proliferating cells expressing K8/K18. Importantly, in the absence of p63, corneal-epithelial commitment (which occurs at embryonic day 12.5 of mouse embryogenesis) was hampered 3 weeks before corneal stem cell renewal (that begins at P14). Taken together, these data illustrate the significant role of p63 in epithelial embryogenesis, before and independently of other functions of p63 in adult stem cells regulation. Transcriptome analysis of laser captured-embryonic tissues confirmed the latter hypothesis, demonstrating that a battery of epidermal genes that were activated in wild-type epidermis remained silent in p63-null tissues. Furthermore, we defined a subset of novel bona fide p63-induced genes orchestrating first epidermal stratification and a subset of p63-repressed mesodermal-specific genes. These data highlight the earliest recognized action of ΔNp63 in the induction epidermal morphogenesis at E11.5. In the absence of p63, a mesodermal program is activated while epidermal morphogenesis does not initiate. PMID:21127502

Efficient Generation of Functional Hepatocytes From Human Embryonic Stem Cells and Induced Pluripotent Stem Cells by HNF4α Transduction

PubMed Central

Takayama, Kazuo; Inamura, Mitsuru; Kawabata, Kenji; Katayama, Kazufumi; Higuchi, Maiko; Tashiro, Katsuhisa; Nonaka, Aki; Sakurai, Fuminori; Hayakawa, Takao; Kusuda Furue, Miho; Mizuguchi, Hiroyuki

2012-01-01

Hepatocyte-like cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are expected to be a useful source of cells drug discovery. Although we recently reported that hepatic commitment is promoted by transduction of SOX17 and HEX into human ESC- and iPSC-derived cells, these hepatocyte-like cells were not sufficiently mature for drug screening. To promote hepatic maturation, we utilized transduction of the hepatocyte nuclear factor 4α (HNF4α) gene, which is known as a master regulator of liver-specific gene expression. Adenovirus vector-mediated overexpression of HNF4α in hepatoblasts induced by SOX17 and HEX transduction led to upregulation of epithelial and mature hepatic markers such as cytochrome P450 (CYP) enzymes, and promoted hepatic maturation by activating the mesenchymal-to-epithelial transition (MET). Thus HNF4α might play an important role in the hepatic differentiation from human ESC-derived hepatoblasts by activating the MET. Furthermore, the hepatocyte like-cells could catalyze the toxication of several compounds. Our method would be a valuable tool for the efficient generation of functional hepatocytes derived from human ESCs and iPSCs, and the hepatocyte-like cells could be used for predicting drug toxicity. PMID:22068426

Early induction of a prechondrogenic population allows efficient generation of stable chondrocytes from human induced pluripotent stem cells.

PubMed

Lee, Jieun; Taylor, Sarah E B; Smeriglio, Piera; Lai, Janice; Maloney, William J; Yang, Fan; Bhutani, Nidhi

2015-08-01

Regeneration of human cartilage is inherently inefficient; an abundant autologous source, such as human induced pluripotent stem cells (hiPSCs), is therefore attractive for engineering cartilage. We report a growth factor-based protocol for differentiating hiPSCs into articular-like chondrocytes (hiChondrocytes) within 2 weeks, with an overall efficiency >90%. The hiChondrocytes are stable and comparable to adult articular chondrocytes in global gene expression, extracellular matrix production, and ability to generate cartilage tissue in vitro and in immune-deficient mice. Molecular characterization identified an early SRY (sex-determining region Y) box (Sox)9(low) cluster of differentiation (CD)44(low)CD140(low) prechondrogenic population during hiPSC differentiation. In addition, 2 distinct Sox9-regulated gene networks were identified in the Sox9(low) and Sox9(high) populations providing novel molecular insights into chondrogenic fate commitment and differentiation. Our findings present a favorable method for generating hiPSC-derived articular-like chondrocytes. The hiChondrocytes are an attractive cell source for cartilage engineering because of their abundance, autologous nature, and potential to generate articular-like cartilage rather than fibrocartilage. In addition, hiChondrocytes can be excellent tools for modeling human musculoskeletal diseases in a dish and for rapid drug screening. © FASEB.

The paradox of Foxd3: how does it function in pluripotency and differentiation of embryonic stem cells?

PubMed Central

Plank-Bazinet, Jennifer L.

2016-01-01

Uncommitted cells of the early mammalian embryo transition through distinct stages of pluripotency, including establishment of ground state “naïve” pluripotency in the early epiblast, transition to a post-implantation “primed” state, and subsequent lineage commitment of the gastrulating epiblast. Previous transcriptional profiling of in vitro models to recapitulate early to late epiblast transition and differentiation suggest that distinct gene regulatory networks are likely to function in each of these states. While the mechanisms underlying transition between pluripotent states are poorly understood, the forkhead family transcription factor Foxd3 has emerged as a key regulatory factor. Foxd3 is required to maintain pluripotent cells of the murine epiblast and for survival, self-renewal and pluripotency of embryonic stem cells (ESCs). Two recent, simultaneous studies have shed light on how Foxd3 regulates gene expression in early cell fate transitions of progenitor cells. While the two publications shared some common findings, they also presented some conflicting results and suggest different models for the mechanisms underlying Foxd3 function. Here, we discuss the key similarities and differences between the publications, highlight data from the literature relevant to their findings, and hypothesize a potential mechanism of Foxd3 action. PMID:27868055

Central adiponectin administration reveals new regulatory mechanisms of bone metabolism in mice

PubMed Central

Wu, Yuwei; Tu, Qisheng; Valverde, Paloma; Zhang, Jin; Murray, Dana; Dong, Lily Q.; Cheng, Jessica; Jiang, Hua; Rios, Maribel; Morgan, Elise; Tang, Zhihui

2014-01-01

Adiponectin (APN), the most abundant adipocyte-secreted adipokine, regulates energy homeostasis and exerts well-characterized insulin-sensitizing properties. The peripheral or central effects of APN regulating bone metabolism are beginning to be explored but are still not clearly understood. In the present study, we found that APN-knockout (APN-KO) mice fed a normal diet exhibited decreased trabecular structure and mineralization and increased bone marrow adiposity compared with wild-type (WT) mice. APN intracerebroventricular infusions decreased uncoupling protein 1 (UCP1) expression in brown adipose tissue, epinephrine and norepinephrine serum levels, and osteoclast numbers, whereas osteoblast osteogenic marker expression and trabecular bone mass increased in APN-KO and WT mice. In addition, centrally administered APN increased hypothalamic tryptophan hydroxylase 2 (TPH2), cocaine- and amphetamine-regulated transcript (CART), and 5-hydroxytryptamine (serotonin) receptor 2C (Htr2C) expressions but decreased hypothalamic cannabinoid receptor-1 expression. Treatment of immortalized mouse neurons with APN demonstrated that APN-mediated effects on TPH2, CART, and Htr2C expression levels were abolished by downregulating adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif (APPL)-1 expression. Pharmacological increase in sympathetic activity stimulated adipogenic differentiation of bone marrow stromal cells (BMSC) and reversed APN-induced expression of the lysine-specific demethylases involved in regulating their commitment to the osteoblastic lineage. In conclusion, we found that APN regulates bone metabolism via central and peripheral mechanisms to decrease sympathetic tone, inhibit osteoclastic differentiation, and promote osteoblastic commitment of BMSC. PMID:24780611

The gsdf gene locus harbors evolutionary conserved and clustered genes preferentially expressed in fish previtellogenic oocytes.

PubMed

Gautier, Aude; Le Gac, Florence; Lareyre, Jean-Jacques

2011-02-01

The gonadal soma-derived factor (GSDF) belongs to the transforming growth factor-β superfamily and is conserved in teleostean fish species. Gsdf is specifically expressed in the gonads, and gene expression is restricted to the granulosa and Sertoli cells in trout and medaka. The gsdf gene expression is correlated to early testis differentiation in medaka and was shown to stimulate primordial germ cell and spermatogonia proliferation in trout. In the present study, we show that the gsdf gene localizes to a syntenic chromosomal fragment conserved among vertebrates although no gsdf-related gene is detected on the corresponding genomic region in tetrapods. We demonstrate using quantitative RT-PCR that most of the genes localized in the synteny are specifically expressed in medaka gonads. Gsdf is the only gene of the synteny with a much higher expression in the testis compared to the ovary. In contrast, gene expression pattern analysis of the gsdf surrounding genes (nup54, aff1, klhl8, sdad1, and ptpn13) indicates that these genes are preferentially expressed in the female gonads. The tissue distribution of these genes is highly similar in medaka and zebrafish, two teleostean species that have diverged more than 110 million years ago. The cellular localization of these genes was determined in medaka gonads using the whole-mount in situ hybridization technique. We confirm that gsdf gene expression is restricted to Sertoli and granulosa cells in contact with the premeiotic and meiotic cells. The nup54 gene is expressed in spermatocytes and previtellogenic oocytes. Transcripts corresponding to the ovary-specific genes (aff1, klhl8, and sdad1) are detected only in previtellogenic oocytes. No expression was detected in the gonocytes in 10 dpf embryos. In conclusion, we show that the gsdf gene localizes to a syntenic chromosomal fragment harboring evolutionary conserved genes in vertebrates. These genes are preferentially expressed in previtelloogenic oocytes, and thus, they display a different cellular localization compared to that of the gsdf gene indicating that the later gene is not co-regulated. Interestingly, our study identifies new clustered genes that are specifically expressed in previtellogenic oocytes (nup54, aff1, klhl8, sdad1). Copyright © 2010 Elsevier B.V. All rights reserved.

Single cell transcriptome analysis of MCF-7 reveals consistently and inconsistently expressed gene groups each associated with distinct cellular localization and functions

PubMed Central

Chen, Tzu-Han; Shiau, Hsin-Chieh

2018-01-01

Single cell transcriptome (SCT) analysis provides superior resolution to illustrate tumor cell heterogeneity for clinical implications. We characterized four SCTs of MCF-7 using 143 housekeeping genes (HKGs) as control, of which lactate dehydrogenase B (LDHB) expression is silenced. These SCT libraries mapped to 11,423, 11,486, 10,380, and 11,306 RefSeq genes (UCSC), respectively. High consistency in HKG expression levels across all four SCTs, along with transcriptional silencing of LDHB, was observed, suggesting a high sensitivity and reproducibility of the SCT analysis. Cross-library comparison on expression levels by scatter plotting revealed a linear correlation and an 83–94% overlap in transcript isoforms and expressed genes were also observed. To gain insight of transcriptional diversity among the SCTs, expressed genes were split into consistently expressed (CE) (expressed in all SCTs) and inconsistently expressed (IE) (expressed in some but not all SCTs) genes for further characterization, along with the 142 expressed HKGs as a reference. Distinct transcriptional strengths were found among these groups, with averages of 1,612.0, 88.0 and 1.2 FPKM for HKGs, CE and IE, respectively. Comparison between CE and IE groups further indicated that expressions of CE genes vary more significantly than that of IE genes. Gene Ontology analysis indicated that proteins encoded by CE genes are mainly involved in fundamental intracellular activities, while proteins encoded by IE genes are mainly for extracellular activities, especially acting as receptors or ion channels. The diversified gene expressions, especially for those encoded by IE genes, may contribute to cancer drug resistance. PMID:29920548

Identification of reference genes for quantitative expression analysis using large-scale RNA-seq data of Arabidopsis thaliana and model crop plants.

PubMed

Kudo, Toru; Sasaki, Yohei; Terashima, Shin; Matsuda-Imai, Noriko; Takano, Tomoyuki; Saito, Misa; Kanno, Maasa; Ozaki, Soichi; Suwabe, Keita; Suzuki, Go; Watanabe, Masao; Matsuoka, Makoto; Takayama, Seiji; Yano, Kentaro

2016-10-13

In quantitative gene expression analysis, normalization using a reference gene as an internal control is frequently performed for appropriate interpretation of the results. Efforts have been devoted to exploring superior novel reference genes using microarray transcriptomic data and to evaluating commonly used reference genes by targeting analysis. However, because the number of specifically detectable genes is totally dependent on probe design in the microarray analysis, exploration using microarray data may miss some of the best choices for the reference genes. Recently emerging RNA sequencing (RNA-seq) provides an ideal resource for comprehensive exploration of reference genes since this method is capable of detecting all expressed genes, in principle including even unknown genes. We report the results of a comprehensive exploration of reference genes using public RNA-seq data from plants such as Arabidopsis thaliana (Arabidopsis), Glycine max (soybean), Solanum lycopersicum (tomato) and Oryza sativa (rice). To select reference genes suitable for the broadest experimental conditions possible, candidates were surveyed by the following four steps: (1) evaluation of the basal expression level of each gene in each experiment; (2) evaluation of the expression stability of each gene in each experiment; (3) evaluation of the expression stability of each gene across the experiments; and (4) selection of top-ranked genes, after ranking according to the number of experiments in which the gene was expressed stably. Employing this procedure, 13, 10, 12 and 21 top candidates for reference genes were proposed in Arabidopsis, soybean, tomato and rice, respectively. Microarray expression data confirmed that the expression of the proposed reference genes under broad experimental conditions was more stable than that of commonly used reference genes. These novel reference genes will be useful for analyzing gene expression profiles across experiments carried out under various experimental conditions.

Near-isogenic cotton germplasm lines that differ in fiber-bundle strength have temporal differences in fiber gene expression patterns as revealed by comparative high-throughput profiling.

PubMed

Hinchliffe, Doug J; Meredith, William R; Yeater, Kathleen M; Kim, Hee Jin; Woodward, Andrew W; Chen, Z Jeffrey; Triplett, Barbara A

2010-05-01

Gene expression profiles of developing cotton (Gossypium hirsutum L.) fibers from two near-isogenic lines (NILs) that differ in fiber-bundle strength, short-fiber content, and in fewer than two genetic loci were compared using an oligonucleotide microarray. Fiber gene expression was compared at five time points spanning fiber elongation and secondary cell wall (SCW) biosynthesis. Fiber samples were collected from field plots in a randomized, complete block design, with three spatially distinct biological replications for each NIL at each time point. Microarray hybridizations were performed in a loop experimental design that allowed comparisons of fiber gene expression profiles as a function of time between the two NILs. Overall, developmental expression patterns revealed by the microarray experiment agreed with previously reported cotton fiber gene expression patterns for specific genes. Additionally, genes expressed coordinately with the onset of SCW biosynthesis in cotton fiber correlated with gene expression patterns of other SCW-producing plant tissues. Functional classification and enrichment analysis of differentially expressed genes between the two NILs revealed that genes associated with SCW biosynthesis were significantly up-regulated in fibers of the high-fiber quality line at the transition stage of cotton fiber development. For independent corroboration of the microarray results, 15 genes were selected for quantitative reverse transcription PCR analysis of fiber gene expression. These analyses, conducted over multiple field years, confirmed the temporal difference in fiber gene expression between the two NILs. We hypothesize that the loci conferring temporal differences in fiber gene expression between the NILs are important regulatory sequences that offer the potential for more targeted manipulation of cotton fiber quality.

Evolutionary conservation of vertebrate notochord genes in the ascidian Ciona intestinalis.

PubMed

Kugler, Jamie E; Passamaneck, Yale J; Feldman, Taya G; Beh, Jeni; Regnier, Todd W; Di Gregorio, Anna

2008-11-01

To reconstruct a minimum complement of notochord genes evolutionarily conserved across chordates, we scanned the Ciona intestinalis genome using the sequences of 182 genes reported to be expressed in the notochord of different vertebrates and identified 139 candidate notochord genes. For 66 of these Ciona genes expression data were already available, hence we analyzed the expression of the remaining 73 genes and found notochord expression for 20. The predicted products of the newly identified notochord genes range from the transcription factors Ci-XBPa and Ci-miER1 to extracellular matrix proteins. We examined the expression of the newly identified notochord genes in embryos ectopically expressing Ciona Brachyury (Ci-Bra) and in embryos expressing a repressor form of this transcription factor in the notochord, and we found that while a subset of the genes examined are clearly responsive to Ci-Bra, other genes are not affected by alterations in its levels. We provide a first description of notochord genes that are not evidently influenced by the ectopic expression of Ci-Bra and we propose alternative regulatory mechanisms that might control their transcription. Copyright 2008 Wiley-Liss, Inc.

Mining microbial metatranscriptomes for expression of antibiotic resistance genes under natural conditions.

PubMed

Versluis, Dennis; D'Andrea, Marco Maria; Ramiro Garcia, Javier; Leimena, Milkha M; Hugenholtz, Floor; Zhang, Jing; Öztürk, Başak; Nylund, Lotta; Sipkema, Detmer; van Schaik, Willem; de Vos, Willem M; Kleerebezem, Michiel; Smidt, Hauke; van Passel, Mark W J

2015-07-08

Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse, and also included β-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance.

Clustering cancer gene expression data by projective clustering ensemble

PubMed Central

Yu, Xianxue; Yu, Guoxian

2017-01-01

Gene expression data analysis has paramount implications for gene treatments, cancer diagnosis and other domains. Clustering is an important and promising tool to analyze gene expression data. Gene expression data is often characterized by a large amount of genes but with limited samples, thus various projective clustering techniques and ensemble techniques have been suggested to combat with these challenges. However, it is rather challenging to synergy these two kinds of techniques together to avoid the curse of dimensionality problem and to boost the performance of gene expression data clustering. In this paper, we employ a projective clustering ensemble (PCE) to integrate the advantages of projective clustering and ensemble clustering, and to avoid the dilemma of combining multiple projective clusterings. Our experimental results on publicly available cancer gene expression data show PCE can improve the quality of clustering gene expression data by at least 4.5% (on average) than other related techniques, including dimensionality reduction based single clustering and ensemble approaches. The empirical study demonstrates that, to further boost the performance of clustering cancer gene expression data, it is necessary and promising to synergy projective clustering with ensemble clustering. PCE can serve as an effective alternative technique for clustering gene expression data. PMID:28234920

Mining microbial metatranscriptomes for expression of antibiotic resistance genes under natural conditions

NASA Astrophysics Data System (ADS)

Versluis, Dennis; D'Andrea, Marco Maria; Ramiro Garcia, Javier; Leimena, Milkha M.; Hugenholtz, Floor; Zhang, Jing; Öztürk, Başak; Nylund, Lotta; Sipkema, Detmer; Schaik, Willem Van; de Vos, Willem M.; Kleerebezem, Michiel; Smidt, Hauke; Passel, Mark W. J. Van

2015-07-01

Antibiotic resistance genes are found in a broad range of ecological niches associated with complex microbiota. Here we investigated if resistance genes are not only present, but also transcribed under natural conditions. Furthermore, we examined the potential for antibiotic production by assessing the expression of associated secondary metabolite biosynthesis gene clusters. Metatranscriptome datasets from intestinal microbiota of four human adults, one human infant, 15 mice and six pigs, of which only the latter have received antibiotics prior to the study, as well as from sea bacterioplankton, a marine sponge, forest soil and sub-seafloor sediment, were investigated. We found that resistance genes are expressed in all studied ecological niches, albeit with niche-specific differences in relative expression levels and diversity of transcripts. For example, in mice and human infant microbiota predominantly tetracycline resistance genes were expressed while in human adult microbiota the spectrum of expressed genes was more diverse, and also included β-lactam, aminoglycoside and macrolide resistance genes. Resistance gene expression could result from the presence of natural antibiotics in the environment, although we could not link it to expression of corresponding secondary metabolites biosynthesis clusters. Alternatively, resistance gene expression could be constitutive, or these genes serve alternative roles besides antibiotic resistance.

Involvement of Resveratrol and ω-3 Polyunsaturated Fatty Acids on Sirtuin 1 Gene Expression in THP1 Cells.

PubMed

Tsuchiya, Takafumi; Endo, Ayano; Tsujikado, Kyoko; Inukai, Toshihiko

2017-10-01

Resveratrol, a kind of polyphenol, has the potential to activate the longevity gene in several cells, in the same manner as calorie restriction. We investigated the effect of resveratrol and ω-3-line polyunsaturated fatty acid on surtuin 1 (SIRT1) gene expression in human monocytes (THP1) cells. We examined the gene expression of THP1 cells using real-time polymerase chain reaction and Western blotting analysis. Resveratol, eicosapentaenoic acid (EPA) and docosahexaeanoic acid (DHA) as n-3 polyunsaturated fatty acid were added on THP1 cells. We observed the changes in the SIRT1 gene expression in those cells, under various doses of agents and in time courses. Then, we examined the interaction of glucose and mannitol on those agents׳ effect of the gene expression. The concentration range of glucose and mannitol was from 5-20mM, respectively. The SIRT1 gene expression could be defined in 24 and 48 hours both in real-time polymerase chain reaction analysis and in Western blotting. Resveratrol showed SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. Although EPA at 10μM showed marked increase in SIRT1 gene expression compared to control condition in Western blotting, this phenomenon was not in dose-dependent manner. DHA did not exhibit any augmentation of SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. We refined the dose-dependent inhibition of the SIRT1 gene expression within 20mM glucose medium. Although 20mM did not exhibit any inhibition, 10μM resveratrol induced the gene expression compared to control medium. Both 5 and 15mM mannitol medium did not significantly alter basic gene expression and 10μM resveratrol-induced gene expression. The present results suggest that resveratrol and EPA, but not DHA, markedly activated the SIRT1 gene expression in THP1 cells, and that high glucose medium could inhibit the basic gene expression, but not powerful resveratrol-induced gene expression, in those cells. Copyright © 2017 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.

Influence of Genetic Variations in Selenoprotein Genes on the Pattern of Gene Expression after Supplementation with Brazil Nuts

PubMed Central

Rogero, Marcelo M.; Hesketh, John

2017-01-01

Selenium (Se) is an essential micronutrient for human health. Its beneficial effects are exerted by selenoproteins, which can be quantified in blood and used as molecular biomarkers of Se status. We hypothesize that the presence of genetic polymorphisms in selenoprotein genes may: (1) influence the gene expression of specific selenoproteins and (2) influence the pattern of global gene expression after Brazil nut supplementation. The study was conducted with 130 healthy volunteers in Sao Paulo, Brazil, who consumed one Brazil nut (300 μg/Se) a day for eight weeks. Gene expression of GPX1 and SELENOP and genotyping were measured by real-time PCR using TaqMan Assays. Global gene expression was assessed by microarray using Illumina HumanHT-12 v4 BeadChips. Brazil nut supplementation significantly increased GPX1 mRNA expression only in subjects with CC genotype at rs1050450 (p < 0.05). SELENOP mRNA expression was significantly higher in A-carriers at rs7579 either before or after supplementation (p < 0.05). Genotype for rs713041 in GPX4 affected the pattern of blood cell global gene expression. Genetic variations in selenoprotein genes modulated both GPX1 and SELENOP selenoprotein gene expression and global gene expression in response to Brazil nut supplementation. PMID:28696394

GEOGLE: context mining tool for the correlation between gene expression and the phenotypic distinction.

PubMed

Yu, Yao; Tu, Kang; Zheng, Siyuan; Li, Yun; Ding, Guohui; Ping, Jie; Hao, Pei; Li, Yixue

2009-08-25

In the post-genomic era, the development of high-throughput gene expression detection technology provides huge amounts of experimental data, which challenges the traditional pipelines for data processing and analyzing in scientific researches. In our work, we integrated gene expression information from Gene Expression Omnibus (GEO), biomedical ontology from Medical Subject Headings (MeSH) and signaling pathway knowledge from sigPathway entries to develop a context mining tool for gene expression analysis - GEOGLE. GEOGLE offers a rapid and convenient way for searching relevant experimental datasets, pathways and biological terms according to multiple types of queries: including biomedical vocabularies, GDS IDs, gene IDs, pathway names and signature list. Moreover, GEOGLE summarizes the signature genes from a subset of GDSes and estimates the correlation between gene expression and the phenotypic distinction with an integrated p value. This approach performing global searching of expression data may expand the traditional way of collecting heterogeneous gene expression experiment data. GEOGLE is a novel tool that provides researchers a quantitative way to understand the correlation between gene expression and phenotypic distinction through meta-analysis of gene expression datasets from different experiments, as well as the biological meaning behind. The web site and user guide of GEOGLE are available at: http://omics.biosino.org:14000/kweb/workflow.jsp?id=00020.

Genetic background of extreme violent behavior

PubMed Central

Tiihonen, J; Rautiainen, M-R; Ollila, HM; Repo-Tiihonen, E; Virkkunen, M; Palotie, A; Pietiläinen, O; Kristiansson, K; Joukamaa, M; Lauerma, H; Saarela, J; Tyni, S; Vartiainen, H; Paananen, J; Goldman, D; Paunio, T

2015-01-01

In developed countries, the majority of all violent crime is committed by a small group of antisocial recidivistic offenders, but no genes have been shown to contribute to recidivistic violent offending or severe violent behavior, such as homicide. Our results, from two independent cohorts of Finnish prisoners, revealed that a monoamine oxidase A (MAOA) low-activity genotype (contributing to low dopamine turnover rate) as well as the CDH13 gene (coding for neuronal membrane adhesion protein) are associated with extremely violent behavior (at least 10 committed homicides, attempted homicides or batteries). No substantial signal was observed for either MAOA or CDH13 among non-violent offenders, indicating that findings were specific for violent offending, and not largely attributable to substance abuse or antisocial personality disorder. These results indicate both low monoamine metabolism and neuronal membrane dysfunction as plausible factors in the etiology of extreme criminal violent behavior, and imply that at least about 5–10% of all severe violent crime in Finland is attributable to the aforementioned MAOA and CDH13 genotypes. PMID:25349169

Dissociation of retinoblastoma gene protein hyperphosphorylation and commitment to enter S phase.

PubMed Central

Ogryzko, V V; Hirai, T H; Shih, C E; Howard, B H

1994-01-01

Mitogenic activities of simian virus 40 large T and small t antigens were studied in serum-deprived human diploid fibroblasts. Wild-type large T and small t cooperated in stimulating DNA synthesis and in inducing hyperphosphorylation of the Rb gene product (pRb). In contrast, a T antigen mutant defective for pRb binding (Rb- T) possessed no detectable mitogenic activity alone and failed to complement small t in stimulating DNA synthesis. Surprisingly, Rb- T and small t cooperated as strongly as wild-type T and small t with respect to pRb hyperphosphorylation. As a consequence, in two closely related conditions (i.e., stimulation by small t plus wild-type T versus small t plus Rb- T), the fraction of pRb in hyperphosphorylated forms dissociated from the fraction of cells in the S phase. These results indicate that pRb hyperphosphorylation is not always tightly coupled with a commitment to initiate DNA replication. Images PMID:8189510

Genetic background of extreme violent behavior.

PubMed

Tiihonen, J; Rautiainen, M-R; Ollila, H M; Repo-Tiihonen, E; Virkkunen, M; Palotie, A; Pietiläinen, O; Kristiansson, K; Joukamaa, M; Lauerma, H; Saarela, J; Tyni, S; Vartiainen, H; Paananen, J; Goldman, D; Paunio, T

2015-06-01

In developed countries, the majority of all violent crime is committed by a small group of antisocial recidivistic offenders, but no genes have been shown to contribute to recidivistic violent offending or severe violent behavior, such as homicide. Our results, from two independent cohorts of Finnish prisoners, revealed that a monoamine oxidase A (MAOA) low-activity genotype (contributing to low dopamine turnover rate) as well as the CDH13 gene (coding for neuronal membrane adhesion protein) are associated with extremely violent behavior (at least 10 committed homicides, attempted homicides or batteries). No substantial signal was observed for either MAOA or CDH13 among non-violent offenders, indicating that findings were specific for violent offending, and not largely attributable to substance abuse or antisocial personality disorder. These results indicate both low monoamine metabolism and neuronal membrane dysfunction as plausible factors in the etiology of extreme criminal violent behavior, and imply that at least about 5-10% of all severe violent crime in Finland is attributable to the aforementioned MAOA and CDH13 genotypes.

Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.)

PubMed Central

2010-01-01

Background Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. Results Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2) were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L.) samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h), a moderately, but stably expressed eEF1A (s), and combined expression of multigene eEF1A (m). NormFinder identified eEF1A (s) and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. Conclusions This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples, helping pave the way to conduct gene expression studies in perennial biomass crops under field-conditions. From our study several stably expressed reference genes have been validated. This provides useful candidates for reference gene selection in perennial ryegrass under conditions other than those tested here. PMID:20089196

DNMT3B modulates the expression of cancer-related genes and downregulates the expression of the gene VAV3 via methylation

PubMed Central

Peralta-Arrieta, Irlanda; Hernández-Sotelo, Daniel; Castro-Coronel, Yaneth; Leyva-Vázquez, Marco Antonio; Illades-Aguiar, Berenice

2017-01-01

Altered promoter DNA methylation is one of the most important epigenetic abnormalities in human cancer. DNMT3B, de novo methyltransferase, is clearly related to abnormal methylation of tumour suppressor genes, DNA repair genes and its overexpression contributes to oncogenic processes and tumorigenesis in vivo. The purpose of this study was to assess the effect of the overexpression of DNMT3B in HaCaT cells on global gene expression and on the methylation of selected genes to the identification of genes that can be target of DNMT3B. We found that the overexpression of DNMT3B in HaCaT cells, modulate the expression of genes related to cancer, downregulated the expression of 151 genes with CpG islands and downregulated the expression of the VAV3 gene via methylation of its promoter. These results highlight the importance of DNMT3B in gene expression and human cancer. PMID:28123849

DNMT3B modulates the expression of cancer-related genes and downregulates the expression of the gene VAV3 via methylation.

PubMed

Peralta-Arrieta, Irlanda; Hernández-Sotelo, Daniel; Castro-Coronel, Yaneth; Leyva-Vázquez, Marco Antonio; Illades-Aguiar, Berenice

2017-01-01

Altered promoter DNA methylation is one of the most important epigenetic abnormalities in human cancer. DNMT3B, de novo methyltransferase, is clearly related to abnormal methylation of tumour suppressor genes, DNA repair genes and its overexpression contributes to oncogenic processes and tumorigenesis in vivo . The purpose of this study was to assess the effect of the overexpression of DNMT3B in HaCaT cells on global gene expression and on the methylation of selected genes to the identification of genes that can be target of DNMT3B. We found that the overexpression of DNMT3B in HaCaT cells, modulate the expression of genes related to cancer, downregulated the expression of 151 genes with CpG islands and downregulated the expression of the VAV3 gene via methylation of its promoter. These results highlight the importance of DNMT3B in gene expression and human cancer.

Biophysical Constraints Arising from Compositional Context in Synthetic Gene Networks.

PubMed

Yeung, Enoch; Dy, Aaron J; Martin, Kyle B; Ng, Andrew H; Del Vecchio, Domitilla; Beck, James L; Collins, James J; Murray, Richard M

2017-07-26

Synthetic gene expression is highly sensitive to intragenic compositional context (promoter structure, spacing regions between promoter and coding sequences, and ribosome binding sites). However, much less is known about the effects of intergenic compositional context (spatial arrangement and orientation of entire genes on DNA) on expression levels in synthetic gene networks. We compare expression of induced genes arranged in convergent, divergent, or tandem orientations. Induction of convergent genes yielded up to 400% higher expression, greater ultrasensitivity, and dynamic range than divergent- or tandem-oriented genes. Orientation affects gene expression whether one or both genes are induced. We postulate that transcriptional interference in divergent and tandem genes, mediated by supercoiling, can explain differences in expression and validate this hypothesis through modeling and in vitro supercoiling relaxation experiments. Treatment with gyrase abrogated intergenic context effects, bringing expression levels within 30% of each other. We rebuilt the toggle switch with convergent genes, taking advantage of supercoiling effects to improve threshold detection and switch stability. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential gene expression in queen–worker caste determination in bumble-bees

PubMed Central

Pereboom, Jeffrey J. M; Jordan, William C; Sumner, Seirian; Hammond, Robert L; Bourke, Andrew F. G

2005-01-01

Investigating how differential gene expression underlies caste determination in the social Hymenoptera is central to understanding how variation in gene expression underlies adaptive phenotypic diversity. We investigated for the first time the association between differential gene expression and queen–worker caste determination in the bumble-bee Bombus terrestris. Using suppression subtractive hybridization we isolated 12 genes that were differentially expressed in queen- and worker-destined larvae. We found that the sets of genes underlying caste differences in larvae and adults failed to overlap greatly. We also found that B. terrestris shares some of the genes whose differential expression is associated with caste determination in the honeybee, Apis mellifera, but their expression patterns were not identical. Instead, we found B. terrestris to exhibit a novel pattern, whereby most genes upregulated (i.e. showing relatively higher levels of expression) in queen-destined larvae early in development were upregulated in worker-destined larvae late in development. Overall, our results suggest that caste determination in B. terrestris involves a difference not so much in the identity of genes expressed by queen- and worker-destined larvae, but primarily in the relative timing of their expression. This conclusion is of potential importance in the further study of phenotypic diversification via differential gene expression. PMID:16024376

Regulatory systems for hypoxia-inducible gene expression in ischemic heart disease gene therapy.

PubMed

Kim, Hyun Ah; Rhim, Taiyoun; Lee, Minhyung

2011-07-18

Ischemic heart diseases are caused by narrowed coronary arteries that decrease the blood supply to the myocardium. In the ischemic myocardium, hypoxia-responsive genes are up-regulated by hypoxia-inducible factor-1 (HIF-1). Gene therapy for ischemic heart diseases uses genes encoding angiogenic growth factors and anti-apoptotic proteins as therapeutic genes. These genes increase blood supply into the myocardium by angiogenesis and protect cardiomyocytes from cell death. However, non-specific expression of these genes in normal tissues may be harmful, since growth factors and anti-apoptotic proteins may induce tumor growth. Therefore, tight gene regulation is required to limit gene expression to ischemic tissues, to avoid unwanted side effects. For this purpose, various gene expression strategies have been developed for ischemic-specific gene expression. Transcriptional, post-transcriptional, and post-translational regulatory strategies have been developed and evaluated in ischemic heart disease animal models. The regulatory systems can limit therapeutic gene expression to ischemic tissues and increase the efficiency of gene therapy. In this review, recent progresses in ischemic-specific gene expression systems are presented, and their applications to ischemic heart diseases are discussed. Copyright © 2011 Elsevier B.V. All rights reserved.

Expression patterns of WRKY genes in di-haploid Populus simonii × P. nigra in response to salinity stress revealed by quantitative real-time PCR and RNA sequencing.

PubMed

Wang, Shengji; Wang, Jiying; Yao, Wenjing; Zhou, Boru; Li, Renhua; Jiang, Tingbo

2014-10-01

Spatio-temporal expression patterns of 13 out of 119 poplar WRKY genes indicated dynamic and tissue-specific roles of WRKY family proteins in salinity stress tolerance. To understand the expression patterns of poplar WRKY genes under salinity stress, 51 of the 119 WRKY genes were selected from di-haploid Populus simonii × P. nigra by quantitative real-time PCR (qRT-PCR). We used qRT-PCR to profile the expression of the top 13 genes under salinity stress across seven time points, and employed RNA-Seq platforms to cross-validate it. Results demonstrated that all the 13 WRKY genes were expressed in root, stem, and leaf tissues, but their expression levels and overall patterns varied notably in these tissues. Regarding overall gene expression in roots, the 13 genes were significantly highly expressed at all six time points after the treatment, reaching the plateau of expression at hour 9. In leaves, the 13 genes were similarly up-regulated from 3 to 12 h in response to NaCl treatment. In stems, however, expression levels of the 13 genes did not show significant changes after the NaCl treatment. Regarding individual gene expression across the time points and the three tissues, the 13 genes can be classified into three clusters: the lowly expressed Cluster 1 containing PthWRKY28, 45 and 105; intermediately expressed Clusters 2 including PthWRKY56, 88 and 116; and highly expressed Cluster 3 consisting of PthWRKY41, 44, 51, 61, 62, 75 and 106. In general, genes in Cluster 2 and 3 displayed a dynamic pattern of "induced amplification-recovering", suggesting that these WRKY genes and corresponding pathways may play a critical role in mediating salt response and tolerance in a dynamic and tissue-specific manner.

Hox gene expression during postlarval development of the polychaete Alitta virens.

PubMed

Bakalenko, Nadezhda I; Novikova, Elena L; Nesterenko, Alexander Y; Kulakova, Milana A

2013-05-01

Hox genes are the family of transcription factors that play a key role in the patterning of the anterior-posterior axis of all bilaterian animals. These genes display clustered organization and colinear expression. Expression boundaries of individual Hox genes usually correspond with morphological boundaries of the body. Previously, we studied Hox gene expression during larval development of the polychaete Alitta virens (formerly Nereis virens) and discovered that Hox genes are expressed in nereid larva according to the spatial colinearity principle. Adult Alitta virens consist of multiple morphologically similar segments, which are formed sequentially in the growth zone. Since the worm grows for most of its life, postlarval segments constantly change their position along the anterior-posterior axis. We studied the expression dynamics of the Hox cluster during postlarval development of the nereid Alitta virens and found that 8 out of 11 Hox genes are transcribed as wide gene-specific gradients in the ventral nerve cord, ectoderm, and mesoderm. The expression domains constantly shift in accordance with the changing proportions of the growing worm, so expression domains of most Hox genes do not have stable anterior or/and posterior boundaries.In the course of our study, we revealed long antisense RNA (asRNA) for some Hox genes. Expression patterns of two of these genes were analyzed using whole-mount in-situ hybridization. This is the first discovery of antisense RNA for Hox genes in Lophotrochozoa. Hox gene expression in juvenile A. virens differs significantly from Hox gene expression patterns both in A. virens larva and in other Bilateria.We suppose that the postlarval function of the Hox genes in this polychaete is to establish and maintain positional coordinates in a constantly growing body, as opposed to creating morphological difference between segments.

Genomic DNA-based absolute quantification of gene expression in Vitis

USDA-ARS?s Scientific Manuscript database

Many studies in which gene expression is quantified by polymerase chain reaction represent the expression of a gene of interest (GOI) relative to that of a reference gene (RG). Relative expression is founded on the assumptions that RG expression is stable across samples, treatments, organs, etc., an...

Changes in proto-oncogene expression associated with reversal of macrophage-like differentiation of HL 60 cells.

PubMed

Studzinski, G P; Brelvi, Z S

1987-07-01

Prolonged exposure to 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] of 2 sublines (AB-2 and AB-26) of human promyelocytic HL 60 leukemia cells produced increased adherence of the cells to the culture substratum. Advantage was taken of this property to separate physically a population of cells highly enriched in macrophage-like forms. When these differentiated cells were placed in culture medium free of 1,25(OH)2D3, there was a rapid reversal of the features of the differentiated phenotype, monitored by the loss of alpha-naphthyl butyrate esterase activity and the loss of adherence to the substrate. The reversal was accompanied by the resumption of normal rates of DNA synthesis, mitosis, and reaccumulation of c-myc and c-myb transcripts. The levels of transcripts of oncogenes c-fos and c-fms, which became abundant in the phenotypically differentiated cultures, declined along with the loss of adhesiveness and reversion to more primitive myeloblastic forms. These changes in proto-oncogene expression became evident before cell proliferation resumed, thereby excluding the diluting effect of the outgrowth of undifferentiated cells. It is concluded that in this system there is no firm commitment to terminal, as opposed to early, differentiation in the great majority of the cells and that the expression of the monocytic maturation-associated genes c-fos and c-fms is down-regulated when macrophage-like cells dedifferentiate. This strengthens the case for an association between macrophage differentiation and the expression of oncogenes c-fos and c-fms.