scholarly journals Genomic positional conservation identifies topological anchor point (tap)RNAs linked to developmental loci

2016 ◽  
Author(s):  
Paulo P. Amaral ◽  
Tommaso Leonardi ◽  
Namshik Han ◽  
Emmanuelle Viré ◽  
Dennis K. Gascoigne ◽  
...  
Keyword(s):  
Noncoding Rnas ◽  
Conserved Sequence ◽  
Positional Conservation ◽  
Large Numbers ◽  
Human Genomes ◽  
Mammalian Genomes ◽  
Recognition Motifs ◽  
Definition Of

The mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider positional conservation across mammalian genomes as an indicator of functional commonality. We identify 665 conserved lncRNA promoters in mouse and human genomes that are preserved in genomic position relative to orthologous coding genes. The identified ‘positionally conserved’ lncRNA genes are primarily associated with developmental transcription factor loci with which they are co-expressed in a tissue-specific manner. Strikingly, over half of all positionally conserved RNAs in this set are linked to distinct chromatin organization structures, overlapping the binding sites for the CTCF chromatin organizer and located at chromatin loop anchor points and borders of topologically associating domains (TADs). These topological anchor point (tap)RNAs possess conserved sequence domains that are enriched in potential recognition motifs for Zinc Finger proteins. Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each other’s expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Thus, interrogation of positionally conserved lncRNAs identifies a new subset of tapRNAs with shared functional properties. These results provide a large dataset of lncRNAs that conform to the “extended gene” model, in which conserved developmental genes are genomically and functionally linked to regulatory lncRNA loci across mammalian evolution.

scholarly journals Further characterization of the circulating cell in chronic lymphocytic leukemia

Blood ◽  
1976 ◽  
Vol 48 (2) ◽  
pp. 223-234 ◽  
Author(s):  
EF Schultz ◽  
S Davis ◽  
AD Rubin
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Maximal Response ◽  
Delayed Response ◽  
Peripheral Lymphocytes ◽  
Large Numbers ◽  
Normal Individuals ◽  
Circulating Lymphocytes

Abstract Peripheral lymphocytes from normal individuals and from patients with chronic lymphocytic leukemia (CLL) were cultured in vitro for 1–7 days. The growth response to phytohemagglutinin (PHA) was quantitated by the incorporation of tritiated uridine into RNA nucleotide during a 2-hr pulse with the radioisotope. While the maximum response in PHA- stimulated normal cultures appeared at 2–3 days, CLL cultures required 5–7 days to develop their maximal response, which was 50%-60% of the normal magnitude. Dilution of the number of normally reactive lymphocytes by culturing them with totally unreactive, mitomycin- treated cells produced a normal 72-hr maximal response, no matter what proportion of unreactive cells was included in the PHA-stimulated cultures. In addition, the response of peripheral lymphocytes from patients with myeloblastic leukemia, where large numbers of unreactive myeloblasts diluted the normal small lymphocytes, a depressed reaction occurred at the anticipated 2–3 days. Nylon fiber-adherent lymphocytes consisting of 85% immunoglobulin (Ig)-bearing cells responded minimally to PHA, but showed no evidence of a delay. When isolated from CLL patients, both fiber-adherent cells (Ig-bearing) as well as non-fiber- adherent (sheep erythrocyterosetting) cells responded to PHA in a delayed fashion. Similarly, a case of CLL, in which 93.5% of the circulating lymphocytes bore sheep red blood cell receptors, showed its peak response to PHA at 7 days. Therefore, using surface marker criteria considered characteristic of normal T cells and B cells, the delayed response to PHA on the part of CLL lymphocytes was independent of thymic or nonthymic origin.

scholarly journals Applicability and Limitations in the Characterization of Poly-Dispersed Engineered Nanomaterials in Cell Media by Dynamic Light Scattering (DLS)

Materials ◽  
2019 ◽  
Vol 12 (23) ◽  
pp. 3833 ◽  
Author(s):  
Arianna Marucco ◽  
Elisabetta Aldieri ◽  
Riccardo Leinardi ◽  
Enrico Bergamaschi ◽  
Chiara Riganti ◽  
...  
Keyword(s):  
Light Scattering ◽  
Dynamic Light Scattering ◽  
Culture Media ◽  
Raw 264.7 ◽  
Pre Treatment ◽  
The Stability ◽  
Definition Of ◽  
Cellular Tests

The dispersion protocol used to administer nanomaterials (NMs) in in vitro cellular tests might affect their toxicity. For this reason, several dispersion procedures have been proposed to harmonize the toxicological methods, allowing for the comparison of the data that were obtained by different laboratories. At the same time, several techniques and methods are available to monitor the identity of the NMs in the cell media. However, while the characterization of suspensions of engineered NMs having narrow size distribution may be easily performed, the description of aggregated NMs forming polydispersions is still challenging. In the present study, sub-micrometric/nanometric TiO2, SiO2, and CeO2 were dispersed in cell media by using two different dispersion protocols, with and without albumin (0.5%) and with different sonication procedures. Dynamic Light Scattering (DLS) was used to characterize NMs in stock solutions and culture media. Pitfalls that affect DLS measurements were identified and, guidance on a critical analysis of the results provided. The NMs were then tested for their cytotoxicity (LDH leakage) toward murine macrophages (RAW 264.7) and PMA-activated human monocytes (THP-1). As markers of pro-inflammatory response, nitric oxide (NO) and cytokine IL-1β production were measured on RAW 264.7 and THP-1 cells, respectively. The pre-treatment with albumin added to a strong sonication treatment increases the stability and homogeneity of the suspensions of nanometric samples, but not of the submicrometric-samples. Nevertheless, while TiO2 and CeO2 were non-cytotoxic in any conditions, differences in cytotoxicity, NO, and IL-1β releases were found for the SiO2, depending upon the protocol. Overall, the results suggest that there is no one-fits-all method valid for all NMs, since each class of NMs respond differently. The definition of validated procedures and parameters for the selection of the most appropriate method of dispersion for each class of NM appears to be a more efficacious strategy for the harmonization of the dispersion protocols.

scholarly journals THE RELATIVE EXTENSIBILITY OF CELL SURFACES

1963 ◽  
Vol 17 (2) ◽  
pp. 289-297 ◽  
Author(s):  
Murray D. Rosenberg
Keyword(s):  
Cell Culture ◽  
Mechanical Deformation ◽  
Culture Chamber ◽  
Cell Surfaces ◽  
Statistical Index ◽  
Degree Of Deformation ◽  
Large Numbers ◽  
Dissociated Cells ◽  
Definition Of

Observations have been made on the response, in vitro, of cultured and freshly dissociated cells to mechanical deformation. Large numbers of individual cells were studied by means of a special culture chamber bounded by two parallel glass coverslips whose spacing could be reduced from 140 to 2 microns in steps of roughly 0.5 micron. The degree of deformation required for herniation of the cell surface was measured. These measurements lead to the definition of a statistical index characteristic of the extensibility of cell surfaces. This index has been shown to be distinctive for several types of cells; to alter with certain stages of embryonic development; and to be stable with respect to the culturing of cells and certain alterations in the method of cell culture.

scholarly journals Elucidating the Essential Role of the A14 Phosphoprotein in Vaccinia Virus Morphogenesis: Construction and Characterization of a Tetracycline-Inducible Recombinant

2000 ◽  
Vol 74 (8) ◽  
pp. 3682-3695 ◽  
Author(s):  
Paula Traktman ◽  
Ke Liu ◽  
Joseph DeMasi ◽  
Robert Rollins ◽  
Sophy Jesty ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Protein A ◽  
Viral Gene ◽  
Large Numbers ◽  
Viral Morphogenesis ◽  
Translational Start

ABSTRACT We have previously reported the construction and characterization of vindH1, an inducible recombinant in which expression of the vaccinia virus H1 phosphatase is regulated experimentally by IPTG (isopropyl-β-d-thiogalactopyranoside) (35). In the absence of H1 expression, the transcriptional competence and infectivity of nascent virions are severely compromised. We have sought to identify H1 substrates by characterizing proteins that are hyperphosphorylated in H1-deficient virions. Here, we demonstrate that the A14 protein, a component of the virion membrane, is indeed an H1 phosphatase substrate in vivo and in vitro. A14 is hyperphosphorylated on serine residues in the absence of H1 expression. To enable a genetic analysis of A14's function during the viral life cycle, we have adopted the regulatory components of the tetracycline (TET) operon and created new reagents for the construction of TET-inducible vaccinia virus recombinants. In the context of a virus expressing the TET repressor (tetR), insertion of the TET operator between the transcriptional and translational start sites of a late viral gene enables its expression to be tightly regulated by TET. We constructed a TET-inducible recombinant for the A14 gene, vindA14. In the absence of TET, vindA14 fails to form plaques and the 24-h yield of infectious progeny is reduced by 3 orders of magnitude. The infection arrests early during viral morphogenesis, with the accumulation of large numbers of vesicles and the appearance of “empty” crescents that appear to adhere only loosely to virosomes. This phenotype corresponds closely to that observed for an IPTG-inducible A14 recombinant whose construction and characterization were reported while our work was ongoing (47). The consistency in the phenotypes seen for the IPTG- and TET-inducible recombinants confirms the efficacy of the TET-inducible system and reinforces the value of having a second, independent system available for generating inducible recombinants.

scholarly journals Further characterization of the circulating cell in chronic lymphocytic leukemia

Blood ◽  
1976 ◽  
Vol 48 (2) ◽  
pp. 223-234
Author(s):  
EF Schultz ◽  
S Davis ◽  
AD Rubin
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Maximal Response ◽  
Delayed Response ◽  
Peripheral Lymphocytes ◽  
Large Numbers ◽  
Normal Individuals ◽  
Circulating Lymphocytes

Peripheral lymphocytes from normal individuals and from patients with chronic lymphocytic leukemia (CLL) were cultured in vitro for 1–7 days. The growth response to phytohemagglutinin (PHA) was quantitated by the incorporation of tritiated uridine into RNA nucleotide during a 2-hr pulse with the radioisotope. While the maximum response in PHA- stimulated normal cultures appeared at 2–3 days, CLL cultures required 5–7 days to develop their maximal response, which was 50%-60% of the normal magnitude. Dilution of the number of normally reactive lymphocytes by culturing them with totally unreactive, mitomycin- treated cells produced a normal 72-hr maximal response, no matter what proportion of unreactive cells was included in the PHA-stimulated cultures. In addition, the response of peripheral lymphocytes from patients with myeloblastic leukemia, where large numbers of unreactive myeloblasts diluted the normal small lymphocytes, a depressed reaction occurred at the anticipated 2–3 days. Nylon fiber-adherent lymphocytes consisting of 85% immunoglobulin (Ig)-bearing cells responded minimally to PHA, but showed no evidence of a delay. When isolated from CLL patients, both fiber-adherent cells (Ig-bearing) as well as non-fiber- adherent (sheep erythrocyterosetting) cells responded to PHA in a delayed fashion. Similarly, a case of CLL, in which 93.5% of the circulating lymphocytes bore sheep red blood cell receptors, showed its peak response to PHA at 7 days. Therefore, using surface marker criteria considered characteristic of normal T cells and B cells, the delayed response to PHA on the part of CLL lymphocytes was independent of thymic or nonthymic origin.

scholarly journals Protein kinases display minimal interpositional dependence on substrate sequence: potential implications for the evolution of signalling networks

2012 ◽  
Vol 367 (1602) ◽  
pp. 2574-2583 ◽  
Author(s):  
Brian A. Joughin ◽  
Chengcheng Liu ◽  
Douglas A. Lauffenburger ◽  
Christopher W. V. Hogue ◽  
Michael B. Yaffe
Keyword(s):  
Substrate Specificity ◽  
Protein Kinases ◽  
Kinase Substrate ◽  
Binding Domains ◽  
Large Numbers ◽  
Cellular Context ◽  
Kinase Substrates

Characterization of in vitro substrates of protein kinases by peptide library screening provides a wealth of information on the substrate specificity of kinases for amino acids at particular positions relative to the site of phosphorylation, but provides no information concerning interdependence among positions. High-throughput techniques have recently made it feasible to identify large numbers of in vivo kinase substrates. We used data from experiments on the kinases ATM/ATR and CDK1, and curated CK2 substrates to evaluate the prevalence of interactions between substrate positions within a motif and the utility of these interactions in predicting kinase substrates. Among these data, evidence of interpositional sequence dependencies is strikingly rare, and what dependency exists does little to aid in the prediction of novel kinase substrates. Significant increases in the ability of models to predict kinase–substrate specificity beyond position-independent models must come largely from inclusion of elements of biological and cellular context, rather than further analysis of substrate sequences alone. Our results suggest that, evolutionarily, kinase substrate fitness exists in a smooth energetic landscape. Taken with results from others indicating that phosphopeptide-binding domains do exhibit interpositional dependence, our data suggest that incorporation of new substrate molecules into phospho-signalling networks may be rate-limited by the evolution of suitability for binding by phosphopeptide-binding domains.

scholarly journals Toward a definition of “fresh” whole blood: an in vitro characterization of coagulation properties in refrigerated whole blood for transfusion

Transfusion ◽  
2011 ◽  
Vol 51 (1) ◽  
pp. 43-51 ◽  
Author(s):  
David Jobes ◽  
Yanika Wolfe ◽  
Daniel O'Neill ◽  
Jennifer Calder ◽  
Lisa Jones ◽  
...  
Keyword(s):  
Whole Blood ◽  
In Vitro Characterization ◽  
Definition Of

scholarly journals Genomic and Transcriptomic Characterization of the New Human Glioblastoma Cell Line AHOL1

2019 ◽  
Author(s):  
Wallax Augusto Silva Ferreira ◽  
Carolina Koury Nassar Amorim ◽  
Rommel Rodriguez Burbano ◽  
Rolando André Rios Villacis ◽  
Fabio Albuquerque Machi ◽  
...  
Keyword(s):  
Cell Line ◽  
Cancer Biology ◽  
Copy Number Alteration ◽  
Transcriptome Profiling ◽  
In Vitro Models ◽  
Altered Expression ◽  
Large Numbers ◽  
Druggable Targets

Cancer cell lines are widely used as in vitro models of tumorigenesis, facilitating fundamental discoveries in cancer biology and translational medicine. Currently, there are few options for glioblastoma (GBM) treatment and limited in vitro models with accurate genomic and transcriptomic characterization. Here, a detailed characterization of a new GBM cell line, namely AHOL1, was conducted in order to fully characterize its molecular composition based on its copy number alteration (CNA) and transcriptome profiling, followed by the validation of key elements associated with GBM tumorigenesis. Large numbers of CNAs and differentially expressed genes (DEGs) were identified. CNAs were distributed throughout the genome, including gains at Xq11.1-q28, Xp22.33-p11.1, Xq21.1-q21.33, 4p15.1-p14, 8q23.2-q23.3 and losses at Yq11.21-q12, Yp11.31-p11.2 and 12p13.31 positions. Nine druggable genes were identified, including HCRTR2, ETV1, PTPRD, PRKX, STS, RPS6KA6, ZFY, USP9Y and KDM5D. By integrating DEGs and CNAs, we identified 57 overlapping genes enriched in fourteen pathways. Altered expression of several cancer-related candidates found in the DEGs-CNA dataset was confirmed by RT-qPCR. Taken together, this first comprehensive genomic and transcriptomic landscape of AHOL1 provides unique resources for further studies and identifies several druggable targets that may be useful for therapeutics and biologic and molecular investigation of GBM.

scholarly journals APOBEC3A deaminates transiently exposed single-strand DNA during LINE-1 retrotransposition

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Sandra R Richardson ◽  
Iñigo Narvaiza ◽  
Randy A Planegger ◽  
Matthew D Weitzman ◽  
John V Moran
Keyword(s):  
Cytidine Deaminase ◽  
Mechanistic Explanation ◽  
Single Strand ◽  
Human Genomes ◽  
Cell Assays ◽  
Long Interspersed Element ◽  
Uridine Nucleotides

Long INterspersed Element-1 (LINE-1 or L1) retrotransposition poses a mutagenic threat to human genomes. Human cells have therefore evolved strategies to regulate L1 retrotransposition. The APOBEC3 (A3) gene family consists of seven enzymes that catalyze deamination of cytidine nucleotides to uridine nucleotides (C-to-U) in single-strand DNA substrates. Among these enzymes, APOBEC3A (A3A) is the most potent inhibitor of L1 retrotransposition in cultured cell assays. However, previous characterization of L1 retrotransposition events generated in the presence of A3A did not yield evidence of deamination. Thus, the molecular mechanism by which A3A inhibits L1 retrotransposition has remained enigmatic. Here, we have used in vitro and in vivo assays to demonstrate that A3A can inhibit L1 retrotransposition by deaminating transiently exposed single-strand DNA that arises during the process of L1 integration. These data provide a mechanistic explanation of how the A3A cytidine deaminase protein can inhibit L1 retrotransposition.

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