cell type identity: Topics by Science.gov

Sample records for cell type identity

Heterogeneity of neuroblastoma cell identity defined by transcriptional circuitries.

PubMed

Boeva, Valentina; Louis-Brennetot, Caroline; Peltier, Agathe; Durand, Simon; Pierre-Eugène, Cécile; Raynal, Virginie; Etchevers, Heather C; Thomas, Sophie; Lermine, Alban; Daudigeos-Dubus, Estelle; Geoerger, Birgit; Orth, Martin F; Grünewald, Thomas G P; Diaz, Elise; Ducos, Bertrand; Surdez, Didier; Carcaboso, Angel M; Medvedeva, Irina; Deller, Thomas; Combaret, Valérie; Lapouble, Eve; Pierron, Gaelle; Grossetête-Lalami, Sandrine; Baulande, Sylvain; Schleiermacher, Gudrun; Barillot, Emmanuel; Rohrer, Hermann; Delattre, Olivier; Janoueix-Lerosey, Isabelle

2017-09-01

Neuroblastoma is a tumor of the peripheral sympathetic nervous system, derived from multipotent neural crest cells (NCCs). To define core regulatory circuitries (CRCs) controlling the gene expression program of neuroblastoma, we established and analyzed the neuroblastoma super-enhancer landscape. We discovered three types of identity in neuroblastoma cell lines: a sympathetic noradrenergic identity, defined by a CRC module including the PHOX2B, HAND2 and GATA3 transcription factors (TFs); an NCC-like identity, driven by a CRC module containing AP-1 TFs; and a mixed type, further deconvoluted at the single-cell level. Treatment of the mixed type with chemotherapeutic agents resulted in enrichment of NCC-like cells. The noradrenergic module was validated by ChIP-seq. Functional studies demonstrated dependency of neuroblastoma with noradrenergic identity on PHOX2B, evocative of lineage addiction. Most neuroblastoma primary tumors express TFs from the noradrenergic and NCC-like modules. Our data demonstrate a previously unknown aspect of tumor heterogeneity relevant for neuroblastoma treatment strategies.
Control of Cell Identity in Pancreas Development and Regeneration

PubMed Central

Stanger, Ben Z.; Hebrok, Matthias

2013-01-01

The endocrine and exocrine cells in the adult pancreas are not static, but can change differentiation state in response to injury or stress. This concept of cells in flux means that there may be ways to generate certain types of cells (such as insulin-producing β-cells) and prevent formation of others (such as transformed, neoplastic cells). We review different aspects of cell identity in the pancreas, discussing how cells achieve their identity during embryonic development and maturation, and how this identity remains plastic, even in the adult pancreas. PMID:23622126
Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity

PubMed Central

Song, Lingyun; Zhang, Zhancheng; Grasfeder, Linda L.; Boyle, Alan P.; Giresi, Paul G.; Lee, Bum-Kyu; Sheffield, Nathan C.; Gräf, Stefan; Huss, Mikael; Keefe, Damian; Liu, Zheng; London, Darin; McDaniell, Ryan M.; Shibata, Yoichiro; Showers, Kimberly A.; Simon, Jeremy M.; Vales, Teresa; Wang, Tianyuan; Winter, Deborah; Zhang, Zhuzhu; Clarke, Neil D.; Birney, Ewan; Iyer, Vishwanath R.; Crawford, Gregory E.; Lieb, Jason D.; Furey, Terrence S.

2011-01-01

The human body contains thousands of unique cell types, each with specialized functions. Cell identity is governed in large part by gene transcription programs, which are determined by regulatory elements encoded in DNA. To identify regulatory elements active in seven cell lines representative of diverse human cell types, we used DNase-seq and FAIRE-seq (Formaldehyde Assisted Isolation of Regulatory Elements) to map “open chromatin.” Over 870,000 DNaseI or FAIRE sites, which correspond tightly to nucleosome-depleted regions, were identified across the seven cell lines, covering nearly 9% of the genome. The combination of DNaseI and FAIRE is more effective than either assay alone in identifying likely regulatory elements, as judged by coincidence with transcription factor binding locations determined in the same cells. Open chromatin common to all seven cell types tended to be at or near transcription start sites and to be coincident with CTCF binding sites, while open chromatin sites found in only one cell type were typically located away from transcription start sites and contained DNA motifs recognized by regulators of cell-type identity. We show that open chromatin regions bound by CTCF are potent insulators. We identified clusters of open regulatory elements (COREs) that were physically near each other and whose appearance was coordinated among one or more cell types. Gene expression and RNA Pol II binding data support the hypothesis that COREs control gene activity required for the maintenance of cell-type identity. This publicly available atlas of regulatory elements may prove valuable in identifying noncoding DNA sequence variants that are causally linked to human disease. PMID:21750106
Metabolic Stress and Compromised Identity of Pancreatic Beta Cells

PubMed Central

Swisa, Avital; Glaser, Benjamin; Dor, Yuval

2017-01-01

Beta cell failure is a central feature of type 2 diabetes (T2D), but the molecular underpinnings of the process remain only partly understood. It has been suggested that beta cell failure in T2D involves massive cell death. Other studies ascribe beta cell failure to cell exhaustion, due to chronic oxidative or endoplasmic reticulum stress leading to cellular dysfunction. More recently it was proposed that beta cells in T2D may lose their differentiated identity, possibly even gaining features of other islet cell types. The loss of beta cell identity appears to be driven by glucotoxicity inhibiting the activity of key beta cell transcription factors including Pdx1, Nkx6.1, MafA and Pax6, thereby silencing beta cell genes and derepressing alternative islet cell genes. The loss of beta cell identity is at least partly reversible upon normalization of glycemia, with implications for the reversibility of T2D, although it is not known if beta cell failure reaches eventually a point of no return. In this review we discuss current evidence for metabolism-driven compromised beta cell identity, key knowledge gaps and opportunities for utility in the treatment of T2D. PMID:28270834
Metabolic Stress and Compromised Identity of Pancreatic Beta Cells.

PubMed

Swisa, Avital; Glaser, Benjamin; Dor, Yuval

2017-01-01

Beta cell failure is a central feature of type 2 diabetes (T2D), but the molecular underpinnings of the process remain only partly understood. It has been suggested that beta cell failure in T2D involves massive cell death. Other studies ascribe beta cell failure to cell exhaustion, due to chronic oxidative or endoplasmic reticulum stress leading to cellular dysfunction. More recently it was proposed that beta cells in T2D may lose their differentiated identity, possibly even gaining features of other islet cell types. The loss of beta cell identity appears to be driven by glucotoxicity inhibiting the activity of key beta cell transcription factors including Pdx1, Nkx6.1, MafA and Pax6, thereby silencing beta cell genes and derepressing alternative islet cell genes. The loss of beta cell identity is at least partly reversible upon normalization of glycemia, with implications for the reversibility of T2D, although it is not known if beta cell failure reaches eventually a point of no return. In this review we discuss current evidence for metabolism-driven compromised beta cell identity, key knowledge gaps and opportunities for utility in the treatment of T2D.
Revealing the vectors of cellular identity with single-cell genomics

PubMed Central

Wagner, Allon; Regev, Aviv; Yosef, Nir

2017-01-01

Single-cell genomics has now made it possible to create a comprehensive atlas of human cells. At the same time, it has reopened definitions of a cell’s identity and type and of the ways in which they are regulated by the cell’s molecular circuitry. Emerging computational analysis methods, especially in single-cell RNA sequencing (scRNA-seq), have already begun to reveal, in a data-driven way, the diverse simultaneous facets of a cell’s identity, from a taxonomy of discrete cell types to continuous dynamic transitions and spatial locations. These developments will eventually allow a cell to be represented as a superposition of ‘basis vectors’, each determining a different (but possibly dependent) aspect of cellular organization and function. However, computational methods must also overcome considerable challenges—from handling technical noise and data scale to forming new abstractions of biology. As the scale of single-cell experiments continues to increase, new computational approaches will be essential for constructing and characterizing a reference map of cell identities. PMID:27824854
The neuron identity problem: form meets function.

PubMed

Fishell, Gord; Heintz, Nathaniel

2013-10-30

A complete understanding of nervous system function cannot be achieved without the identification of its component cell types. In this Perspective, we explore a series of related issues surrounding cell identity and how revolutionary methods for labeling and probing specific neuronal types have clarified this question. Specifically, we ask the following questions: what is the purpose of such diversity, how is it generated, how is it maintained, and, ultimately, how can one unambiguously identity one cell type from another? We suggest that each cell type can be defined by a unique and conserved molecular ground state that determines its capabilities. We believe that gaining an understanding of these molecular barcodes will advance our ability to explore brain function, enhance our understanding of the biochemical basis of CNS disorders, and aid in the development of novel therapeutic strategies. Copyright © 2013 Elsevier Inc. All rights reserved.
Allelic exchange of pheromones and their receptors reprograms sexual identity in Cryptococcus neoformans.

PubMed

Stanton, Brynne C; Giles, Steven S; Staudt, Mark W; Kruzel, Emilia K; Hull, Christina M

2010-02-26

Cell type specification is a fundamental process that all cells must carry out to ensure appropriate behaviors in response to environmental stimuli. In fungi, cell identity is critical for defining "sexes" known as mating types and is controlled by components of mating type (MAT) loci. MAT-encoded genes function to define sexes via two distinct paradigms: 1) by controlling transcription of components common to both sexes, or 2) by expressing specially encoded factors (pheromones and their receptors) that differ between mating types. The human fungal pathogen Cryptococcus neoformans has two mating types (a and alpha) that are specified by an extremely unusual MAT locus. The complex architecture of this locus makes it impossible to predict which paradigm governs mating type. To identify the mechanism by which the C. neoformans sexes are determined, we created strains in which the pheromone and pheromone receptor from one mating type (a) replaced the pheromone and pheromone receptor of the other (alpha). We discovered that these "alpha(a)" cells effectively adopt a new mating type (that of a cells); they sense and respond to alpha factor, they elicit a mating response from alpha cells, and they fuse with alpha cells. In addition, alpha(a) cells lose the alpha cell type-specific response to pheromone and do not form germ tubes, instead remaining spherical like a cells. Finally, we discovered that exogenous expression of the diploid/dikaryon-specific transcription factor Sxi2a could then promote complete sexual development in crosses between alpha and alpha(a) strains. These data reveal that cell identity in C. neoformans is controlled fully by three kinds of MAT-encoded proteins: pheromones, pheromone receptors, and homeodomain proteins. Our findings establish the mechanisms for maintenance of distinct cell types and subsequent developmental behaviors in this unusual human fungal pathogen.
Evolution of petal epidermal micromorphology in Leguminosae and its use as a marker of petal identity.

PubMed

Ojeda, Isidro; Francisco-Ortega, Javier; Cronk, Quentin C B

2009-11-01

The legume flower is highly variable in symmetry and differentiation of petal types. Most papilionoid flowers are zygomorphic with three types of petals: one dorsal, two lateral and two ventral petals. Mimosoids have radial flowers with reduced petals while caesalpinioids display a range from strongly zygomorphic to nearly radial symmetry. The aims are to characterize the petal micromorphology relative to flower morphology and evolution within the family and assess its use as a marker of petal identity (whether dorsal, lateral or ventral) as determined by the expression of developmental genes. Petals were analysed using the scanning electron microscope and light microscope. A total of 175 species were studied representing 26 tribes and 89 genera in all three subfamilies of the Leguminosae. The papilionoids have the highest degree of variation of epidermal types along the dorsiventral axis within the flower. In Loteae and genistoids, in particular, it is common for each petal type to have a different major epidermal micromorphology. Papillose conical cells are mainly found on dorsal and lateral petals. Tabular rugose cells are mainly found on lateral petals and tabular flat cells are found only in ventral petals. Caesalpinioids lack strong micromorphological variation along this axis and usually have only a single major epidermal type within a flower, although the type maybe either tabular rugose cells, papillose conical cells or papillose knobby rugose cells, depending on the species. Strong micromorphological variation between different petals in the flower is exclusive to the subfamily Papilionoideae. Both major and minor epidermal types can be used as micromorphological markers of petal identity, at least in papilionoids, and they are important characters of flower evolution in the whole family. The molecular developmental pathway between specific epidermal micromorphology and the expression of petal identity genes has yet to be established.
The mating type-like loci of Candida glabrata.

PubMed

Yáñez-Carrillo, Patricia; Robledo-Márquez, Karina A; Ramírez-Zavaleta, Candy Y; De Las Peñas, Alejandro; Castaño, Irene

2014-01-01

Candida glabrata, a haploid and opportunistic fungal pathogen that has not known sexual cycle, has conserved the majority of the genes required for mating and cell type identity. The C. glabrata genome contains three mating-type-like loci called MTL1, MTL2 and MTL3. The three loci encode putative transcription factors, a1, α1 and α2 that regulate cell type identity and sexual reproduction in other fungi like the closely related Saccharomyces cerevisiae. MTL1 can contain either a or α information. MTL2, which contains a information and MTL3 with α information, are relatively close to two telomeres. MTL1 and MTL2 are transcriptionally active, while MTL3 is subject to an incomplete silencing nucleated at the telomere that depends on the silencing proteins Sir2, Sir3, Sir4, yKu70/80, Rif1, Rap1 and Sum1. C. glabrata does not seem to maintain cell type identity, as cell type-specific genes are expressed regardless of the type (or even absence) of mating information. These data highlight important differences in the control of mating and cell type identity between the non-pathogenic yeast S. cerevisiae and C. glabrata, which might explain the absence of a sexual cycle in C. glabrata. The fact that C. glabrata has conserved the vast majority of the genes involved in mating might suggest that some of these genes perhaps have been rewired to control other processes important for the survival inside the host as a commensal or as a human pathogen. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). Copyright © 2013 Revista Iberoamericana de Micología. Published by Elsevier Espana. All rights reserved.
Transcriptional Architecture of Synaptic Communication Delineates GABAergic Neuron Identity.

PubMed

Paul, Anirban; Crow, Megan; Raudales, Ricardo; He, Miao; Gillis, Jesse; Huang, Z Josh

2017-10-19

Understanding the organizational logic of neural circuits requires deciphering the biological basis of neuronal diversity and identity, but there is no consensus on how neuron types should be defined. We analyzed single-cell transcriptomes of a set of anatomically and physiologically characterized cortical GABAergic neurons and conducted a computational genomic screen for transcriptional profiles that distinguish them from one another. We discovered that cardinal GABAergic neuron types are delineated by a transcriptional architecture that encodes their synaptic communication patterns. This architecture comprises 6 categories of ∼40 gene families, including cell-adhesion molecules, transmitter-modulator receptors, ion channels, signaling proteins, neuropeptides and vesicular release components, and transcription factors. Combinatorial expression of select members across families shapes a multi-layered molecular scaffold along the cell membrane that may customize synaptic connectivity patterns and input-output signaling properties. This molecular genetic framework of neuronal identity integrates cell phenotypes along multiple axes and provides a foundation for discovering and classifying neuron types. Copyright © 2017 Elsevier Inc. All rights reserved.
β-Cell Deficit in Obese Type 2 Diabetes, a Minor Role of β-Cell Dedifferentiation and Degranulation.

PubMed

Butler, Alexandra E; Dhawan, Sangeeta; Hoang, Jonathan; Cory, Megan; Zeng, Kylie; Fritsch, Helga; Meier, Juris J; Rizza, Robert A; Butler, Peter C

2016-02-01

Type 2 diabetes is characterized by a β-cell deficit and a progressive defect in β-cell function. It has been proposed that the deficit in β-cells may be due to β-cell degranulation and transdifferentiation to other endocrine cell types. The objective of the study was to establish the potential impact of β-cell dedifferentiation and transdifferentiation on β-cell deficit in type 2 diabetes and to consider the alternative that cells with an incomplete identity may be newly forming rather than dedifferentiated. Pancreata obtained at autopsy were evaluated from 14 nondiabetic and 13 type 2 diabetic individuals, from four fetal cases, and from 10 neonatal cases. Whereas there was a slight increase in islet endocrine cells expressing no hormone in type 2 diabetes (0.11 ± 0.03 cells/islet vs 0.03 ± 0.01 cells/islet, P < .01), the impact on the β-cell deficit would be minimal. Furthermore, we established that the deficit in β-cells per islet cannot be accounted for by an increase in other endocrine cell types. The distribution of hormone negative endocrine cells in type 2 diabetes (most abundant in cells scattered in the exocrine pancreas) mirrors that in developing (embryo and neonatal) pancreas, implying that these may represent newly forming cells. Therefore, although we concur that in type 2 diabetes there are endocrine cells with altered cell identity, this process does not account for the deficit in β-cells in type 2 diabetes but may reflect, in part, attempted β-cell regeneration.
PAX6 maintains β cell identity by repressing genes of alternative islet cell types.

PubMed

Swisa, Avital; Avrahami, Dana; Eden, Noa; Zhang, Jia; Feleke, Eseye; Dahan, Tehila; Cohen-Tayar, Yamit; Stolovich-Rain, Miri; Kaestner, Klaus H; Glaser, Benjamin; Ashery-Padan, Ruth; Dor, Yuval

2017-01-03

Type 2 diabetes is thought to involve a compromised β cell differentiation state, but the mechanisms underlying this dysfunction remain unclear. Here, we report a key role for the TF PAX6 in the maintenance of adult β cell identity and function. PAX6 was downregulated in β cells of diabetic db/db mice and in WT mice treated with an insulin receptor antagonist, revealing metabolic control of expression. Deletion of Pax6 in β cells of adult mice led to lethal hyperglycemia and ketosis that were attributed to loss of β cell function and expansion of α cells. Lineage-tracing, transcriptome, and chromatin analyses showed that PAX6 is a direct activator of β cell genes, thus maintaining mature β cell function and identity. In parallel, we found that PAX6 binds promoters and enhancers to repress alternative islet cell genes including ghrelin, glucagon, and somatostatin. Chromatin analysis and shRNA-mediated gene suppression experiments indicated a similar function of PAX6 in human β cells. We conclude that reduced expression of PAX6 in metabolically stressed β cells may contribute to β cell failure and α cell dysfunction in diabetes.
PAX6 maintains β cell identity by repressing genes of alternative islet cell types

PubMed Central

Swisa, Avital; Avrahami, Dana; Eden, Noa; Zhang, Jia; Feleke, Eseye; Dahan, Tehila; Cohen-Tayar, Yamit; Stolovich-Rain, Miri; Kaestner, Klaus H.; Glaser, Benjamin; Ashery-Padan, Ruth

2016-01-01

Type 2 diabetes is thought to involve a compromised β cell differentiation state, but the mechanisms underlying this dysfunction remain unclear. Here, we report a key role for the TF PAX6 in the maintenance of adult β cell identity and function. PAX6 was downregulated in β cells of diabetic db/db mice and in WT mice treated with an insulin receptor antagonist, revealing metabolic control of expression. Deletion of Pax6 in β cells of adult mice led to lethal hyperglycemia and ketosis that were attributed to loss of β cell function and expansion of α cells. Lineage-tracing, transcriptome, and chromatin analyses showed that PAX6 is a direct activator of β cell genes, thus maintaining mature β cell function and identity. In parallel, we found that PAX6 binds promoters and enhancers to repress alternative islet cell genes including ghrelin, glucagon, and somatostatin. Chromatin analysis and shRNA-mediated gene suppression experiments indicated a similar function of PAX6 in human β cells. We conclude that reduced expression of PAX6 in metabolically stressed β cells may contribute to β cell failure and α cell dysfunction in diabetes. PMID:27941241
Evolution of petal epidermal micromorphology in Leguminosae and its use as a marker of petal identity

PubMed Central

Ojeda, Isidro; Francisco-Ortega, Javier; Cronk, Quentin C. B.

2009-01-01

Background and Aims The legume flower is highly variable in symmetry and differentiation of petal types. Most papilionoid flowers are zygomorphic with three types of petals: one dorsal, two lateral and two ventral petals. Mimosoids have radial flowers with reduced petals while caesalpinioids display a range from strongly zygomorphic to nearly radial symmetry. The aims are to characterize the petal micromorphology relative to flower morphology and evolution within the family and assess its use as a marker of petal identity (whether dorsal, lateral or ventral) as determined by the expression of developmental genes. Methods Petals were analysed using the scanning electron microscope and light microscope. A total of 175 species were studied representing 26 tribes and 89 genera in all three subfamilies of the Leguminosae. Key Results The papilionoids have the highest degree of variation of epidermal types along the dorsiventral axis within the flower. In Loteae and genistoids, in particular, it is common for each petal type to have a different major epidermal micromorphology. Papillose conical cells are mainly found on dorsal and lateral petals. Tabular rugose cells are mainly found on lateral petals and tabular flat cells are found only in ventral petals. Caesalpinioids lack strong micromorphological variation along this axis and usually have only a single major epidermal type within a flower, although the type maybe either tabular rugose cells, papillose conical cells or papillose knobby rugose cells, depending on the species. Conclusions Strong micromorphological variation between different petals in the flower is exclusive to the subfamily Papilionoideae. Both major and minor epidermal types can be used as micromorphological markers of petal identity, at least in papilionoids, and they are important characters of flower evolution in the whole family. The molecular developmental pathway between specific epidermal micromorphology and the expression of petal identity genes has yet to be established. PMID:19789174
Biological computational approaches: new hopes to improve (re)programming robustness, regenerative medicine and cancer therapeutics.

PubMed

Ebrahimi, Behnam

2016-01-01

Hundreds of transcription factors (TFs) are expressed and work in each cell type, but the identity of the cells is defined and maintained through the activity of a small number of core TFs. Existing reprogramming strategies predominantly focus on the ectopic expression of core TFs of an intended fate in a given cell type regardless of the state of native/somatic gene regulatory networks (GRNs) of the starting cells. Interestingly, an important point is that how much products of the reprogramming, transdifferentiation and differentiation (programming) are identical to their in vivo counterparts. There is evidence that shows that direct fate conversions of somatic cells are not complete, with target cell identity not fully achieved. Manipulation of core TFs provides a powerful tool for engineering cell fate in terms of extinguishment of native GRNs, the establishment of a new GRN, and preventing installation of aberrant GRNs. Conventionally, core TFs are selected to convert one cell type into another mostly based on literature and the experimental identification of genes that are differentially expressed in one cell type compared to the specific cell types. Currently, there is not a universal standard strategy for identifying candidate core TFs. Remarkably, several biological computational platforms are developed, which are capable of evaluating the fidelity of reprogramming methods and refining existing protocols. The current review discusses some deficiencies of reprogramming technologies in the production of a pure population of authentic target cells. Furthermore, it reviews the role of computational approaches (e.g. CellNet, KeyGenes, Mogrify, etc.) in improving (re)programming methods and consequently in regenerative medicine and cancer therapeutics. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.
A Single-Cell Roadmap of Lineage Bifurcation in Human ESC Models of Embryonic Brain Development.

PubMed

Yao, Zizhen; Mich, John K; Ku, Sherman; Menon, Vilas; Krostag, Anne-Rachel; Martinez, Refugio A; Furchtgott, Leon; Mulholland, Heather; Bort, Susan; Fuqua, Margaret A; Gregor, Ben W; Hodge, Rebecca D; Jayabalu, Anu; May, Ryan C; Melton, Samuel; Nelson, Angelique M; Ngo, N Kiet; Shapovalova, Nadiya V; Shehata, Soraya I; Smith, Michael W; Tait, Leah J; Thompson, Carol L; Thomsen, Elliot R; Ye, Chaoyang; Glass, Ian A; Kaykas, Ajamete; Yao, Shuyuan; Phillips, John W; Grimley, Joshua S; Levi, Boaz P; Wang, Yanling; Ramanathan, Sharad

2017-01-05

During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders. Copyright © 2017 Elsevier Inc. All rights reserved.
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
Micropattern differentiation of mouse pluripotent stem cells recapitulates embryo regionalized cell fate patterning

PubMed Central

Morgani, Sophie M; Metzger, Jakob J; Nichols, Jennifer

2018-01-01

During gastrulation epiblast cells exit pluripotency as they specify and spatially arrange the three germ layers of the embryo. Similarly, human pluripotent stem cells (PSCs) undergo spatially organized fate specification on micropatterned surfaces. Since in vivo validation is not possible for the human, we developed a mouse PSC micropattern system and, with direct comparisons to mouse embryos, reveal the robust specification of distinct regional identities. BMP, WNT, ACTIVIN and FGF directed mouse epiblast-like cells to undergo an epithelial-to-mesenchymal transition and radially pattern posterior mesoderm fates. Conversely, WNT, ACTIVIN and FGF patterned anterior identities, including definitive endoderm. By contrast, epiblast stem cells, a developmentally advanced state, only specified anterior identities, but without patterning. The mouse micropattern system offers a robust scalable method to generate regionalized cell types present in vivo, resolve how signals promote distinct identities and generate patterns, and compare mechanisms operating in vivo and in vitro and across species. PMID:29412136
The Evolution of Human Cells in Terms of Protein Innovation

PubMed Central

Sardar, Adam J.; Oates, Matt E.; Fang, Hai; Forrest, Alistair R.R.; Kawaji, Hideya; Gough, Julian; Rackham, Owen J.L.

2014-01-01

Humans are composed of hundreds of cell types. As the genomic DNA of each somatic cell is identical, cell type is determined by what is expressed and when. Until recently, little has been reported about the determinants of human cell identity, particularly from the joint perspective of gene evolution and expression. Here, we chart the evolutionary past of all documented human cell types via the collective histories of proteins, the principal product of gene expression. FANTOM5 data provide cell-type–specific digital expression of human protein-coding genes and the SUPERFAMILY resource is used to provide protein domain annotation. The evolutionary epoch in which each protein was created is inferred by comparison with domain annotation of all other completely sequenced genomes. Studying the distribution across epochs of genes expressed in each cell type reveals insights into human cellular evolution in terms of protein innovation. For each cell type, its history of protein innovation is charted based on the genes it expresses. Combining the histories of all cell types enables us to create a timeline of cell evolution. This timeline identifies the possibility that our common ancestor Coelomata (cavity-forming animals) provided the innovation required for the innate immune system, whereas cells which now form the brain of human have followed a trajectory of continually accumulating novel proteins since Opisthokonta (boundary of animals and fungi). We conclude that exaptation of existing domain architectures into new contexts is the dominant source of cell-type–specific domain architectures. PMID:24692656

Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity

PubMed Central

Omori, Yoshihiro; Kubo, Shun; Kon, Tetsuo; Furuhashi, Mayu; Narita, Hirotaka; Kominami, Taro; Ueno, Akiko; Tsutsumi, Ryotaro; Chaya, Taro; Yamamoto, Haruka; Suetake, Isao; Ueno, Shinji; Koseki, Haruhiko; Furukawa, Takahisa

2017-01-01

Precise transcriptional regulation controlled by a transcription factor network is known to be crucial for establishing correct neuronal cell identities and functions in the CNS. In the retina, the expression of various cone and rod photoreceptor cell genes is regulated by multiple transcription factors; however, the role of epigenetic regulation in photoreceptor cell gene expression has been poorly understood. Here, we found that Samd7, a rod-enriched sterile alpha domain (SAM) domain protein, is essential for silencing nonrod gene expression through H3K27me3 regulation in rod photoreceptor cells. Samd7-null mutant mice showed ectopic expression of nonrod genes including S-opsin in rod photoreceptor cells and rod photoreceptor cell dysfunction. Samd7 physically interacts with Polyhomeotic homologs (Phc proteins), components of the Polycomb repressive complex 1 (PRC1), and colocalizes with Phc2 and Ring1B in Polycomb bodies. ChIP assays showed a significant decrease of H3K27me3 in the genes up-regulated in the Samd7-deficient retina, showing that Samd7 deficiency causes the derepression of nonrod gene expression in rod photoreceptor cells. The current study suggests that Samd7 is a cell type-specific PRC1 component epigenetically defining rod photoreceptor cell identity. PMID:28900001
Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity.

PubMed

Omori, Yoshihiro; Kubo, Shun; Kon, Tetsuo; Furuhashi, Mayu; Narita, Hirotaka; Kominami, Taro; Ueno, Akiko; Tsutsumi, Ryotaro; Chaya, Taro; Yamamoto, Haruka; Suetake, Isao; Ueno, Shinji; Koseki, Haruhiko; Nakagawa, Atsushi; Furukawa, Takahisa

2017-09-26

Precise transcriptional regulation controlled by a transcription factor network is known to be crucial for establishing correct neuronal cell identities and functions in the CNS. In the retina, the expression of various cone and rod photoreceptor cell genes is regulated by multiple transcription factors; however, the role of epigenetic regulation in photoreceptor cell gene expression has been poorly understood. Here, we found that Samd7, a rod-enriched sterile alpha domain (SAM) domain protein, is essential for silencing nonrod gene expression through H3K27me3 regulation in rod photoreceptor cells. Samd7- null mutant mice showed ectopic expression of nonrod genes including S-opsin in rod photoreceptor cells and rod photoreceptor cell dysfunction. Samd7 physically interacts with Polyhomeotic homologs (Phc proteins), components of the Polycomb repressive complex 1 (PRC1), and colocalizes with Phc2 and Ring1B in Polycomb bodies. ChIP assays showed a significant decrease of H3K27me3 in the genes up-regulated in the Samd7 -deficient retina, showing that Samd7 deficiency causes the derepression of nonrod gene expression in rod photoreceptor cells. The current study suggests that Samd7 is a cell type-specific PRC1 component epigenetically defining rod photoreceptor cell identity.
Biogeographic and phylogenetic diversity of thermoacidophilic cyanidiales in Yellowstone National Park, Japan, and New Zealand.

PubMed

Toplin, J A; Norris, T B; Lehr, C R; McDermott, T R; Castenholz, R W

2008-05-01

Members of the rhodophytan order Cyanidiales are unique among phototrophs in their ability to live in extreme environments that combine low pH levels ( approximately 0.2 to 4.0) and moderately high temperatures of 40 to 56 degrees C. These unicellular algae occur in far-flung volcanic areas throughout the earth. Three genera (Cyanidium, Galdieria, and Cyanidioschyzon) are recognized. The phylogenetic diversity of culture isolates of the Cyanidiales from habitats throughout Yellowstone National Park (YNP), three areas in Japan, and seven regions in New Zealand was examined by using the chloroplast RuBisCO large subunit gene (rbcL) and the 18S rRNA gene. Based on the nucleotide sequences of both genes, the YNP isolates fall into two groups, one with high identity to Galdieria sulphuraria (type II) and another that is by far the most common and extensively distributed Yellowstone type (type IA). The latter is a spherical, walled cell that reproduces by internal divisions, with a subsequent release of smaller daughter cells. This type, nevertheless, shows a 99 to 100% identity to Cyanidioschyzon merolae (type IB), which lacks a wall, divides by "fission"-like cytokinesis into two daughter cells, and has less than 5% of the cell volume of type IA. The evolutionary and taxonomic ramifications of this disparity are discussed. Although the 18S rRNA and rbcL genes did not reveal diversity among the numerous isolates of type IA, chloroplast short sequence repeats did show some variation by location within YNP. In contrast, Japanese and New Zealand strains showed considerable diversity when we examined only the sequences of 18S and rbcL genes. Most exhibited identities closer to Galdieria maxima than to other strains, but these identities were commonly as low as 91 to 93%. Some of these Japanese and New Zealand strains probably represent undescribed species that diverged after long-term geographic isolation.
Ontology based molecular signatures for immune cell types via gene expression analysis

PubMed Central

2013-01-01

Background New technologies are focusing on characterizing cell types to better understand their heterogeneity. With large volumes of cellular data being generated, innovative methods are needed to structure the resulting data analyses. Here, we describe an ‘Ontologically BAsed Molecular Signature’ (OBAMS) method that identifies novel cellular biomarkers and infers biological functions as characteristics of particular cell types. This method finds molecular signatures for immune cell types based on mapping biological samples to the Cell Ontology (CL) and navigating the space of all possible pairwise comparisons between cell types to find genes whose expression is core to a particular cell type’s identity. Results We illustrate this ontological approach by evaluating expression data available from the Immunological Genome project (IGP) to identify unique biomarkers of mature B cell subtypes. We find that using OBAMS, candidate biomarkers can be identified at every strata of cellular identity from broad classifications to very granular. Furthermore, we show that Gene Ontology can be used to cluster cell types by shared biological processes in order to find candidate genes responsible for somatic hypermutation in germinal center B cells. Moreover, through in silico experiments based on this approach, we have identified genes sets that represent genes overexpressed in germinal center B cells and identify genes uniquely expressed in these B cells compared to other B cell types. Conclusions This work demonstrates the utility of incorporating structured ontological knowledge into biological data analysis – providing a new method for defining novel biomarkers and providing an opportunity for new biological insights. PMID:24004649
Transcription factor Etv5 is essential for the maintenance of alveolar type II cells.

PubMed

Zhang, Zhen; Newton, Kim; Kummerfeld, Sarah K; Webster, Joshua; Kirkpatrick, Donald S; Phu, Lilian; Eastham-Anderson, Jeffrey; Liu, Jinfeng; Lee, Wyne P; Wu, Jiansheng; Li, Hong; Junttila, Melissa R; Dixit, Vishva M

2017-04-11

Alveolar type II (AT2) cell dysfunction contributes to a number of significant human pathologies including respiratory distress syndrome, lung adenocarcinoma, and debilitating fibrotic diseases, but the critical transcription factors that maintain AT2 cell identity are unknown. Here we show that the E26 transformation-specific (ETS) family transcription factor Etv5 is essential to maintain AT2 cell identity. Deletion of Etv5 from AT2 cells produced gene and protein signatures characteristic of differentiated alveolar type I (AT1) cells. Consistent with a defect in the AT2 stem cell population, Etv5 deficiency markedly reduced recovery following bleomycin-induced lung injury. Lung tumorigenesis driven by mutant KrasG12D was also compromised by Etv5 deficiency. ERK activation downstream of Ras was found to stabilize Etv5 through inactivation of the cullin-RING ubiquitin ligase CRL4 COP1/DET1 that targets Etv5 for proteasomal degradation. These findings identify Etv5 as a critical output of Ras signaling in AT2 cells, contributing to both lung homeostasis and tumor initiation.
SEA: a super-enhancer archive.

PubMed

Wei, Yanjun; Zhang, Shumei; Shang, Shipeng; Zhang, Bin; Li, Song; Wang, Xinyu; Wang, Fang; Su, Jianzhong; Wu, Qiong; Liu, Hongbo; Zhang, Yan

2016-01-04

Super-enhancers are large clusters of transcriptional enhancers regarded as having essential roles in driving the expression of genes that control cell identity during development and tumorigenesis. The construction of a genome-wide super-enhancer database is urgently needed to better understand super-enhancer-directed gene expression regulation for a given biology process. Here, we present a specifically designed web-accessible database, Super-Enhancer Archive (SEA, http://sea.edbc.org). SEA focuses on integrating super-enhancers in multiple species and annotating their potential roles in the regulation of cell identity gene expression. The current release of SEA incorporates 83 996 super-enhancers computationally or experimentally identified in 134 cell types/tissues/diseases, including human (75 439, three of which were experimentally identified), mouse (5879, five of which were experimentally identified), Drosophila melanogaster (1774) and Caenorhabditis elegans (904). To facilitate data extraction, SEA supports multiple search options, including species, genome location, gene name, cell type/tissue and super-enhancer name. The response provides detailed (epi)genetic information, incorporating cell type specificity, nearby genes, transcriptional factor binding sites, CRISPR/Cas9 target sites, evolutionary conservation, SNPs, H3K27ac, DNA methylation, gene expression and TF ChIP-seq data. Moreover, analytical tools and a genome browser were developed for users to explore super-enhancers and their roles in defining cell identity and disease processes in depth. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
Cells of origin in the embryonic nerve roots for NF1-associated plexiform neurofibroma

PubMed Central

Chen, Zhiguo; Liu, Chiachi; Patel, Amish J.; Liao, Chung-Ping; Wang, Yong; Le, Lu Q.

2014-01-01

Summary Neurofibromatosis type 1 is a tumor-predisposing genetic disorder. Plexiform neurofibromas are common NF1 tumors carrying a risk of malignant transformation, which is typically fatal. Little is known about mechanisms mediating initiation and identity of specific cell-type that gives rise to neurofibromas. Using cell-lineage tracing, we identify a population of GAP43+ PLP+ precursors in embryonic nerve roots as the cells of origin for these tumors and report a non-germline model of neurofibroma for preclinical drug screening to identify effective therapies. The identity of tumor cell-of-origin and facility for isolation and expansion provides fertile ground for continued analysis to define intrinsic and extrinsic factors critical for neurofibromagenesis. It also provides unique approaches to develop therapies to prevent neurofibroma formation in NF1 patients. PMID:25446898
Specifying pancreatic endocrine cell fates.

PubMed

Collombat, Patrick; Hecksher-Sørensen, Jacob; Serup, Palle; Mansouri, Ahmed

2006-07-01

Cell replacement therapy could represent an attractive alternative to insulin injections for the treatment of diabetes. However, this approach requires a thorough understanding of the molecular switches controlling the specification of the different pancreatic cell-types in vivo. These are derived from an apparently identical pool of cells originating from the early gut endoderm, which are successively specified towards the pancreatic, endocrine, and hormone-expressing cell lineages. Numerous studies have outlined the crucial roles exerted by transcription factors in promoting the cell destiny, defining the cell identity and maintaining a particular cell fate. This review focuses on the mechanisms regulating the morphogenesis of the pancreas with particular emphasis on recent findings concerning the transcription factor hierarchy orchestrating endocrine cell fate allocation.
Loss of Trem2 in microglia leads to widespread disruption of cell coexpression networks in mouse brain.

PubMed

Carbajosa, Guillermo; Malki, Karim; Lawless, Nathan; Wang, Hong; Ryder, John W; Wozniak, Eva; Wood, Kristie; Mein, Charles A; Dobson, Richard J B; Collier, David A; O'Neill, Michael J; Hodges, Angela K; Newhouse, Stephen J

2018-05-17

Rare heterozygous coding variants in the triggering receptor expressed in myeloid cells 2 (TREM2) gene, conferring increased risk of developing late-onset Alzheimer's disease, have been identified. We examined the transcriptional consequences of the loss of Trem2 in mouse brain to better understand its role in disease using differential expression and coexpression network analysis of Trem2 knockout and wild-type mice. We generated RNA-Seq data from cortex and hippocampus sampled at 4 and 8 months. Using brain cell-type markers and ontology enrichment, we found subnetworks with cell type and/or functional identity. We primarily discovered changes in an endothelial gene-enriched subnetwork at 4 months, including a shift toward a more central role for the amyloid precursor protein gene, coupled with widespread disruption of other cell-type subnetworks, including a subnetwork with neuronal identity. We reveal an unexpected potential role of Trem2 in the homeostasis of endothelial cells that goes beyond its known functions as a microglial receptor and signaling hub, suggesting an underlying link between immune response and vascular disease in dementia. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
Investigating Evolutionary Conservation of Dendritic Cell Subset Identity and Functions

PubMed Central

Vu Manh, Thien-Phong; Bertho, Nicolas; Hosmalin, Anne; Schwartz-Cornil, Isabelle; Dalod, Marc

2015-01-01

Dendritic cells (DCs) were initially defined as mononuclear phagocytes with a dendritic morphology and an exquisite efficiency for naïve T-cell activation. DC encompass several subsets initially identified by their expression of specific cell surface molecules and later shown to excel in distinct functions and to develop under the instruction of different transcription factors or cytokines. Very few cell surface molecules are expressed in a specific manner on any immune cell type. Hence, to identify cell types, the sole use of a small number of cell surface markers in classical flow cytometry can be deceiving. Moreover, the markers currently used to define mononuclear phagocyte subsets vary depending on the tissue and animal species studied and even between laboratories. This has led to confusion in the definition of DC subset identity and in their attribution of specific functions. There is a strong need to identify a rigorous and consensus way to define mononuclear phagocyte subsets, with precise guidelines potentially applicable throughout tissues and species. We will discuss the advantages, drawbacks, and complementarities of different methodologies: cell surface phenotyping, ontogeny, functional characterization, and molecular profiling. We will advocate that gene expression profiling is a very rigorous, largely unbiased and accessible method to define the identity of mononuclear phagocyte subsets, which strengthens and refines surface phenotyping. It is uniquely powerful to yield new, experimentally testable, hypotheses on the ontogeny or functions of mononuclear phagocyte subsets, their molecular regulation, and their evolutionary conservation. We propose defining cell populations based on a combination of cell surface phenotyping, expression analysis of hallmark genes, and robust functional assays, in order to reach a consensus and integrate faster the huge but scattered knowledge accumulated by different laboratories on different cell types, organs, and species. PMID:26082777
Brain tumor specifies intermediate progenitor cell identity by attenuating β-catenin/Armadillo activity

PubMed Central

Komori, Hideyuki; Xiao, Qi; McCartney, Brooke M.; Lee, Cheng-Yu

2014-01-01

During asymmetric stem cell division, both the daughter stem cell and the presumptive intermediate progenitor cell inherit cytoplasm from their parental stem cell. Thus, proper specification of intermediate progenitor cell identity requires an efficient mechanism to rapidly extinguish the activity of self-renewal factors, but the mechanisms remain unknown in most stem cell lineages. During asymmetric division of a type II neural stem cell (neuroblast) in the Drosophila larval brain, the Brain tumor (Brat) protein segregates unequally into the immature intermediate neural progenitor (INP), where it specifies INP identity by attenuating the function of the self-renewal factor Klumpfuss (Klu), but the mechanisms are not understood. Here, we report that Brat specifies INP identity through its N-terminal B-boxes via a novel mechanism that is independent of asymmetric protein segregation. Brat-mediated specification of INP identity is critically dependent on the function of the Wnt destruction complex, which attenuates the activity of β-catenin/Armadillo (Arm) in immature INPs. Aberrantly increasing Arm activity in immature INPs further exacerbates the defects in the specification of INP identity and enhances the supernumerary neuroblast mutant phenotype in brat mutant brains. By contrast, reducing Arm activity in immature INPs suppresses supernumerary neuroblast formation in brat mutant brains. Finally, reducing Arm activity also strongly suppresses supernumerary neuroblasts induced by overexpression of klu. Thus, the Brat-dependent mechanism extinguishes the function of the self-renewal factor Klu in the presumptive intermediate progenitor cell by attenuating Arm activity, balancing stem cell maintenance and progenitor cell specification. PMID:24257623
Cell-assembly coding in several memory processes.

PubMed

Sakurai, Y

1998-01-01

The present paper discusses why the cell assembly, i.e., an ensemble population of neurons with flexible functional connections, is a tenable view of the basic code for information processes in the brain. The main properties indicating the reality of cell-assembly coding are neurons overlaps among different assemblies and connection dynamics within and among the assemblies. The former can be detected as multiple functions of individual neurons in processing different kinds of information. Individual neurons appear to be involved in multiple information processes. The latter can be detected as changes of functional synaptic connections in processing different kinds of information. Correlations of activity among some of the recorded neurons appear to change in multiple information processes. Recent experiments have compared several different memory processes (tasks) and detected these two main properties, indicating cell-assembly coding of memory in the working brain. The first experiment compared different types of processing of identical stimuli, i.e., working memory and reference memory of auditory stimuli. The second experiment compared identical processes of different types of stimuli, i.e., discriminations of simple auditory, simple visual, and configural auditory-visual stimuli. The third experiment compared identical processes of different types of stimuli with or without temporal processing of stimuli, i.e., discriminations of elemental auditory, configural auditory-visual, and sequential auditory-visual stimuli. Some possible features of the cell-assembly coding, especially "dual coding" by individual neurons and cell assemblies, are discussed for future experimental approaches. Copyright 1998 Academic Press.
Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity

PubMed Central

Mazzoni, Esteban O; Mahony, Shaun; Closser, Michael; Morrison, Carolyn A; Nedelec, Stephane; Williams, Damian J; An, Disi; Gifford, David K; Wichterle, Hynek

2013-01-01

Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation–sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type–specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types. PMID:23872598
H3K4me3 breadth is linked to cell identity and transcriptional consistency.

PubMed

Benayoun, Bérénice A; Pollina, Elizabeth A; Ucar, Duygu; Mahmoudi, Salah; Karra, Kalpana; Wong, Edith D; Devarajan, Keerthana; Daugherty, Aaron C; Kundaje, Anshul B; Mancini, Elena; Hitz, Benjamin C; Gupta, Rakhi; Rando, Thomas A; Baker, Julie C; Snyder, Michael P; Cherry, J Michael; Brunet, Anne

2014-07-31

Trimethylation of histone H3 at lysine 4 (H3K4me3) is a chromatin modification known to mark the transcription start sites of active genes. Here, we show that H3K4me3 domains that spread more broadly over genes in a given cell type preferentially mark genes that are essential for the identity and function of that cell type. Using the broadest H3K4me3 domains as a discovery tool in neural progenitor cells, we identify novel regulators of these cells. Machine learning models reveal that the broadest H3K4me3 domains represent a distinct entity, characterized by increased marks of elongation. The broadest H3K4me3 domains also have more paused polymerase at their promoters, suggesting a unique transcriptional output. Indeed, genes marked by the broadest H3K4me3 domains exhibit enhanced transcriptional consistency and [corrected] increased transcriptional levels, and perturbation of H3K4me3 breadth leads to changes in transcriptional consistency. Thus, H3K4me3 breadth contains information that could ensure transcriptional precision at key cell identity/function genes. Copyright © 2014 Elsevier Inc. All rights reserved.
Origin and specification of type II neuroblasts in the Drosophila embryo.

PubMed

Álvarez, José-Andrés; Díaz-Benjumea, Fernando J

2018-04-05

In Drosophila , neural stem cells or neuroblasts (NBs) acquire different identities according to their site of origin in the embryonic neuroectoderm. Their identity determines the number of times they will divide and the types of daughter cells they will generate. All NBs divide asymmetrically, with type I NBs undergoing self-renewal and generating another cell that will divide only once more. By contrast, a small set of NBs in the larval brain, type II NBs, divides differently, undergoing self-renewal and generating an intermediate neural progenitor (INP) that continues to divide asymmetrically several more times, generating larger lineages. In this study, we have analysed the origin of type II NBs and how they are specified. Our results indicate that these cells originate in three distinct clusters in the dorsal protocerebrum during stage 12 of embryonic development. Moreover, it appears that their specification requires the combined action of EGFR signalling and the activity of the related genes buttonhead and Drosophila Sp1 In addition, we also show that the INPs generated in the embryo enter quiescence at the end of embryogenesis, resuming proliferation during the larval stage. © 2018. Published by The Company of Biologists Ltd.
Development of technologies for welding interconnects to fifty-micron thick silicon solar cells

NASA Technical Reports Server (NTRS)

Patterson, R. E.

1982-01-01

A program was conducted to develop technologies for welding interconnects to 50 microns thick, 2 by 2 cm solar cells. The cells were characterized with respect to electrical performance, cell thickness, silver contact thickness, contact waviness, bowing, and fracture strength. Weld schedules were independently developed for each of the three cell types and were coincidentally identical. Thermal shock tests (100 cycles from 100 C to -180 C) were performed on 16 cell coupons for each cell type without any weld joint failures or electrical degradation. Three 48 cell modules (one for each cell type) were assembled with 50 microns thick cells, frosted fused silica covers, silver clad Invar interconnectors, and Kapton substrates.
Welding interconnects to 50-micron silicon solar cells

NASA Technical Reports Server (NTRS)

Patterson, R. E.; Mesch, H. G.

1983-01-01

A program was conducted to develop technologies for welding interconnects to 50-micron thick, 2 by 2 cm solar cells obtained from three suppliers. The cells were characterized with respect to electrical performance, cell thickness, silver contact thickness, contact waviness, bowing, and fracture strength. Weld schedules were independently developed for each of the three cell types and were coincidentally identical. Thermal shock tests (100 cycles from 100 deg to -180 deg C) were performed on 16-cell coupons for each cell type without any weld joint failures or electrical degradation. Three 48-cell modules (one for each cell type) were assembled with 50-micron thick cells, frosted fused silica covers, silver clad Invar interconnectors, and Kapton substrates.
Label-free haemogram using wavelength modulated Raman spectroscopy for identifying immune-cell subset

NASA Astrophysics Data System (ADS)

Ashok, Praveen C.; Praveen, Bavishna B.; Campbell, Elaine C.; Dholakia, Kishan; Powis, Simon J.

2014-03-01

Leucocytes in the blood of mammals form a powerful protective system against a wide range of dangerous pathogens. There are several types of immune cells that has specific role in the whole immune system. The number and type of immune cells alter in the disease state and identifying the type of immune cell provides information about a person's state of health. There are several immune cell subsets that are essentially morphologically identical and require external labeling to enable discrimination. Here we demonstrate the feasibility of using Wavelength Modulated Raman Spectroscopy (WMRS) with suitable machine learning algorithms as a label-free method to distinguish between different closely lying immune cell subset. Principal Component Analysis (PCA) was performed on WMRS data from single cells, obtained using confocal Raman microscopy for feature reduction, followed by Support Vector Machine (SVM) for binary discrimination of various cell subset, which yielded an accuracy >85%. The method was successful in discriminating between untouched and unfixed purified populations of CD4+CD3+ and CD8+CD3+ T lymphocyte subsets, and CD56+CD3- natural killer cells with a high degree of specificity. It was also proved sensitive enough to identify unique Raman signatures that allow clear discrimination between dendritic cell subsets, comprising CD303+CD45+ plasmacytoid and CD1c+CD141+ myeloid dendritic cells. The results of this study clearly show that WMRS is highly sensitive and can distinguish between cell types that are morphologically identical.
The LIM and POU homeobox genes ttx-3 and unc-86 act as terminal selectors in distinct cholinergic and serotonergic neuron types.

PubMed

Zhang, Feifan; Bhattacharya, Abhishek; Nelson, Jessica C; Abe, Namiko; Gordon, Patricia; Lloret-Fernandez, Carla; Maicas, Miren; Flames, Nuria; Mann, Richard S; Colón-Ramos, Daniel A; Hobert, Oliver

2014-01-01

Transcription factors that drive neuron type-specific terminal differentiation programs in the developing nervous system are often expressed in several distinct neuronal cell types, but to what extent they have similar or distinct activities in individual neuronal cell types is generally not well explored. We investigate this problem using, as a starting point, the C. elegans LIM homeodomain transcription factor ttx-3, which acts as a terminal selector to drive the terminal differentiation program of the cholinergic AIY interneuron class. Using a panel of different terminal differentiation markers, including neurotransmitter synthesizing enzymes, neurotransmitter receptors and neuropeptides, we show that ttx-3 also controls the terminal differentiation program of two additional, distinct neuron types, namely the cholinergic AIA interneurons and the serotonergic NSM neurons. We show that the type of differentiation program that is controlled by ttx-3 in different neuron types is specified by a distinct set of collaborating transcription factors. One of the collaborating transcription factors is the POU homeobox gene unc-86, which collaborates with ttx-3 to determine the identity of the serotonergic NSM neurons. unc-86 in turn operates independently of ttx-3 in the anterior ganglion where it collaborates with the ARID-type transcription factor cfi-1 to determine the cholinergic identity of the IL2 sensory and URA motor neurons. In conclusion, transcription factors operate as terminal selectors in distinct combinations in different neuron types, defining neuron type-specific identity features.
Reprogramming cellular identity for regenerative medicine

PubMed Central

Cherry, Anne B.C.; Daley, George Q.

2012-01-01

The choreographed development of over 200 distinct differentiated cell types from a single zygote is a complex and poorly understood process. Whereas development leads unidirectionally towards more restricted cell fates, recent work in cellular reprogramming has proven that striking conversions of one cellular identity into another can be engineered, promising countless applications in biomedical research and paving the way for modeling disease with patient-derived stem cells. To date, there has been little discussion of which disease models are likely to be most informative. We here review evidence demonstrating that because environmental influences and epigenetic signatures are largely erased during reprogramming, patient-specific models of diseases with strong genetic bases and high penetrance are likely to prove most informative in the near term. However, manipulating in vitro culture conditions may ultimately enable cell-based models to recapitulate gene-environment interactions. Here, we discuss the implications of the new reprogramming paradigm in biomedicine and outline how reprogramming of cell identities is enhancing our understanding of cell differentiation and prospects for cellular therapies and in vivo regeneration. PMID:22424223

Gene expression profiling of bovine ovarian follicular and luteal cells provides insight into cellular identities and functions

USDA-ARS?s Scientific Manuscript database

After ovulation, somatic cells of the ovarian follicle (theca and granulosa cells) become the small and large luteal cells of the corpus luteum. Aside from known cell type-specific receptors and steroidogenic enzymes, little is known about the differences in the gene expression profiles of these fou...
The coordination of ploidy and cell size differs between cell layers in leaves

PubMed Central

Katagiri, Yohei; Hasegawa, Junko; Fujikura, Ushio; Hoshino, Rina; Matsunaga, Sachihiro; Tsukaya, Hirokazu

2016-01-01

Growth and developmental processes are occasionally accompanied by multiple rounds of DNA replication, known as endoreduplication. Coordination between endoreduplication and cell size regulation often plays a crucial role in proper organogenesis and cell differentiation. Here, we report that the level of correlation between ploidy and cell volume is different in the outer and inner cell layers of leaves of Arabidopsis thaliana using a novel imaging technique. Although there is a well-known, strong correlation between ploidy and cell volume in pavement cells of the epidermis, this correlation was extremely weak in palisade mesophyll cells. Induction of epidermis cell identity based on the expression of the homeobox gene ATML1 in mesophyll cells enhanced the level of correlation between ploidy and cell volume to near that of wild-type epidermal cells. We therefore propose that the correlation between ploidy and cell volume is regulated by cell identity. PMID:26903507
All Tumor Cells Are Not Created Equal | Center for Cancer Research

Cancer.gov

Cell division is commonly thought of as a process whereby one cell gives rise to two identical daughter cells. However, rare cell divisions are asymmetric, generating daughter cells that may differ in size, developmental potential, or even DNA content. The ability of stem cells to undergo asymmetric division allows them to self-renew while simultaneously generate daughter cells committed to differentiating into specialized cell types.
Establishing neural crest identity: a gene regulatory recipe

PubMed Central

Simões-Costa, Marcos; Bronner, Marianne E.

2015-01-01

The neural crest is a stem/progenitor cell population that contributes to a wide variety of derivatives, including sensory and autonomic ganglia, cartilage and bone of the face and pigment cells of the skin. Unique to vertebrate embryos, it has served as an excellent model system for the study of cell behavior and identity owing to its multipotency, motility and ability to form a broad array of cell types. Neural crest development is thought to be controlled by a suite of transcriptional and epigenetic inputs arranged hierarchically in a gene regulatory network. Here, we examine neural crest development from a gene regulatory perspective and discuss how the underlying genetic circuitry results in the features that define this unique cell population. PMID:25564621
Evidence for Loss in Identity, De-Differentiation, and Trans-Differentiation of Islet β-Cells in Type 2 Diabetes

PubMed Central

Hunter, Chad S.; Stein, Roland W.

2017-01-01

The two main types of diabetes mellitus have distinct etiologies, yet a similar outcome: loss of islet β-cell function that is solely responsible for the secretion of the insulin hormone to reduce elevated plasma glucose toward euglycemic levels. Type 1 diabetes (T1D) has traditionally been characterized by autoimmune-mediated β-cell death leading to insulin-dependence, whereas type 2 diabetes (T2D) has hallmarks of peripheral insulin resistance, β-cell dysfunction, and cell death. However, a growing body of evidence suggests that, especially during T2D, key components of β-cell failure involves: (1) loss of cell identity, specifically proteins associated with mature cell function (e.g., insulin and transcription factors like MAFA, PDX1, and NKX6.1), as well as (2) de-differentiation, defined by regression to a progenitor or stem cell-like state. New technologies have allowed the field to compare islet cell characteristics from normal human donors to those under pathophysiological conditions by single cell RNA-Sequencing and through epigenetic analysis. This has revealed a remarkable level of heterogeneity among histologically defined “insulin-positive” β-cells. These results not only suggest that these β-cell subsets have different responses to insulin secretagogues, but that defining their unique gene expression and epigenetic modification profiles will offer opportunities to develop cellular therapeutics to enrich/maintain certain subsets for correcting pathological glucose levels. In this review, we will summarize the recent literature describing how β-cell heterogeneity and plasticity may be influenced in T2D, and various possible avenues of therapeutic intervention. PMID:28424732
Functional Dissection of Sugar Signals Affecting Gene Expression in Arabidopsis thaliana

PubMed Central

Kunz, Sabine; Pesquet, Edouard; Kleczkowski, Leszek A.

2014-01-01

Background Sugars modulate expression of hundreds of genes in plants. Previous studies on sugar signaling, using intact plants or plant tissues, were hampered by tissue heterogeneity, uneven sugar transport and/or inter-conversions of the applied sugars. This, in turn, could obscure the identity of a specific sugar that acts as a signal affecting expression of given gene in a given tissue or cell-type. Methodology/Principal Findings To bypass those biases, we have developed a novel biological system, based on stem-cell-like Arabidopsis suspension culture. The cells were grown in a hormone-free medium and were sustained on xylose as the only carbon source. Using functional genomics we have identified 290 sugar responsive genes, responding rapidly (within 1 h) and specifically to low concentration (1 mM) of glucose, fructose and/or sucrose. For selected genes, the true nature of the signaling sugar molecules and sites of sugar perception were further clarified using non-metabolizable sugar analogues. Using both transgenic and wild-type A. thaliana seedlings, it was shown that the expression of selected sugar-responsive genes was not restricted to a specific tissue or cell type and responded to photoperiod-related changes in sugar availability. This suggested that sugar-responsiveness of genes identified in the cell culture system was not biased toward heterotrophic background and resembled that in whole plants. Conclusions Altogether, our research strategy, using a combination of cell culture and whole plants, has provided an unequivocal evidence for the identity of sugar-responsive genes and the identity of the sugar signaling molecules, independently from their inter-conversions or use for energy metabolism. PMID:24950222
MiRNAs in β-Cell Development, Identity, and Disease

PubMed Central

Martinez-Sanchez, Aida; Rutter, Guy A.; Latreille, Mathieu

2017-01-01

Pancreatic β-cells regulate glucose metabolism by secreting insulin, which in turn stimulates the utilization or storage of the sugar by peripheral tissues. Insulin insufficiency and a prolonged period of insulin resistance are usually the core components of type 2 diabetes (T2D). Although, decreased insulin levels in T2D have long been attributed to a decrease in β-cell function and/or mass, this model has recently been refined with the recognition that a loss of β-cell “identity” and dedifferentiation also contribute to the decline in insulin production. MicroRNAs (miRNAs) are key regulatory molecules that display tissue-specific expression patterns and maintain the differentiated state of somatic cells. During the past few years, great strides have been made in understanding how miRNA circuits impact β-cell identity. Here, we review current knowledge on the role of miRNAs in regulating the acquisition of the β-cell fate during development and in maintaining mature β-cell identity and function during stress situations such as obesity, pregnancy, aging, or diabetes. We also discuss how miRNA function could be harnessed to improve our ability to generate β-cells for replacement therapy for T2D. PMID:28123396
Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells.

PubMed Central

Gauldie, J; Richards, C; Harnish, D; Lansdorp, P; Baumann, H

1987-01-01

One of the oldest and most preserved of the homeostatic responses of the body to injury is the acute phase protein response associated with inflammation. The liver responds to hormone-like mediators by the increased synthesis of a series of plasma proteins called acute phase reactants. In these studies, we examined the relationship of hepatocyte-stimulating factor derived from peripheral blood monocytes to interferon beta 2 (IFN-beta 2), which has been cloned. Antibodies raised against fibroblast-derived IFN-beta having neutralizing activity against both IFN-beta 1 and -beta 2 inhibited the major hepatocyte-stimulating activity derived from monocytes. Fibroblast-derived mediator elicited the identical stimulated response in human HepG2 cells and primary rat hepatocytes as the monocyte cytokine. Finally, recombinant-derived human B-cell stimulatory factor type 2 (IFN-beta 2) from Escherichia coli induced the synthesis of all major acute phase proteins studied in human hepatoma HepG2 and primary rat hepatocyte cultures. These data demonstrate that monocyte-derived hepatocyte-stimulating factor and IFN-beta 2 share immunological and functional identity and that IFN-beta 2, also known as B-cell stimulatory factor and hybridoma plasmacytoma growth factor, has the hepatocyte as a major physiologic target and thereby is essential in controlling the hepatic acute phase response. Images PMID:2444978
rse, a novel receptor-type tyrosine kinase with homology to Axl/Ufo, is expressed at high levels in the brain.

PubMed

Mark, M R; Scadden, D T; Wang, Z; Gu, Q; Goddard, A; Godowski, P J

1994-04-08

We have isolated cDNA clones that encode the human and murine forms of a novel receptor-type tyrosine kinase termed Rse. Sequence analysis indicates that human Rse contains 890 amino acids, with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains. Murine Rse contains 880 amino acids and shares 90% amino acid identity with its human counterpart. Rse is structurally similar to the receptor-type tyrosine kinase Axl/Ufo, and the two proteins have 35 and 63% sequence identity in their extracellular and intracellular domains, respectively. To study the synthesis and activation of this putative receptor-type tyrosine kinase, we constructed a version of Rse (termed gD-Rse, where gD represents glycoprotein D) that contains an NH2-terminal epitope tag. NIH3T3 cells were engineered to express gD-Rse, which could be detected at the cell surface by fluorescence-activated cell sorting. Moreover, gD-Rse was rapidly phosphorylated on tyrosine residues upon incubation of the cells with an antibody directed against the epitope tag, suggesting that rse encodes an active tyrosine kinase. In the human tissues we examined, the highest level of expression of rse mRNA was observed in the brain; rse mRNA was also detected in the premegakaryocytopoietic cell lines CMK11-5 and Dami. The gene for rse was localized to human chromosome 15.
Cell identity regulators link development and stress responses in the Arabidopsis root.

PubMed

Iyer-Pascuzzi, Anjali S; Jackson, Terry; Cui, Hongchang; Petricka, Jalean J; Busch, Wolfgang; Tsukagoshi, Hironaka; Benfey, Philip N

2011-10-18

Stress responses in plants are tightly coordinated with developmental processes, but interaction of these pathways is poorly understood. We used genome-wide assays at high spatiotemporal resolution to understand the processes that link development and stress in the Arabidopsis root. Our meta-analysis finds little evidence for a universal stress response. However, common stress responses appear to exist with many showing cell type specificity. Common stress responses may be mediated by cell identity regulators because mutations in these genes resulted in altered responses to stress. Evidence for a direct role for cell identity regulators came from genome-wide binding profiling of the key regulator SCARECROW, which showed binding to regulatory regions of stress-responsive genes. Coexpression in response to stress was used to identify genes involved in specific developmental processes. These results reveal surprising linkages between stress and development at cellular resolution, and show the power of multiple genome-wide data sets to elucidate biological processes. Copyright © 2011 Elsevier Inc. All rights reserved.
The Self-Identity Protein IdsD Is Communicated between Cells in Swarming Proteus mirabilis Colonies.

PubMed

Saak, Christina C; Gibbs, Karine A

2016-12-15

Proteus mirabilis is a social bacterium that is capable of self (kin) versus nonself recognition. Swarming colonies of this bacterium expand outward on surfaces to centimeter-scale distances due to the collective motility of individual cells. Colonies of genetically distinct populations remain separate, while those of identical populations merge. Ids proteins are essential for this recognition behavior. Two of these proteins, IdsD and IdsE, encode identity information for each strain. These two proteins bind in vitro in an allele-restrictive manner. IdsD-IdsE binding is correlated with the merging of populations, whereas a lack of binding is correlated with the separation of populations. Key questions remained about the in vivo interactions of IdsD and IdsE, specifically, whether IdsD and IdsE bind within single cells or whether IdsD-IdsE interactions occur across neighboring cells and, if so, which of the two proteins is exchanged. Here we demonstrate that IdsD must originate from another cell to communicate identity and that this nonresident IdsD interacts with IdsE resident in the recipient cell. Furthermore, we show that unbound IdsD in recipient cells does not cause cell death and instead appears to contribute to a restriction in the expansion radius of the swarming colony. We conclude that P. mirabilis communicates IdsD between neighboring cells for nonlethal kin recognition, which suggests that the Ids proteins constitute a type of cell-cell communication. We demonstrate that self (kin) versus nonself recognition in P. mirabilis entails the cell-cell communication of an identity-encoding protein that is exported from one cell and received by another. We further show that this intercellular exchange affects swarm colony expansion in a nonlethal manner, which adds social communication to the list of potential swarm-related regulatory factors. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
The Self-Identity Protein IdsD Is Communicated between Cells in Swarming Proteus mirabilis Colonies

PubMed Central

Saak, Christina C.

2016-01-01

ABSTRACT Proteus mirabilis is a social bacterium that is capable of self (kin) versus nonself recognition. Swarming colonies of this bacterium expand outward on surfaces to centimeter-scale distances due to the collective motility of individual cells. Colonies of genetically distinct populations remain separate, while those of identical populations merge. Ids proteins are essential for this recognition behavior. Two of these proteins, IdsD and IdsE, encode identity information for each strain. These two proteins bind in vitro in an allele-restrictive manner. IdsD-IdsE binding is correlated with the merging of populations, whereas a lack of binding is correlated with the separation of populations. Key questions remained about the in vivo interactions of IdsD and IdsE, specifically, whether IdsD and IdsE bind within single cells or whether IdsD-IdsE interactions occur across neighboring cells and, if so, which of the two proteins is exchanged. Here we demonstrate that IdsD must originate from another cell to communicate identity and that this nonresident IdsD interacts with IdsE resident in the recipient cell. Furthermore, we show that unbound IdsD in recipient cells does not cause cell death and instead appears to contribute to a restriction in the expansion radius of the swarming colony. We conclude that P. mirabilis communicates IdsD between neighboring cells for nonlethal kin recognition, which suggests that the Ids proteins constitute a type of cell-cell communication. IMPORTANCE We demonstrate that self (kin) versus nonself recognition in P. mirabilis entails the cell-cell communication of an identity-encoding protein that is exported from one cell and received by another. We further show that this intercellular exchange affects swarm colony expansion in a nonlethal manner, which adds social communication to the list of potential swarm-related regulatory factors. PMID:27672195
The coordination of ploidy and cell size differs between cell layers in leaves.

PubMed

Katagiri, Yohei; Hasegawa, Junko; Fujikura, Ushio; Hoshino, Rina; Matsunaga, Sachihiro; Tsukaya, Hirokazu

2016-04-01

Growth and developmental processes are occasionally accompanied by multiple rounds of DNA replication, known as endoreduplication. Coordination between endoreduplication and cell size regulation often plays a crucial role in proper organogenesis and cell differentiation. Here, we report that the level of correlation between ploidy and cell volume is different in the outer and inner cell layers of leaves of Arabidopsis thaliana using a novel imaging technique. Although there is a well-known, strong correlation between ploidy and cell volume in pavement cells of the epidermis, this correlation was extremely weak in palisade mesophyll cells. Induction of epidermis cell identity based on the expression of the homeobox gene ATML1 in mesophyll cells enhanced the level of correlation between ploidy and cell volume to near that of wild-type epidermal cells. We therefore propose that the correlation between ploidy and cell volume is regulated by cell identity. © 2016. Published by The Company of Biologists Ltd.
Molecular Genetic Evidence for a Common Clonal Origin of Urinary Bladder Small Cell Carcinoma and Coexisting Urothelial Carcinoma

PubMed Central

Cheng, Liang; Jones, Timothy D.; McCarthy, Ryan P.; Eble, John N.; Wang, Mingsheng; MacLennan, Gregory T.; Lopez-Beltran, Antonio; Yang, Ximing J.; Koch, Michael O.; Zhang, Shaobo; Pan, Chong-Xian; Baldridge, Lee Ann

2005-01-01

In most cases, small-cell carcinoma of the urinary bladder is admixed with other histological types of bladder carcinoma. To understand the pathogenetic relationship between the two tumor types, we analyzed histologically distinct tumor cell populations from the same patient for loss of heterozygosity (LOH) and X chromosome inactivation (in female patients). We examined five polymorphic microsatellite markers located on chromosome 3p25-26 (D3S3050), chromosome 9p21 (IFNA and D9S171), chromosome 9q32-33 (D9S177), and chromosome 17p13 (TP53) in 20 patients with small-cell carcinoma of the urinary bladder and concurrent urothelial carcinoma. DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser-assisted microdissection. A nearly identical pattern of allelic loss was observed in the two tumor types in all cases, with an overall frequency of allelic loss of 90% (18 of 20 cases). Three patients showed different allelic loss patterns in the two tumor types at a single locus; however, the LOH patterns at the remaining loci were identical. Similarly, the same pattern of nonrandom X chromosome inactivation was present in both carcinoma components in the four cases analyzed. Concordant genetic alterations and X chromosome inactivation between small-cell carcinoma and coexisting urothelial carcinoma suggest that both tumor components originate from the same cells in the urothelium. PMID:15855652
Nucleotide sequence of wild-type hepatitis A virus GBM in comparison with two cell culture-adapted variants.

PubMed Central

Graff, J; Normann, A; Feinstone, S M; Flehmig, B

1994-01-01

In order to study cell tropism and attenuation of hepatitis A virus (HAV), the genome of HAV wild-type GBM and two cell culture-adapted variants, GBM/FRhK and GBM/HFS, were cloned and sequenced after amplification by reverse transcriptase-PCR. During virus cultivation, the HAV variant GBM/FRhK had a strict host range for FRhK-4 cells, in contrast to GBM/HFS, which can be grown in HFS and FRhK-4 cells. The HAV variant GBM/HFS was shown to be attenuated when inoculated into chimpanzees (B. Flehmig, R. F. Mauler, G. Noll, E. Weinmann, and J. P. Gregerson, p. 87-90, in A. Zuckerman, ed., Viral Hepatitis and Liver Disease, 1988). On the basis of this biological background, the comparison of the nucleotide sequences of these three HAV GBM variants should elucidate differences which may be of importance for cell tropism and attenuation. The comparison of the genome between the GBM wild type and HAV wild types HM175 (J. I. Cohen, J. R. Ticehurst, R. H. Purcell, A. Buckler-White, and B. M. Baroudy, J. Virol. 61:50-59, 1987) and HAV-LA (R. Najarian, O. Caput, W. Gee, S. J. Potter, A. Renard, J. Merryweather, G. Van Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985) showed a 92 to 96.3% identity, whereas the identity was 99.3 to 99.6% between the GBM variants. Nucleotide differences between the wild-type and the cell culture-adapted variants, which were identical in both cell culture-adapted GBM variants, were localized in the 5' noncoding region; in 2B, 3B, and 3D; and in the 3' noncoding region. Our result concerning the 2B/2C region confirms a mutation at position 3889 (C-->T, alanine to valine), which had been shown to be of importance for cell culture adaptation (S. U. Emerson, C. McRill, B. Rosenblum, S. M. Feinstone, and R. H. Purcell, J. Virol. 65:4882-4886, 1991; S. U. Emerson, Y. K. Huang, C. McRill, M. Lewis, and R. H. Purcell, J. Virol. 66:650-654, 1992), whereas other mutations differ from published HAV sequence data and may be cell specific. Further comparison of the two cell culture-adapted GBM variants showed cell-specific mutations resulting in deletions of six amino acids in the VP1 region and three amino acids in the 3A region of the GBM variant GBM/FRhK. PMID:8254770
Neurogenin 3 is essential for the proper specification of gastric enteroendocrine cells and the maintenance of gastric epithelial cell identity

PubMed Central

Lee, Catherine S.; Perreault, Nathalie; Brestelli, John E.; Kaestner, Klaus H.

2002-01-01

The notch signaling pathway is essential for the endocrine cell fate in various tissues including the enteroendocrine system of the gastrointestinal tract. Enteroendocrine cells are one of the four major cell types found in the gastric epithelium of the glandular stomach. To understand the molecular basis of enteroendocrine cell development, we have used gene targeting in mouse embryonic stem cells to derive an EGFP-marked null allele of the bHLH transcription factor, neurogenin 3 (ngn3). In ngn3−/− mice, glucagon secreting A-cells, somatostatin secreting D-cells, and gastrin secreting G-cells are absent from the epithelium of the glandular stomach, whereas the number of serotonin-expressing enterochromaffin (EC) cells is decreased dramatically. In addition, ngn3−/− mice display intestinal metaplasia of the gastric epithelium. Thus, ngn3 is required for the differentiation of enteroendocrine cells in the stomach and the maintenance of gastric epithelial cell identity. PMID:12080087
Fluctuations of the transcription factor ATML1 generate the pattern of giant cells in the Arabidopsis sepal

PubMed Central

Meyer, Heather M; Teles, José; Formosa-Jordan, Pau; Refahi, Yassin; San-Bento, Rita; Ingram, Gwyneth; Jönsson, Henrik; Locke, James C W; Roeder, Adrienne H K

2017-01-01

Multicellular development produces patterns of specialized cell types. Yet, it is often unclear how individual cells within a field of identical cells initiate the patterning process. Using live imaging, quantitative image analyses and modeling, we show that during Arabidopsis thaliana sepal development, fluctuations in the concentration of the transcription factor ATML1 pattern a field of identical epidermal cells to differentiate into giant cells interspersed between smaller cells. We find that ATML1 is expressed in all epidermal cells. However, its level fluctuates in each of these cells. If ATML1 levels surpass a threshold during the G2 phase of the cell cycle, the cell will likely enter a state of endoreduplication and become giant. Otherwise, the cell divides. Our results demonstrate a fluctuation-driven patterning mechanism for how cell fate decisions can be initiated through a random yet tightly regulated process. DOI: http://dx.doi.org/10.7554/eLife.19131.001 PMID:28145865
Producing primate embryonic stem cells by somatic cell nuclear transfer.

PubMed

Byrne, J A; Pedersen, D A; Clepper, L L; Nelson, M; Sanger, W G; Gokhale, S; Wolf, D P; Mitalipov, S M

2007-11-22

Derivation of embryonic stem (ES) cells genetically identical to a patient by somatic cell nuclear transfer (SCNT) holds the potential to cure or alleviate the symptoms of many degenerative diseases while circumventing concerns regarding rejection by the host immune system. However, the concept has only been achieved in the mouse, whereas inefficient reprogramming and poor embryonic development characterizes the results obtained in primates. Here, we used a modified SCNT approach to produce rhesus macaque blastocysts from adult skin fibroblasts, and successfully isolated two ES cell lines from these embryos. DNA analysis confirmed that nuclear DNA was identical to donor somatic cells and that mitochondrial DNA originated from oocytes. Both cell lines exhibited normal ES cell morphology, expressed key stem-cell markers, were transcriptionally similar to control ES cells and differentiated into multiple cell types in vitro and in vivo. Our results represent successful nuclear reprogramming of adult somatic cells into pluripotent ES cells and demonstrate proof-of-concept for therapeutic cloning in primates.
A-type potassium currents in smooth muscle.

PubMed

Amberg, Gregory C; Koh, Sang Don; Imaizumi, Yuji; Ohya, Susumu; Sanders, Kenton M

2003-03-01

A-type currents are voltage-gated, calcium-independent potassium (Kv) currents that undergo rapid activation and inactivation. Commonly associated with neuronal and cardiac cell-types, A-type currents have also been identified and characterized in vascular, genitourinary, and gastrointestinal smooth muscle cells. This review examines the molecular identity, biophysical properties, pharmacology, regulation, and physiological function of smooth muscle A-type currents. In general, this review is intended to facilitate the comparison of A-type currents present in different smooth muscles by providing a comprehensive report of the literature to date. This approach should also aid in the identification of areas of research requiring further attention.
Positive and Negative Regulation of Muscle Cell Identity by Members of the hedgehog and TGF-β Gene Families

PubMed Central

Du, Shao Jun; Devoto, Stephen H.; Westerfield, Monte; Moon, Randall T.

1997-01-01

We have examined whether the development of embryonic muscle fiber type is regulated by competing influences between Hedgehog and TGF-β signals, as previously shown for development of neuronal cell identity in the neural tube. We found that ectopic expression of Hedgehogs or inhibition of protein kinase A in zebrafish embryos induces slow muscle precursors throughout the somite but muscle pioneer cells only in the middle of the somite. Ectopic expression in the notochord of Dorsalin-1, a member of the TGF-β superfamily, inhibits the formation of muscle pioneer cells, demonstrating that TGF-β signals can antagonize the induction of muscle pioneer cells by Hedgehog. We propose that a Hedgehog signal first induces the formation of slow muscle precursor cells, and subsequent Hedgehog and TGF-β signals exert competing positive and negative influences on the development of muscle pioneer cells. PMID:9314535

Advances in hepatic stem/progenitor cell biology

PubMed Central

Verhulst, Stefaan; Best, Jan; van Grunsven, Leo A.; Dollé, Laurent

2015-01-01

The liver is famous for its strong regenerative capacity, employing different modes of regeneration according to type and extent of injury. Mature liver cells are able to proliferate in order to replace the damaged tissue allowing the recovery of the parenchymal function. In more severe scenarios hepatocytes are believed to arise also from a facultative liver progenitor cell compartment. In human, severe acute liver failure and liver cirrhosis are also both important clinical targets in which regeneration is impaired, where the role of this stem cell compartment seems more convincing. In animal models, the current state of ambiguity regarding the identity and role of liver progenitor cells in liver physiology dampens the enthusiasm for the potential use of these cells in regenerative medicine. The aim of this review is to give the basics of liver progenitor cell biology and discuss recent results vis-à-vis their identity and contribution to liver regeneration. PMID:26600740
Voltage-matched, monolithic, multi-band-gap devices

DOEpatents

Wanlass, Mark W.; Mascarenhas, Angelo

2006-08-22

Monolithic, tandem, photonic cells include at least a first semiconductor layer and a second semiconductor layer, wherein each semiconductor layer includes an n-type region, a p-type region, and a given band-gap energy. Formed within each semiconductor layer is a sting of electrically connected photonic sub-cells. By carefully selecting the numbers of photonic sub-cells in the first and second layer photonic sub-cell string(s), and by carefully selecting the manner in which the sub-cells in a first and second layer photonic sub-cell string(s) are electrically connected, each of the first and second layer sub-cell strings may be made to achieve one or more substantially identical electrical characteristics.
Voltage-Matched, Monolithic, Multi-Band-Gap Devices

DOEpatents

Wanlass, M. W.; Mascarenhas, A.

2006-08-22

Monolithic, tandem, photonic cells include at least a first semiconductor layer and a second semiconductor layer, wherein each semiconductor layer includes an n-type region, a p-type region, and a given band-gap energy. Formed within each semiconductor layer is a string of electrically connected photonic sub-cells. By carefully selecting the numbers of photonic sub-cells in the first and second layer photonic sub-cell string(s), and by carefully selecting the manner in which the sub-cells in a first and second layer photonic sub-cell string(s) are electrically connected, each of the first and second layer sub-cell strings may be made to achieve one or more substantially identical electrical characteristics.
Presynaptic (Type III) cells in mouse taste buds sense sour (acid) taste.

PubMed

Huang, Yijen A; Maruyama, Yutaka; Stimac, Robert; Roper, Stephen D

2008-06-15

Taste buds contain two types of cells that directly participate in taste transduction - receptor (Type II) cells and presynaptic (Type III) cells. Receptor cells respond to sweet, bitter and umami taste stimulation but until recently the identity of cells that respond directly to sour (acid) tastants has only been inferred from recordings in situ, from behavioural studies, and from immunostaining for putative sour transduction molecules. Using calcium imaging on single isolated taste cells and with biosensor cells to identify neurotransmitter release, we show that presynaptic (Type III) cells specifically respond to acid taste stimulation and release serotonin. By recording responses in cells isolated from taste buds and in taste cells in lingual slices to acetic acid titrated to different acid levels (pH), we also show that the active stimulus for acid taste is the membrane-permeant, uncharged acetic acid moiety (CH(3)COOH), not free protons (H(+)). That observation is consistent with the proximate stimulus for acid taste being intracellular acidification, not extracellular protons per se. These findings may also have implications for other sensory receptors that respond to acids, such as nociceptors.
Acquisition of granule neuron precursor identity is a critical determinant of progenitor cell competence to form Hedgehog-induced medulloblastoma

PubMed Central

Schüller, Ulrich; Heine, Vivi M.; Mao, Junhao; Kho, Alvin T.; Dillon, Allison K.; Han, Young-Goo; Huillard, Emmanuelle; Sun, Tao; Ligon, Azra H.; Qian, Ying; Ma, Qiufu; Alvarez-Buylla, Arturo; McMahon, Andrew P.; Rowitch, David H.; Ligon, Keith L.

2008-01-01

Origins of the brain tumor, medulloblastoma, from stem cells or restricted progenitor cells are unclear. To investigate this, we activated oncogenic Hedgehog (Hh) signaling in multipotent and lineage-restricted CNS progenitors. We observed that normal unipotent cerebellar granule neuron precursors (CGNP) derive from hGFAP+ and Olig2+ RL progenitors. Hh activation in a spectrum of early and late stage CNS progenitors generated similar medulloblastomas, but not other brain cancers, indicating that acquisition of CGNP identity is essential for tumorigenesis. We show in human and mouse medulloblastoma that cells expressing the glia-associated markers Gfap and Olig2 are neoplastic and that they retain features of embryonic-type granule lineage progenitors. Thus, oncogenic Hh signaling promotes medulloblastoma from lineage-restricted granule cell progenitors. PMID:18691547
Converting adult pancreatic islet α-cells into β-cells by targeting both Dnmt1 and Arx

PubMed Central

Chakravarthy, Harini; Gu, Xueying; Enge, Martin; Dai, Xiaoqing; Wang, Yong; Damond, Nicolas; Downie, Carolina; Liu, Kathy; Wang, Jing; Xing, Yuan; Chera, Simona; Thorel, Fabrizio; Quake, Stephen; Oberholzer, Jose; MacDonald, Patrick E.; Herrera, Pedro L.; Kim, Seung K.

2017-01-01

Summary Insulin-producing pancreatic β-cells in mice can slowly regenerate from glucagon-producing α-cells in settings like β-cell loss, but the basis of this conversion is unknown. Moreover it remains unclear if this intra-islet cell conversion is relevant to diseases like type 1 diabetes (T1D). We show that the α-cell regulators Aristaless-related homeobox (Arx) and DNA methyltransferase 1 (Dnmt1) maintain α-cell identity in mice. Within 3 months of Dnmt1 and Arx loss, lineage tracing and single cell RNA sequencing revealed extensive α-cell conversion into progeny resembling native β-cells. Physiological studies demonstrated that converted α-cells acquire hallmark β-cell electrophysiology, and show glucose-stimulated insulin secretion. In T1D patients, subsets of Glucagon-expressing cells show loss of DNMT1 and ARX, and produce Insulin and other β-cell factors, suggesting that DNMT1 and ARX maintain α-cell identity in humans. Our work reveals pathways regulated by Arx and Dnmt1 sufficient for achieving targeted generation of β-cells from adult pancreatic α-cells. PMID:28215845
Control of Cell Morphology: Signalling by the Receptor Notch.

DTIC Science & Technology

1996-10-01

missense mutations or small deletions at the extreme C-terminus of NOTCH, and lie within the minimal region that includes the C-terminal binding site for...20 Figure 4. Genetic interaction of null and hypomorphic alleles of Notch with abl mutations ...wide variety of cell types during Drosophila embryogenesis [1, 2]. Mutations in the Notch gene lead to severe defects in cell identity in the nervous
Does epigenetic dysregulation of pancreatic islets contribute to impaired insulin secretion and type 2 diabetes?

PubMed

Dayeh, Tasnim; Ling, Charlotte

2015-10-01

β cell dysfunction is central to the development and progression of type 2 diabetes (T2D). T2D develops when β cells are not able to compensate for the increasing demand for insulin caused by insulin resistance. Epigenetic modifications play an important role in establishing and maintaining β cell identity and function in physiological conditions. On the other hand, epigenetic dysregulation can cause a loss of β cell identity, which is characterized by reduced expression of genes that are important for β cell function, ectopic expression of genes that are not supposed to be expressed in β cells, and loss of genetic imprinting. Consequently, this may lead to β cell dysfunction and impaired insulin secretion. Risk factors that can cause epigenetic dysregulation include parental obesity, an adverse intrauterine environment, hyperglycemia, lipotoxicity, aging, physical inactivity, and mitochondrial dysfunction. These risk factors can affect the epigenome at different time points throughout the lifetime of an individual and even before an individual is conceived. The plasticity of the epigenome enables it to change in response to environmental factors such as diet and exercise, and also makes the epigenome a good target for epigenetic drugs that may be used to enhance insulin secretion and potentially treat diabetes.
Sound insulation property of membrane-type acoustic metamaterials carrying different masses at adjacent cells

NASA Astrophysics Data System (ADS)

Zhang, Yuguang; Wen, Jihong; Zhao, Honggang; Yu, Dianlong; Cai, Li; Wen, Xisen

2013-08-01

We present the experimental realization and theoretical understanding of membrane-type acoustic metamaterials embedded with different masses at adjacent cells, capable of increasing the transmission loss at low frequency. Owing to the reverse vibration of adjacent cells, Transmission loss (TL) peaks appear, and the magnitudes of the TL peaks exceed the predicted results of the composite wall. Compared with commonly used configuration, i.e., all cells carrying with identical mass, the nonuniformity of attaching masses causes another much low TL peak. Finite element analysis was employed to validate and provide insights into the TL behavior of the structure.
Maintenance of the marginal-zone B cell compartment specifically requires the RNA-binding protein ZFP36L1.

PubMed

Newman, Rebecca; Ahlfors, Helena; Saveliev, Alexander; Galloway, Alison; Hodson, Daniel J; Williams, Robert; Besra, Gurdyal S; Cook, Charlotte N; Cunningham, Adam F; Bell, Sarah E; Turner, Martin

2017-06-01

RNA-binding proteins of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU-rich elements in the 3' untranslated region and promoting mRNA decay. Here we identified an indispensable role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal-zone B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene-expression program related to signaling, cell adhesion and locomotion; it achieved this in part by limiting expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RNA-binding proteins for maintaining cellular identity among closely related cell types.
Maintenance of the marginal zone B cell compartment specifically requires the RNA-binding protein ZFP36L1

PubMed Central

Newman, Rebecca; Ahlfors, Helena; Saveliev, Alexander; Galloway, Alison; Hodson, Daniel J; Williams, Robert; Besra, Gurdyal S.; Cook, Charlotte N; Cunningham, Adam F; Bell, Sarah E; Turner, Martin

2017-01-01

RNA binding proteins (RBP) of the ZFP36 family are best known for inhibiting the expression of cytokines through binding to AU rich elements in the 3’UTR and promoting mRNA decay. Here we show an indispensible role for ZFP36L1 as the regulator of a post-transcriptional hub that determined the identity of marginal zone (MZ) B cells by promoting their proper localization and survival. ZFP36L1 controlled a gene expression program related to signaling, cell-adhesion and locomotion, in part by limiting the expression of the transcription factors KLF2 and IRF8, which are known to enforce the follicular B cell phenotype. These mechanisms emphasize the importance of integrating transcriptional and post-transcriptional processes by RBP for maintaining cellular identity between closely related cell types. PMID:28394372
Criteria for robustness of heteroclinic cycles in neural microcircuits

PubMed Central

2011-01-01

We introduce a test for robustness of heteroclinic cycles that appear in neural microcircuits modeled as coupled dynamical cells. Robust heteroclinic cycles (RHCs) can appear as robust attractors in Lotka-Volterra-type winnerless competition (WLC) models as well as in more general coupled and/or symmetric systems. It has been previously suggested that RHCs may be relevant to a range of neural activities, from encoding and binding to spatio-temporal sequence generation. The robustness or otherwise of such cycles depends both on the coupling structure and the internal structure of the neurons. We verify that robust heteroclinic cycles can appear in systems of three identical cells, but only if we require perturbations to preserve some invariant subspaces for the individual cells. On the other hand, heteroclinic attractors can appear robustly in systems of four or more identical cells for some symmetric coupling patterns, without restriction on the internal dynamics of the cells. PMID:22656192
Progress in developing ultrathin solar cell blanket technology

NASA Technical Reports Server (NTRS)

Patterson, R. E.; Mesch, H. G.; Scott-Monck, J.

1984-01-01

A program was conducted to develop technologies for welding interconnects to three types of 50-micron-thick, 2 by 2-cm solar cells. Parallel-gap resistance welding was used for interconnect attachment. Weld schedules were independently developed for each of the three cell types and were coincidentally identical. Six 48-cell modules were assembled with 50-micron (nominal) thick cells, frosted fused-silica covers, silver-plated Invar interconnectors, and four different substrate designs. Three modules (one for each cell type) have single-layer Kapton (50-micron-thick) substrates. The other three modules each have a different substrate (Kapton-Kevlar-Kapton, Kapton-graphite-Kapton, and Kapton-graphite-aluminum honeycomb-graphite). All six modules were subjected to 4112 thermal cycles from -175 to 65 C (corresponding to over 40 years of simulated geosynchronous orbit thermal cycling) and experienced only negligible electrical degradation (1.1 percent average of six 48-cell modules).
Ectopic KNOX Expression Affects Plant Development by Altering Tissue Cell Polarity and Identity[OPEN

PubMed Central

Rebocho, Alexandra B.

2016-01-01

Plant development involves two polarity types: tissue cell (asymmetries within cells are coordinated across tissues) and regional (identities vary spatially across tissues) polarity. Both appear altered in the barley (Hordeum vulgare) Hooded mutant, in which ectopic expression of the KNOTTED1-like Homeobox (KNOX) gene, BKn3, causes inverted polarity of differentiated hairs and ectopic flowers, in addition to wing-shaped outgrowths. These lemma-specific effects allow the spatiotemporal analysis of events following ectopic BKn3 expression, determining the relationship between KNOXs, polarity, and shape. We show that tissue cell polarity, based on localization of the auxin transporter SISTER OF PINFORMED1 (SoPIN1), dynamically reorients as ectopic BKn3 expression increases. Concurrently, ectopic expression of the auxin importer LIKE AUX1 and boundary gene NO APICAL MERISTEM is activated. The polarity of hairs reflects SoPIN1 patterns, suggesting that tissue cell polarity underpins oriented cell differentiation. Wing cell files reveal an anisotropic growth pattern, and computational modeling shows how polarity guiding growth can account for this pattern and wing emergence. The inverted ectopic flower orientation does not correlate with SoPIN1, suggesting that this form of regional polarity is not controlled by tissue cell polarity. Overall, the results suggest that KNOXs trigger different morphogenetic effects through interplay between tissue cell polarity, identity, and growth. PMID:27553356
The function and molecular identity of inward rectifier channels in vestibular hair cells of the mouse inner ear

PubMed Central

Levin, Michaela E.

2012-01-01

Inner ear hair cells respond to mechanical stimuli with graded receptor potentials. These graded responses are modulated by a host of voltage-dependent currents that flow across the basolateral membrane. Here, we examine the molecular identity and the function of a class of voltage-dependent ion channels that carries the potassium-selective inward rectifier current known as IK1. IK1 has been identified in vestibular hair cells of various species, but its molecular composition and functional contributions remain obscure. We used quantitative RT-PCR to show that the inward rectifier gene, Kir2.1, is highly expressed in mouse utricle between embryonic day 15 and adulthood. We confirmed Kir2.1 protein expression in hair cells by immunolocalization. To examine the molecular composition of IK1, we recorded voltage-dependent currents from type II hair cells in response to 50-ms steps from −124 to −54 in 10-mV increments. Wild-type cells had rapidly activating inward currents with reversal potentials close to the K+ equilibrium potential and a whole-cell conductance of 4.8 ± 1.5 nS (n = 46). In utricle hair cells from Kir2.1-deficient (Kir2.1−/−) mice, IK1 was absent at all stages examined. To identify the functional contribution of Kir2.1, we recorded membrane responses in current-clamp mode. Hair cells from Kir2.1−/− mice had significantly (P < 0.001) more depolarized resting potentials and larger, slower membrane responses than those of wild-type cells. These data suggest that Kir2.1 is required for IK1 in type II utricle hair cells and contributes to hyperpolarized resting potentials and fast, small amplitude receptor potentials in response to current inputs, such as those evoked by hair bundle deflections. PMID:22496522
Therapeutic cloning and cellular reprogramming.

PubMed

Rodriguez, Ramon M; Ross, Pablo J; Cibelli, Jose B

2012-01-01

Embryonic stem cells are capable of differentiating into any cell-type present in an adult organism, and constitute a renewable source of tissue for regenerative therapies. The transplant of allogenic stem cells is challenging due to the risk of immune rejection. Nevertheless, somatic cell reprogramming techniques allow the generation of isogenic embryonic stem cells, genetically identical to the patient. In this chapter we will discuss the cellular reprogramming techniques in the context of regenerative therapy and the biological and technical barriers that they will need to overcome before clinical use.
An immunohistochemical identification key for cell types in adult mouse prostatic and urethral tissue sections

PubMed Central

Turco, Anne E.; Gottschalk, Adam; Halberg, Richard B.; Guo, Jinjin; McMahon, Jill A.; McMahon, Andrew P.

2017-01-01

Though many methods can be used to identify cell types contained in complex tissues, most require cell disaggregation and destroy information about where cells reside in relation to their microenvironment. Here, we describe a polytomous key for cell type identification in intact sections of adult mouse prostate and prostatic urethra. The key is organized as a decision tree and initiates with one round of immunostaining for nerve, epithelial, fibromuscular/hematolymphoid, or vascular associated cells. Cell identities are recursively eliminated by subsequent staining events until the remaining pool of potential cell types can be distinguished by direct comparison to other cells. We validated our identification key using wild type adult mouse prostate and urethra tissue sections and it currently resolves sixteen distinct cell populations which include three nerve fiber types as well as four epithelial, five fibromuscular/hematolymphoid, one nerve-associated, and three vascular-associated cell types. We demonstrate two uses of this novel identification methodology. We first used the identification key to characterize prostate stromal cell type changes in response to constitutive phosphatidylinositide-3-kinase activation in prostate epithelium. We then used the key to map cell lineages in a new reporter mouse strain driven by Wnt10aem1(cre/ERT2)Amc. The identification key facilitates rigorous and reproducible cell identification in prostate tissue sections and can be expanded to resolve additional cell types as new antibodies and other resources become available. PMID:29145476
MreB is important for cell shape but not for chromosome segregation of the filamentous cyanobacterium Anabaena sp. PCC 7120.

PubMed

Hu, Bin; Yang, Guohua; Zhao, Weixing; Zhang, Yingjiao; Zhao, Jindong

2007-03-01

MreB is a bacterial actin that plays important roles in determination of cell shape and chromosome partitioning in Escherichia coli and Caulobacter crescentus. In this study, the mreB from the filamentous cyanobacterium Anabaena sp. PCC 7120 was inactivated. Although the mreB null mutant showed a drastic change in cell shape, its growth rate, cell division and the filament length were unaltered. Thus, MreB in Anabaena maintains cell shape but is not required for chromosome partitioning. The wild type and the mutant had eight and 10 copies of chromosomes per cell respectively. We demonstrated that DNA content in two daughter cells after cell division in both strains was not always identical. The ratios of DNA content in two daughter cells had a Gaussian distribution with a standard deviation much larger than a value expected if the DNA content in two daughter cells were identical, suggesting that chromosome partitioning is a random process. The multiple copies of chromosomes in cyanobacteria are likely required for chromosome random partitioning in cell division.
Characterizing the replicability of cell types defined by single cell RNA-sequencing data using MetaNeighbor.

PubMed

Crow, Megan; Paul, Anirban; Ballouz, Sara; Huang, Z Josh; Gillis, Jesse

2018-02-28

Single-cell RNA-sequencing (scRNA-seq) technology provides a new avenue to discover and characterize cell types; however, the experiment-specific technical biases and analytic variability inherent to current pipelines may undermine its replicability. Meta-analysis is further hampered by the use of ad hoc naming conventions. Here we demonstrate our replication framework, MetaNeighbor, that quantifies the degree to which cell types replicate across datasets, and enables rapid identification of clusters with high similarity. We first measure the replicability of neuronal identity, comparing results across eight technically and biologically diverse datasets to define best practices for more complex assessments. We then apply this to novel interneuron subtypes, finding that 24/45 subtypes have evidence of replication, which enables the identification of robust candidate marker genes. Across tasks we find that large sets of variably expressed genes can identify replicable cell types with high accuracy, suggesting a general route forward for large-scale evaluation of scRNA-seq data.
Involvement of a Gardos-type potassium channel in head activator-induced mitosis of BON cells.

PubMed

Kayser, S T; Ulrich, H; Schaller, H C

1998-06-01

The human neuroendocrine cell line BON was used to study second messengers involved in signal transduction for entry into mitosis. BON cells produce the neuropeptide head activator (HA) and use it as autocrine growth factor. HA stimulates BON cell proliferation by triggering entry into mitosis. HA-induced mitosis is mediated by an inhibitory G protein, the action of which is blocked by pertussis toxin. HA signaling requires inhibition of the cAMP pathway, calcium influx, and hyperpolarization of cells. The latter is a very important and sensitive step involving a calcium-activated potassium channel. Cell cycle progression and proliferation of BON cells are most efficiently inhibited with specific inhibitors of this potassium channel. Pharmacology and RNA analysis suggest identity with the recently cloned Gardos-type potassium channel.

Temporal, but not spatial, changes in expression patterns of petal identity genes are associated with loss of papillate conical cells and the shift to bird pollination in Macaronesian Lotus (Leguminosae).

PubMed

Ojeda, D I; Jaén-Molina, R; Santos-Guerra, A; Caujape-Castells, J; Cronk, Q

2017-05-01

In the generally bee-pollinated genus Lotus a group of four species have evolved bird-pollinated flowers. The floral changes in these species include altered petal orientation, shape and texture. In Lotus these characters are associated with dorsiventral petal identity, suggesting that shifts in the expression of dorsal identity genes may be involved in the evolution of bird pollination. Of particular interest is Lotus japonicus CYCLOIDEA 2 (LjCYC2), known to determine the presence of papillate conical cells on the dorsal petal in L. japonicus. Bird-pollinated species are unusual in not having papillate conical cells on the dorsal petal. Using RT-PCR at various stages of flower development, we determined the timing of expression in all petal types for the three putative petal identity genes (CYC-like genes) in different species with contrasting floral morphology and pollination syndromes. In bird-pollinated species the dorsal identity gene, LjCYC2, is not expressed at the floral stage when papillate conical cells are normally differentiating in bee-pollinated species. In contrast, in bee-pollinated species, LjCYC2 is expressed during conical cell development. Changes in the timing of expression of the above two genes are associated with modifications in petal growth and lateralisation of the dorsal and ventral petals in the bird-pollinated species. This study indicates that changes in the timing, rather than spatial distribution, of expression likely contribute to the modifications of petal micromorphology and petal size during the transition from bee to bird pollination in Macaronesian Lotus species. © 2017 German Botanical Society and The Royal Botanical Society of the Netherlands.
THY-1 Receptor Expression Differentiates Cardiosphere-Derived Cells with Divergent Cardiogenic Differentiation Potential

PubMed Central

Gago-Lopez, Nuria; Awaji, Obinna; Zhang, Yiqiang; Ko, Christopher; Nsair, Ali; Liem, David; Stempien-Otero, April; MacLellan, W. Robb

2014-01-01

Summary Despite over a decade of intense research, the identity and differentiation potential of human adult cardiac progenitor cells (aCPC) remains controversial. Cardiospheres have been proposed as a means to expand aCPCs in vitro, but the identity of the progenitor cell within these 3D structures is unknown. We show that clones derived from cardiospheres could be subdivided based on expression of thymocyte differentiation antigen 1 (THY-1/CD90) into two distinct populations that exhibit divergent cardiac differentiation potential. One population, which is CD90+, expressed markers consistent with a mesenchymal/myofibroblast cell. The second clone type was CD90− and could form mature, functional myocytes with sarcomeres albeit at a very low rate. These two populations of cardiogenic clones displayed distinct cell surface markers and unique transcriptomes. Our study suggests that a rare aCPC exists in cardiospheres along with a mesenchymal/myofibroblast cell, which demonstrates incomplete cardiac myocyte differentiation. PMID:24936447
The Role of Dipeptidyl Peptidase IV in Lung Metastasis of Breast Cancer Cells

DTIC Science & Technology

1999-05-01

Our studies focused on (1) cloning and sequencing of wild-type endothelial DPP IV (wtDPP IV) and preparation of truncated DPP IV ( tDPP IV); (2...that was identical to hepatic DPP IV. Acid extraction of rat lung yielded a tDPP IV, which was an effective inhibitor of breast cancer cell adhesion to
Inversion of the chromosomal region between two mating type loci switches the mating type in Hansenula polymorpha.

PubMed

Maekawa, Hiromi; Kaneko, Yoshinobu

2014-11-01

Yeast mating type is determined by the genotype at the mating type locus (MAT). In homothallic (self-fertile) Saccharomycotina such as Saccharomyces cerevisiae and Kluveromyces lactis, high-efficiency switching between a and α mating types enables mating. Two silent mating type cassettes, in addition to an active MAT locus, are essential components of the mating type switching mechanism. In this study, we investigated the structure and functions of mating type genes in H. polymorpha (also designated as Ogataea polymorpha). The H. polymorpha genome was found to harbor two MAT loci, MAT1 and MAT2, that are ∼18 kb apart on the same chromosome. MAT1-encoded α1 specifies α cell identity, whereas none of the mating type genes were required for a identity and mating. MAT1-encoded α2 and MAT2-encoded a1 were, however, essential for meiosis. When present in the location next to SLA2 and SUI1 genes, MAT1 or MAT2 was transcriptionally active, while the other was repressed. An inversion of the MAT intervening region was induced by nutrient limitation, resulting in the swapping of the chromosomal locations of two MAT loci, and hence switching of mating type identity. Inversion-deficient mutants exhibited severe defects only in mating with each other, suggesting that this inversion is the mechanism of mating type switching and homothallism. This chromosomal inversion-based mechanism represents a novel form of mating type switching that requires only two MAT loci.
Sustained and transient calcium currents in horizontal cells of the white bass retina.

PubMed

Sullivan, J M; Lasater, E M

1992-01-01

Calcium currents were recorded from cultured horizontal cells (HCs) isolated from adult white bass retinas, using the whole-cell patch-clamp technique. Ca2+ currents were enhanced using 10 mM extracellular Ca2+, while Na+ and K+ currents were pharmacologically suppressed. Two components of the Ca2+ current, one transient, the other sustained, were found. The large transient component of the Ca2+ current, which has not been seen before in HCs, is similar, but not identical, to the T-type Ca2+ current described previously in a variety of preparations. The sustained component of the Ca2+ current is similar, but not identical, to the L-type current described in other preparations. FTX, a factor isolated from the venom of the funnel-web spider, Agelenopsis aperta, preferentially and irreversibly blocks the sustained component of the Ca2+ current at very dilute concentrations. The sustained component of the Ca2+ current inactivates slowly, over the course of 15-60 s, in some HCs. This inactivation of the sustained Ca2+ current, when present, is primarily voltage dependent rather than Ca2+ dependent.
Sustained and transient calcium currents in horizontal cells of the white bass retina

PubMed Central

1992-01-01

Calcium currents were recorded from cultured horizontal cells (HCs) isolated from adult white bass retinas, using the whole-cell patch- clamp technique. Ca2+ currents were enhanced using 10 mM extracellular Ca2+, while Na+ and K+ currents were pharmacologically suppressed. Two components of the Ca2+ current, one transient, the other sustained, were found. The large transient component of the Ca2+ current, which has not been seen before in HCs, is similar, but not identical, to the T-type Ca2+ current described previously in a variety of preparations. The sustained component of the Ca2+ current is similar, but not identical, to the L-type current described in other preparations. FTX, a factor isolated from the venom of the funnel-web spider, Agelenopsis aperta, preferentially and irreversibly blocks the sustained component of the Ca2+ current at very dilute concentrations. The sustained component of the Ca2+ current inactivates slowly, over the course of 15- 60 s, in some HCs. This inactivation of the sustained Ca2+ current, when present, is primarily voltage dependent rather than Ca2+ dependent. PMID:1371309
Method and apparatus for rebalancing a redox flow cell system

NASA Technical Reports Server (NTRS)

Gahn, Randall F. (Inventor)

1986-01-01

A rebalance cell is provided for a REDOX electrochemical system of the type having anode and cathode fluids which are aqueous HCl solutions with two metal species in each. The rebalance cell has a cathode compartment and a chlorine compartment separated by an ion permeable membrane. By applying an electrical potential to the rebalance cell while circulating cathode fluid through the cathode compartment and while circulating an identical fluid through the chlorine compartment, any significant imbalance of the REDOX system is prevented.
Method and apparatus for rebalancing a REDOX flow cell system

NASA Technical Reports Server (NTRS)

Gahn, R. F. (Inventor)

1985-01-01

A rebalance cell is provided for a REDOX electrochemical system of the type with anode and cathode fluids which are aqueous HC1 solutions with two metal species in each. The rebalance cell has a cathode compartment and a chlorine compartment separated by an ion permeable membrane. By applying an electrical potential to the rebalance cell while circulating cathode fluid through the cathode compartment and while circulating an identical fluid through the chlorine compartment, any significant imbalance of the REDOX system is prevented.
dbSUPER: a database of super-enhancers in mouse and human genome

PubMed Central

Khan, Aziz; Zhang, Xuegong

2016-01-01

Super-enhancers are clusters of transcriptional enhancers that drive cell-type-specific gene expression and are crucial to cell identity. Many disease-associated sequence variations are enriched in super-enhancer regions of disease-relevant cell types. Thus, super-enhancers can be used as potential biomarkers for disease diagnosis and therapeutics. Current studies have identified super-enhancers in more than 100 cell types and demonstrated their functional importance. However, a centralized resource to integrate all these findings is not currently available. We developed dbSUPER (http://bioinfo.au.tsinghua.edu.cn/dbsuper/), the first integrated and interactive database of super-enhancers, with the primary goal of providing a resource for assistance in further studies related to transcriptional control of cell identity and disease. dbSUPER provides a responsive and user-friendly web interface to facilitate efficient and comprehensive search and browsing. The data can be easily sent to Galaxy instances, GREAT and Cistrome web-servers for downstream analysis, and can also be visualized in the UCSC genome browser where custom tracks can be added automatically. The data can be downloaded and exported in variety of formats. Furthermore, dbSUPER lists genes associated with super-enhancers and also links to external databases such as GeneCards, UniProt and Entrez. dbSUPER also provides an overlap analysis tool to annotate user-defined regions. We believe dbSUPER is a valuable resource for the biology and genetic research communities. PMID:26438538
Prosomes. Ubiquity and inter-species structural variation.

PubMed

Martins de Sa, C; Grossi de Sa, M F; Akhayat, O; Broders, F; Scherrer, K; Horsch, A; Schmid, H P

1986-02-20

The "prosomes", a novel type of ubiquitous ribonucleoprotein particle of extraordinary stability and of defined electron microscopical structure, have been characterized in several cell types and species. Identified as a 19 S sub-component of free mRNA-protein complexes, including globin and other repressed mRNA, in the cytoplasm of duck, mouse and HeLa cells, they were previously found to inhibit protein synthesis in vitro. In all cells studied, electron microscopy shows an identical, seemingly ring-like but rather raspberry-shaped particle of 12 nm diameter, resistant to EDTA and 1% (w/v) Sarkosyl. Two-dimensional electrophoretic analysis of prosomal proteins shows a characteristic pattern in the 19,000 to 35,000 Mr range of pI 4 to 7, with an additional 56,000 Mr component specific to avian species. The prosomes found in globin mRNA-protein complexes contain about 25 protein components, 16 of which have identical molecular weight and pI values in duck and mouse, and which are also found in the prosomes of the heterogeneous free mRNPs of HeLa cells. Seral and monoclonal antibodies raised in mice against the prosomes of duck erythroblasts cross-react with some of the proteins of the mouse and HeLa cell particles. Prosomes isolated from duck and mouse globin mRNP, both contain small cytoplasmic RNAs of 70 to 90 nucleotides, which represent about 15% of the particle mass. The molecular weight and the 3'-terminal oligonucleotide of each one of these small cytoplasmic RNAs are identical in the two animal species; fingerprints of their oligonucleotides generated by RNase T1 show that more than 80% of spots are identical. In contrast, the prosomes of HeLa cells, associated with a large population of repressed mRNA, contain at least 12 small cytoplasmic RNA species. All prosomal RNAs tested so far hybridize to mRNA. The data available indicate that prosomes constitute a novel class of ubiquitous cellular ribonucleoprotein complexes, present in the nucleus and cytoplasm that, in its structural variations shown here, reflects function and species.
Genomic tissue typing and optimal antithymocyte globuline dose using unrelated donors results in similar survival and relapse as HLA-identical siblings in haematopoietic stem-cell transplantation for leukaemia.

PubMed

Remberger, Mats; Mattsson, Jonas; Hausenberger, Dan; Schaffer, Marie; Svahn, Britt-Marie; Ringdén, Olle

2008-05-01

Sixty-one leukaemia patients treated with haematopoietic stem cell transplantation (HSCT) from a genomic human leucocyte antigen (HLA)-A, -B and -DRbeta1 matched unrelated donor (MUD) were compared with 121 patients with an HLA-identical sibling donor. All patients received conventional conditioning. We selected all patients with unrelated donors who received optimal antithymocyte globuline (ATG) dose, 6 mg/kg. One hundred and seven patients received stem cells from peripheral blood and 75 patients received bone marrow (BM) cells. The incidences of acute graft-versus-host disease (GVHD) grades II-IV were 33.4% and 34.7% in the MUD and sibling group, respectively. After year 2001, the incidence of chronic GVHD was similar in the two groups (27.8% vs. 25.8%). There was no difference in overall survival (60% vs. 60%), transplant-related mortality (18.6% vs. 16.6%) and relapse (23% vs. 26.4%) between the two groups. Haematopoietic stem cell transplantation with unrelated donors results in similar GVHD, relapse and survival as compared to using sibling donors. Reasons for this may be improved tissue-typing techniques and supportive care and optimisation of the ATG dose.
Sequence of contactin, a 130-kD glycoprotein concentrated in areas of interneuronal contact, defines a new member of the immunoglobulin supergene family in the nervous system

PubMed Central

1988-01-01

The primary amino acid sequence of contactin, a neuronal cell surface glycoprotein of 130 kD that is isolated in association with components of the cytoskeleton (Ranscht, B., D. J. Moss, and C. Thomas. 1984. J. Cell Biol. 99:1803-1813), was deduced from the nucleotide sequence of cDNA clones and is reported here. The cDNA sequence contains an open reading frame for a 1,071-amino acid transmembrane protein with 962 extracellular and 89 cytoplasmic amino acids. In its extracellular portion, the polypeptide features six type 1 and two type 2 repeats. The six amino-terminal type 1 repeats (I-VI) each consist of 81-99 amino acids and contain two cysteine residues that are in the right context to form globular domains as described for molecules with immunoglobulin structure. Within the proposed globular region, contactin shares 31% identical amino acids with the neural cell adhesion molecule NCAM. The two type 2 repeats (I-II) are each composed of 100 amino acids and lack cysteine residues. They are 20-31% identical to fibronectin type III repeats. Both the structural similarity of contactin to molecules of the immunoglobulin supergene family, in particular the amino acid sequence resemblance to NCAM, and its relationship to fibronectin indicate that contactin could be involved in some aspect of cellular adhesion. This suggestion is further strengthened by its localization in neuropil containing axon fascicles and synapses. PMID:3049624
Glass promotes the differentiation of neuronal and non-neuronal cell types in the Drosophila eye

PubMed Central

Morrison, Carolyn A.; Chen, Hao; Cook, Tiffany; Brown, Stuart

2018-01-01

Transcriptional regulators can specify different cell types from a pool of equivalent progenitors by activating distinct developmental programs. The Glass transcription factor is expressed in all progenitors in the developing Drosophila eye, and is maintained in both neuronal and non-neuronal cell types. Glass is required for neuronal progenitors to differentiate as photoreceptors, but its role in non-neuronal cone and pigment cells is unknown. To determine whether Glass activity is limited to neuronal lineages, we compared the effects of misexpressing it in neuroblasts of the larval brain and in epithelial cells of the wing disc. Glass activated overlapping but distinct sets of genes in these neuronal and non-neuronal contexts, including markers of photoreceptors, cone cells and pigment cells. Coexpression of other transcription factors such as Pax2, Eyes absent, Lozenge and Escargot enabled Glass to induce additional genes characteristic of the non-neuronal cell types. Cell type-specific glass mutations generated in cone or pigment cells using somatic CRISPR revealed autonomous developmental defects, and expressing Glass specifically in these cells partially rescued glass mutant phenotypes. These results indicate that Glass is a determinant of organ identity that acts in both neuronal and non-neuronal cells to promote their differentiation into functional components of the eye. PMID:29324767
Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus.

PubMed

García-Betancur, Juan-Carlos; Goñi-Moreno, Angel; Horger, Thomas; Schott, Melanie; Sharan, Malvika; Eikmeier, Julian; Wohlmuth, Barbara; Zernecke, Alma; Ohlsen, Knut; Kuttler, Christina; Lopez, Daniel

2017-09-12

A central question to biology is how pathogenic bacteria initiate acute or chronic infections. Here we describe a genetic program for cell-fate decision in the opportunistic human pathogen Staphylococcus aureus , which generates the phenotypic bifurcation of the cells into two genetically identical but different cell types during the course of an infection. Whereas one cell type promotes the formation of biofilms that contribute to chronic infections, the second type is planktonic and produces the toxins that contribute to acute bacteremia. We identified a bimodal switch in the agr quorum sensing system that antagonistically regulates the differentiation of these two physiologically distinct cell types. We found that extracellular signals affect the behavior of the agr bimodal switch and modify the size of the specialized subpopulations in specific colonization niches. For instance, magnesium-enriched colonization niches causes magnesium binding to S. aureus teichoic acids and increases bacterial cell wall rigidity. This signal triggers a genetic program that ultimately downregulates the agr bimodal switch. Colonization niches with different magnesium concentrations influence the bimodal system activity, which defines a distinct ratio between these subpopulations; this in turn leads to distinct infection outcomes in vitro and in an in vivo murine infection model. Cell differentiation generates physiological heterogeneity in clonal bacterial infections and helps to determine the distinct infection types.
Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus

PubMed Central

García-Betancur, Juan-Carlos; Goñi-Moreno, Angel; Horger, Thomas; Schott, Melanie; Sharan, Malvika; Eikmeier, Julian; Wohlmuth, Barbara; Zernecke, Alma; Ohlsen, Knut; Kuttler, Christina

2017-01-01

A central question to biology is how pathogenic bacteria initiate acute or chronic infections. Here we describe a genetic program for cell-fate decision in the opportunistic human pathogen Staphylococcus aureus, which generates the phenotypic bifurcation of the cells into two genetically identical but different cell types during the course of an infection. Whereas one cell type promotes the formation of biofilms that contribute to chronic infections, the second type is planktonic and produces the toxins that contribute to acute bacteremia. We identified a bimodal switch in the agr quorum sensing system that antagonistically regulates the differentiation of these two physiologically distinct cell types. We found that extracellular signals affect the behavior of the agr bimodal switch and modify the size of the specialized subpopulations in specific colonization niches. For instance, magnesium-enriched colonization niches causes magnesium binding to S. aureusteichoic acids and increases bacterial cell wall rigidity. This signal triggers a genetic program that ultimately downregulates the agr bimodal switch. Colonization niches with different magnesium concentrations influence the bimodal system activity, which defines a distinct ratio between these subpopulations; this in turn leads to distinct infection outcomes in vitro and in an in vivo murine infection model. Cell differentiation generates physiological heterogeneity in clonal bacterial infections and helps to determine the distinct infection types. PMID:28893374
'Who's who' in two different flower types of Calluna vulgaris (Ericaceae): morphological and molecular analyses of flower organ identity

PubMed Central

2009-01-01

Background The ornamental crop Calluna vulgaris is of increasing importance to the horticultural industry in the northern hemisphere due to a flower organ mutation: the flowers of the 'bud-flowering' phenotype remain closed i.e. as buds throughout the total flowering period and thereby maintain more colorful flowers for a longer period of time than the wild-type. This feature is accompanied and presumably caused by the complete lack of stamens. Descriptions of this botanical particularity are inconsistent and partially conflicting. In order to clarify basic questions of flower organ identity in general and stamen loss in detail, a study of the wild-type and the 'bud-flowering' flower type of C. vulgaris was initiated. Results Flowers were examined by macro- and microscopic techniques. Organ development was investigated comparatively in both the wild-type and the 'bud-flowering' type by histological analyses. Analysis of epidermal cell surface structure of vegetative tissues and perianth organs using scanning electron microscopy revealed that in wild-type flowers the outer whorls of colored organs may be identified as sepals, while the inner ones may be identified as petals. In the 'bud-flowering' type, two whorls of sepals are directly followed by the gynoecium. Both, petals and stamens, are completely missing in this flower type. The uppermost whorl of green leaves represents bracts in both flower types. In addition, two MADS-box genes (homologs of AP3/DEF and SEP1/2) were identified in C. vulgaris using RACE-PCR. Expression analysis by qRT-PCR was conducted for both genes in leaves, bracts, sepals and petals. These experiments revealed an expression pattern supporting the organ classification based on morphological characteristics. Conclusions Organ identity in both wild-type and 'bud-flowering' C. vulgaris was clarified using a combination of microscopic and molecular methods. Our results for bract, sepal and petal organ identity are supported by the 'ABCDE model'. However, loss of stamens in the 'bud-flowering' phenotype is an exceptional flower organ modification that cannot be explained by modified spatial expression of known organ identity genes. PMID:20003430
Interferon Lambda: A New Sword in Cancer Immunotherapy

PubMed Central

Lasfar, Ahmed; Abushahba, Walid; Balan, Murugabaskar; Cohen-Solal, Karine A.

2011-01-01

The discovery of the interferon-lambda (IFN-λ) family has considerably contributed to our understanding of the role of interferon not only in viral infections but also in cancer. IFN-λ proteins belong to the new type III IFN group. Type III IFN is structurally similar to type II IFN (IFN-γ) but functionally identical to type I IFN (IFN-α/β). However, in contrast to type I or type II IFNs, the response to type III IFN is highly cell-type specific. Only epithelial-like cells and to a lesser extent some immune cells respond to IFN-λ. This particular pattern of response is controlled by the differential expression of the IFN-λ receptor, which, in contrast to IFN-α, should result in limited side effects in patients. Recently, we and other groups have shown in several animal models a potent antitumor role of IFN-λ that will open a new challenging era for the current IFN therapy. PMID:22190970
The Aquilegia JAGGED homolog promotes proliferation of adaxial cell types in both leaves and stems.

PubMed

Min, Ya; Kramer, Elena M

2017-10-01

In order to explore the functional conservation of JAGGED, a key gene involved in the sculpting of lateral organs in several model species, we identified its ortholog AqJAG in the lower eudicot species Aquilegia coerulea. We analyzed the expression patterns of AqJAG in various tissues and developmental stages, and used RNAi-based methods to generate knockdown phenotypes of AqJAG. AqJAG was strongly expressed in shoot apices, floral meristems, lateral root primordia and all lateral organ primordia. Silencing of AqJAG revealed a wide range of defects in the developing stems, leaves and flowers; strongest phenotypes include severe reduction of leaflet laminae due to a decrease in cell size and number, change of adaxial cell identity, outgrowth of laminar-like tissue on the inflorescence stem, and early arrest of floral meristems and floral organ primordia. Our results indicate that AqJAG plays a critical role in controlling primordia initiation and distal growth of floral organs, and laminar development of leaflets. Most strikingly, we demonstrated that AqJAG disproportionally controls the behavior of cells with adaxial identity in vegetative tissues, providing evidence of how cell proliferation is controlled in an identity-specific manner. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.
A conserved gene family encodes transmembrane proteins with fibronectin, immunoglobulin and leucine-rich repeat domains (FIGLER)

PubMed Central

Munfus, Delicia L; Haga, Christopher L; Burrows, Peter D; Cooper, Max D

2007-01-01

Background In mouse the cytokine interleukin-7 (IL-7) is required for generation of B lymphocytes, but human IL-7 does not appear to have this function. A bioinformatics approach was therefore used to identify IL-7 receptor related genes in the hope of identifying the elusive human cytokine. Results Our database search identified a family of nine gene candidates, which we have provisionally named fibronectin immunoglobulin leucine-rich repeat (FIGLER). The FIGLER 1–9 genes are predicted to encode type I transmembrane glycoproteins with 6–12 leucine-rich repeats (LRR), a C2 type Ig domain, a fibronectin type III domain, a hydrophobic transmembrane domain, and a cytoplasmic domain containing one to four tyrosine residues. Members of this multichromosomal gene family possess 20–47% overall amino acid identity and are differentially expressed in cell lines and primary hematopoietic lineage cells. Genes for FIGLER homologs were identified in macaque, orangutan, chimpanzee, mouse, rat, dog, chicken, toad, and puffer fish databases. The non-human FIGLER homologs share 38–99% overall amino acid identity with their human counterpart. Conclusion The extracellular domain structure and absence of recognizable cytoplasmic signaling motifs in members of the highly conserved FIGLER gene family suggest a trophic or cell adhesion function for these molecules. PMID:17854505
Islet cells share promoter hypomethylation independently of expression, but exhibit cell-type-specific methylation in enhancers.

PubMed

Neiman, Daniel; Moss, Joshua; Hecht, Merav; Magenheim, Judith; Piyanzin, Sheina; Shapiro, A M James; de Koning, Eelco J P; Razin, Aharon; Cedar, Howard; Shemer, Ruth; Dor, Yuval

2017-12-19

DNA methylation at promoters is an important determinant of gene expression. Earlier studies suggested that the insulin gene promoter is uniquely unmethylated in insulin-expressing pancreatic β-cells, providing a classic example of this paradigm. Here we show that islet cells expressing insulin, glucagon, or somatostatin share a lack of methylation at the promoters of the insulin and glucagon genes. This is achieved by rapid demethylation of the insulin and glucagon gene promoters during differentiation of Neurogenin3 + embryonic endocrine progenitors, regardless of the specific endocrine cell-type chosen. Similar methylation dynamics were observed in transgenic mice containing a human insulin promoter fragment, pointing to the responsible cis element. Whole-methylome comparison of human α- and β-cells revealed generality of the findings: genes active in one cell type and silent in the other tend to share demethylated promoters, while methylation differences between α- and β-cells are concentrated in enhancers. These findings suggest an epigenetic basis for the observed plastic identity of islet cell types, and have implications for β-cell reprogramming in diabetes and diagnosis of β-cell death using methylation patterns of circulating DNA. Copyright © 2017 the Author(s). Published by PNAS.

Parallel RNAi screens across different cell lines identify generic and cell type-specific regulators of actin organization and cell morphology.

PubMed

Liu, Tao; Sims, David; Baum, Buzz

2009-01-01

In recent years RNAi screening has proven a powerful tool for dissecting gene functions in animal cells in culture. However, to date, most RNAi screens have been performed in a single cell line, and results then extrapolated across cell types and systems. Here, to dissect generic and cell type-specific mechanisms underlying cell morphology, we have performed identical kinome RNAi screens in six different Drosophila cell lines, derived from two distinct tissues of origin. This analysis identified a core set of kinases required for normal cell morphology in all lines tested, together with a number of kinases with cell type-specific functions. Most significantly, the screen identified a role for minibrain (mnb/DYRK1A), a kinase associated with Down's syndrome, in the regulation of actin-based protrusions in CNS-derived cell lines. This cell type-specific requirement was not due to the peculiarities in the morphology of CNS-derived cells and could not be attributed to differences in mnb expression. Instead, it likely reflects differences in gene expression that constitute the cell type-specific functional context in which mnb/DYRK1A acts. Using parallel RNAi screens and gene expression analyses across cell types we have identified generic and cell type-specific regulators of cell morphology, which include mnb/DYRK1A in the regulation of protrusion morphology in CNS-derived cell lines. This analysis reveals the importance of using different cell types to gain a thorough understanding of gene function across the genome and, in the case of kinases, the difficulties of using the differential gene expression to predict function.
The Survey of Personal and National Identity on Cell Phone Addicts and Non-Addicts

PubMed Central

Alavi, Seyyed Salman; Ghanizadeh, Maryam; Mohammadi, Mohammad Reza; Mohammadi Kalhory, Soroush; Jannatifard, Fereshteh; Sepahbodi, Ghazal

2018-01-01

Objective: Smart phones have rapidly become an integral, and for some, an essential communication device worldwide. The issue of identity has always been a subject of interest among psychologists. The present study was conducted to compare personal and national identity and their subscales between cell phone addicts and non-addicts. Method : In this cross-sectional study, 500 student cell phone users from various universities in Tehran were recruited using stratified sampling. Participants completed cell phone addiction questionnaires including Mobile Phone Problematic Use Scale (MPPUS), Cell Phone Dependency Questionnaire (CPDQ), Personal Identity Development Questionnaire, Extended Objective Measure of Ego Identity Status (EOMEIS) and National Identity Questionnaire. Then, the subscales of these instruments were analyzed using SPSS Version 20. Results: Results of this study revealed significant differences between cell phone addicts and non-addicts in the scores of national identity, personal identity, and most subscales, except for some subscales (P<0.05). In addition, a negative and significant relationship was found between personal and national identity and cell phone addiction (r=-0.35, -0.33, respectively).On the other hand, after controlling for the confounder variables, we found that national identity had an effect on cell phone addiction(OR=0.05, CI=0.92-0.98). Conclusion: The results of this study indicated that cell phone overuse may be correlated with defects in some aspects of national and personal identity. PMID:29892313
The Survey of Personal and National Identity on Cell Phone Addicts and Non-Addicts.

PubMed

Alavi, Seyyed Salman; Ghanizadeh, Maryam; Mohammadi, Mohammad Reza; Mohammadi Kalhory, Soroush; Jannatifard, Fereshteh; Sepahbodi, Ghazal

2018-01-01

Objective: Smart phones have rapidly become an integral, and for some, an essential communication device worldwide. The issue of identity has always been a subject of interest among psychologists. The present study was conducted to compare personal and national identity and their subscales between cell phone addicts and non-addicts. Method : In this cross-sectional study, 500 student cell phone users from various universities in Tehran were recruited using stratified sampling. Participants completed cell phone addiction questionnaires including Mobile Phone Problematic Use Scale (MPPUS), Cell Phone Dependency Questionnaire (CPDQ), Personal Identity Development Questionnaire, Extended Objective Measure of Ego Identity Status (EOMEIS) and National Identity Questionnaire. Then, the subscales of these instruments were analyzed using SPSS Version 20. Results: Results of this study revealed significant differences between cell phone addicts and non-addicts in the scores of national identity, personal identity, and most subscales, except for some subscales (P<0.05). In addition, a negative and significant relationship was found between personal and national identity and cell phone addiction (r=-0.35, -0.33, respectively).On the other hand, after controlling for the confounder variables, we found that national identity had an effect on cell phone addiction(OR=0.05, CI=0.92-0.98). Conclusion: The results of this study indicated that cell phone overuse may be correlated with defects in some aspects of national and personal identity.
Intrinsic physiological properties of rat retinal ganglion cells with a comparative analysis.

PubMed

Wong, Raymond C S; Cloherty, Shaun L; Ibbotson, Michael R; O'Brien, Brendan J

2012-10-01

Mammalian retina contains 15-20 different retinal ganglion cell (RGC) types, each of which is responsible for encoding different aspects of the visual scene. The encoding is defined by a combination of RGC synaptic inputs, the neurotransmitter systems used, and their intrinsic physiological properties. Each cell's intrinsic properties are defined by its morphology and membrane characteristics, including the complement and localization of the ion channels expressed. In this study, we examined the hypothesis that the intrinsic properties of individual RGC types are conserved among mammalian species. To do so, we measured the intrinsic properties of 16 morphologically defined rat RGC types and compared these data with cat RGC types. Our data demonstrate that in the rat different morphologically defined RGC types have distinct patterns of intrinsic properties. Variation in these properties across cell types was comparable to that found for cat RGC types. When presumed morphological homologs in rat and cat retina were compared directly, some RGC types had very similar properties. The rat A2 cell exhibited patterns of intrinsic properties nearly identical to the cat alpha cell. In contrast, rat D2 cells (ON-OFF directionally selective) had a very different pattern of intrinsic properties than the cat iota cell. Our data suggest that the intrinsic properties of RGCs with similar morphology and suspected visual function may be subject to variation due to the behavioral needs of the species.
Multiplexed droplet single-cell RNA-sequencing using natural genetic variation.

PubMed

Kang, Hyun Min; Subramaniam, Meena; Targ, Sasha; Nguyen, Michelle; Maliskova, Lenka; McCarthy, Elizabeth; Wan, Eunice; Wong, Simon; Byrnes, Lauren; Lanata, Cristina M; Gate, Rachel E; Mostafavi, Sara; Marson, Alexander; Zaitlen, Noah; Criswell, Lindsey A; Ye, Chun Jimmie

2018-01-01

Droplet single-cell RNA-sequencing (dscRNA-seq) has enabled rapid, massively parallel profiling of transcriptomes. However, assessing differential expression across multiple individuals has been hampered by inefficient sample processing and technical batch effects. Here we describe a computational tool, demuxlet, that harnesses natural genetic variation to determine the sample identity of each droplet containing a single cell (singlet) and detect droplets containing two cells (doublets). These capabilities enable multiplexed dscRNA-seq experiments in which cells from unrelated individuals are pooled and captured at higher throughput than in standard workflows. Using simulated data, we show that 50 single-nucleotide polymorphisms (SNPs) per cell are sufficient to assign 97% of singlets and identify 92% of doublets in pools of up to 64 individuals. Given genotyping data for each of eight pooled samples, demuxlet correctly recovers the sample identity of >99% of singlets and identifies doublets at rates consistent with previous estimates. We apply demuxlet to assess cell-type-specific changes in gene expression in 8 pooled lupus patient samples treated with interferon (IFN)-β and perform eQTL analysis on 23 pooled samples.
NGSCheckMate: software for validating sample identity in next-generation sequencing studies within and across data types.

PubMed

Lee, Sejoon; Lee, Soohyun; Ouellette, Scott; Park, Woong-Yang; Lee, Eunjung A; Park, Peter J

2017-06-20

In many next-generation sequencing (NGS) studies, multiple samples or data types are profiled for each individual. An important quality control (QC) step in these studies is to ensure that datasets from the same subject are properly paired. Given the heterogeneity of data types, file types and sequencing depths in a multi-dimensional study, a robust program that provides a standardized metric for genotype comparisons would be useful. Here, we describe NGSCheckMate, a user-friendly software package for verifying sample identities from FASTQ, BAM or VCF files. This tool uses a model-based method to compare allele read fractions at known single-nucleotide polymorphisms, considering depth-dependent behavior of similarity metrics for identical and unrelated samples. Our evaluation shows that NGSCheckMate is effective for a variety of data types, including exome sequencing, whole-genome sequencing, RNA-seq, ChIP-seq, targeted sequencing and single-cell whole-genome sequencing, with a minimal requirement for sequencing depth (>0.5X). An alignment-free module can be run directly on FASTQ files for a quick initial check. We recommend using this software as a QC step in NGS studies. https://github.com/parklab/NGSCheckMate. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
Generation of multiple cell types in Bacillus subtilis.

PubMed

Lopez, Daniel; Vlamakis, Hera; Kolter, Roberto

2009-01-01

Bacillus subtilis is a Gram-positive bacterium that is well known for its ability to differentiate into metabolically inactive spores that are highly resistant to environmental stresses. In fact, populations of genetically identical B. subtilis comprise numerous distinct cell types. In addition to spores, cells can become genetically competent, motile, produce extracellular matrix or degradative enzymes, or secrete toxins that allow them to cannibalize their neighbors. Many of the cell fates listed above appear to be mutually exclusive. In this review, we discuss how individual cells within a population control their gene expression to ensure that proper regulation of differentiation occurs. These different cell fates are regulated by an intricate network that relies primarily on the activity of three major transcriptional regulators: Spo0A, DegU, and ComK. While individual cells must choose distinct cell fates, the population as a whole exhibits a spectrum of phenotypes whose diversity may increase fitness.
Genome-Wide Mapping of Collier In Vivo Binding Sites Highlights Its Hierarchical Position in Different Transcription Regulatory Networks

PubMed Central

Dubois, Laurence; Bataillé, Laetitia; Painset, Anaïs; Le Gras, Stéphanie; Jost, Bernard; Crozatier, Michèle; Vincent, Alain

2015-01-01

Collier, the single Drosophila COE (Collier/EBF/Olf-1) transcription factor, is required in several developmental processes, including head patterning and specification of muscle and neuron identity during embryogenesis. To identify direct Collier (Col) targets in different cell types, we used ChIP-seq to map Col binding sites throughout the genome, at mid-embryogenesis. In vivo Col binding peaks were associated to 415 potential direct target genes. Gene Ontology analysis revealed a strong enrichment in proteins with DNA binding and/or transcription-regulatory properties. Characterization of a selection of candidates, using transgenic CRM-reporter assays, identified direct Col targets in dorso-lateral somatic muscles and specific neuron types in the central nervous system. These data brought new evidence that Col direct control of the expression of the transcription regulators apterous and eyes-absent (eya) is critical to specifying neuronal identities. They also showed that cross-regulation between col and eya in muscle progenitor cells is required for specification of muscle identity, revealing a new parallel between the myogenic regulatory networks operating in Drosophila and vertebrates. Col regulation of eya, both in specific muscle and neuronal lineages, may illustrate one mechanism behind the evolutionary diversification of Col biological roles. PMID:26204530
The protein expression landscape of the Arabidopsis root

PubMed Central

Petricka, Jalean J.; Schauer, Monica A.; Megraw, Molly; Breakfield, Natalie W.; Thompson, J. Will; Georgiev, Stoyan; Soderblom, Erik J.; Ohler, Uwe; Moseley, Martin Arthur; Grossniklaus, Ueli; Benfey, Philip N.

2012-01-01

Because proteins are the major functional components of cells, knowledge of their cellular localization is crucial to gaining an understanding of the biology of multicellular organisms. We have generated a protein expression map of the Arabidopsis root providing the identity and cell type-specific localization of nearly 2,000 proteins. Grouping proteins into functional categories revealed unique cellular functions and identified cell type-specific biomarkers. Cellular colocalization provided support for numerous protein–protein interactions. With a binary comparison, we found that RNA and protein expression profiles are weakly correlated. We then performed peak integration at cell type-specific resolution and found an improved correlation with transcriptome data using continuous values. We performed GeLC-MS/MS (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry) proteomic experiments on mutants with ectopic and no root hairs, providing complementary proteomic data. Finally, among our root hair-specific proteins we identified two unique regulators of root hair development. PMID:22447775
Keratin 17 null mice exhibit age- and strain-dependent alopecia.

PubMed

McGowan, Kevin M; Tong, Xuemei; Colucci-Guyon, Emma; Langa, Francina; Babinet, Charles; Coulombe, Pierre A

2002-06-01

Onset of type I keratin 17 (K17) synthesis marks the adoption of an appendageal fate within embryonic ectoderm, and its expression persists in specific cell types within mature hair, glands, and nail. We report that K17 null mice develop severe alopecia during the first week postbirth, correlating with hair fragility, alterations in follicular histology, and apoptosis in matrix cells. These alterations are incompletely penetrant and normalize starting with the first postnatal cycle. Absence of a hair phenotype correlates with a genetic strain-dependent compensation by related keratins, including K16. These findings reveal a crucial role for K17 in the structural integrity of the first hair produced and the survival of hair-producing cells. Given that identical inherited mutations in this gene can cause either pachyonychia congenita or steatocystoma multiplex, the features of this mouse model suggest that this clinical heterogeneity arises from a cell type-specific, genetically determined compensation by related keratins.
Competent for commitment: you've got to have heart!

PubMed

Jain, Rajan; Epstein, Jonathan A

2018-01-01

The mature heart is composed primarily of four different cell types: cardiac myocytes, endothelium, smooth muscle, and fibroblasts. These cell types derive from pluripotent progenitors that become progressively restricted with regard to lineage potential, giving rise to multipotent cardiac progenitor cells and, ultimately, the differentiated cell types of the heart. Recent studies have begun to shed light on the defining characteristics of the intermediary cell types that exist transiently during this developmental process and the extrinsic and cell-autonomous factors that influence cardiac lineage decisions and cellular competence. This information will shape our understanding of congenital and adult cardiac disease and guide regenerative therapeutic approaches. In addition, cardiac progenitor specification can serve as a model for understanding basic mechanisms regulating the acquisition of cellular identity. In this review, we present the concept of "chromatin competence" that describes the potential for three-dimensional chromatin organization to function as the molecular underpinning of the ability of a progenitor cell to respond to inductive lineage cues and summarize recent studies advancing our understanding of cardiac cell specification, gene regulation, and chromatin organization and how they impact cardiac development. © 2018 Jain and Epstein; Published by Cold Spring Harbor Laboratory Press.
Characteristics of allelic gene expression in human brain cells from single-cell RNA-seq data analysis.

PubMed

Zhao, Dejian; Lin, Mingyan; Pedrosa, Erika; Lachman, Herbert M; Zheng, Deyou

2017-11-10

Monoallelic expression of autosomal genes has been implicated in human psychiatric disorders. However, there is a paucity of allelic expression studies in human brain cells at the single cell and genome wide levels. In this report, we reanalyzed a previously published single-cell RNA-seq dataset from several postmortem human brains and observed pervasive monoallelic expression in individual cells, largely in a random manner. Examining single nucleotide variants with a predicted functional disruption, we found that the "damaged" alleles were overall expressed in fewer brain cells than their counterparts, and at a lower level in cells where their expression was detected. We also identified many brain cell type-specific monoallelically expressed genes. Interestingly, many of these cell type-specific monoallelically expressed genes were enriched for functions important for those brain cell types. In addition, function analysis showed that genes displaying monoallelic expression and correlated expression across neuronal cells from different individual brains were implicated in the regulation of synaptic function. Our findings suggest that monoallelic gene expression is prevalent in human brain cells, which may play a role in generating cellular identity and neuronal diversity and thus increasing the complexity and diversity of brain cell functions.
Single cell analysis of voltage-gated potassium channels that determines neuronal types of rat hypothalamic paraventricular nucleus neurons.

PubMed

Lee, S K; Lee, S; Shin, S Y; Ryu, P D; Lee, S Y

2012-03-15

The hypothalamic paraventricular nucleus (PVN), a site for the integration of both the neuroendocrine and autonomic systems, has heterogeneous cell composition. These neurons are classified into type I and type II neurons based on their electrophysiological properties. In the present study, we investigated the molecular identification of voltage-gated K+ (Kv) channels, which determines a distinctive characteristic of type I PVN neurons, by means of single-cell reverse transcription-polymerase chain reaction (RT-PCR) along with slice patch clamp recordings. In order to determine the mRNA expression profiles, firstly, the PVN neurons of male rats were classified into type I and type II neurons, and then, single-cell RT-PCR and single-cell real-time RT-PCR analysis were performed using the identical cell. The single-cell RT-PCR analysis revealed that Kv1.2, Kv1.3, Kv1.4, Kv4.1, Kv4.2, and Kv4.3 were expressed both in type I and in type II neurons, and several Kv channels were co-expressed in a single PVN neuron. However, we found that the expression densities of Kv4.2 and Kv4.3 were significantly higher in type I neurons than in type II neurons. Taken together, several Kv channels encoding A-type K+ currents are present both in type I and in type II neurons, and among those, Kv4.2 and Kv4.3 are the major Kv subunits responsible for determining the distinct electrophysiological properties. Thus these 2 Kv subunits may play important roles in determining PVN cell types and regulating PVN neuronal excitability. This study further provides key molecular mechanisms for differentiating type I and type II PVN neurons. Copyright Â© 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
Molecular characterization of chronic-type adult T-cell leukemia/lymphoma.

PubMed

Yoshida, Noriaki; Karube, Kennosuke; Utsunomiya, Atae; Tsukasaki, Kunihiro; Imaizumi, Yoshitaka; Taira, Naoya; Uike, Naokuni; Umino, Akira; Arita, Kotaro; Suguro, Miyuki; Tsuzuki, Shinobu; Kinoshita, Tomohiro; Ohshima, Koichi; Seto, Masao

2014-11-01

Adult T-cell leukemia/lymphoma (ATL) is a human T-cell leukemia virus type-1-induced neoplasm with four clinical subtypes: acute, lymphoma, chronic, and smoldering. Although the chronic type is regarded as indolent ATL, about half of the cases progress to acute-type ATL. The molecular pathogenesis of acute transformation in chronic-type ATL is only partially understood. In an effort to determine the molecular pathogeneses of ATL, and especially the molecular mechanism of acute transformation, oligo-array comparative genomic hybridization and comprehensive gene expression profiling were applied to 27 and 35 cases of chronic and acute type ATL, respectively. The genomic profile of the chronic type was nearly identical to that of acute-type ATL, although more genomic alterations characteristic of acute-type ATL were observed. Among the genomic alterations frequently observed in acute-type ATL, the loss of CDKN2A, which is involved in cell-cycle deregulation, was especially characteristic of acute-type ATL compared with chronic-type ATL. Furthermore, we found that genomic alteration of CD58, which is implicated in escape from the immunosurveillance mechanism, is more frequently observed in acute-type ATL than in the chronic-type. Interestingly, the chronic-type cases with cell-cycle deregulation and disruption of immunosurveillance mechanism were associated with earlier progression to acute-type ATL. These findings suggested that cell-cycle deregulation and the immune escape mechanism play important roles in acute transformation of the chronic type and indicated that these alterations are good predictive markers for chronic-type ATL. ©2014 American Association for Cancer Research.
Gap junctional coupling in the vertebrate retina: variations on one theme?

PubMed

Völgyi, Béla; Kovács-Oller, Tamás; Atlasz, Tamás; Wilhelm, Márta; Gábriel, Róbert

2013-05-01

Gap junctions connect cells in the bodies of all multicellular organisms, forming either homologous or heterologous (i.e. established between identical or different cell types, respectively) cell-to-cell contacts by utilizing identical (homotypic) or different (heterotypic) connexin protein subunits. Gap junctions in the nervous system serve electrical signaling between neurons, thus they are also called electrical synapses. Such electrical synapses are particularly abundant in the vertebrate retina where they are specialized to form links between neurons as well as glial cells. In this article, we summarize recent findings on retinal cell-to-cell coupling in different vertebrates and identify general features in the light of the evergrowing body of data. In particular, we describe and discuss tracer coupling patterns, connexin proteins, junctional conductances and modulatory processes. This multispecies comparison serves to point out that most features are remarkably conserved across the vertebrate classes, including (i) the cell types connected via electrical synapses; (ii) the connexin makeup and the conductance of each cell-to-cell contact; (iii) the probable function of each gap junction in retinal circuitry; (iv) the fact that gap junctions underlie both electrical and/or tracer coupling between glial cells. These pan-vertebrate features thus demonstrate that retinal gap junctions have changed little during the over 500 million years of vertebrate evolution. Therefore, the fundamental architecture of electrically coupled retinal circuits seems as old as the retina itself, indicating that gap junctions deeply incorporated in retinal wiring from the very beginning of the eye formation of vertebrates. In addition to hard wiring provided by fast synaptic transmitter-releasing neurons and soft wiring contributed by peptidergic, aminergic and purinergic systems, electrical coupling may serve as the 'skeleton' of lateral processing, enabling important functions such as signal averaging and synchronization. 2013 Elsevier Ltd. All rights reserved.
Cell type of origin as well as genetic alterations contribute to breast cancer phenotypes

PubMed Central

West, William W.; Qiu, Fang; Band, Hamid; Band, Vimla

2015-01-01

Breast cancer is classified into different subtypes that are associated with different patient survival outcomes, underscoring the importance of understanding the role of precursor cell and genetic alterations in determining tumor subtypes. In this study, we evaluated the oncogenic phenotype of two distinct mammary stem/progenitor cell types designated as K5+/K19− or K5+/K19+ upon introduction of identical combinations of oncogenes-mutant H-Ras (mRas) and mutant p53 (mp53), together with either wild-type ErbB2(wtErbB2) or wild-type EGFR (wtEGFR). We examined their tumor forming and metastasis potential, using both in-vitro and in-vivo assays. Both the combinations efficiently transformed K5+/K19− or K5+/K19+ cells. Xenograft tumors formed by these cells were histologically heterogeneous, with variable proportions of luminal, basal-like and claudin-low type components depending on the cell types and oncogene combinations. Notably, K5+/K19− cells transformed with mRas/mp53/wtEGFR combination had a significantly longer latency for primary tumor development than other cell lines but more lung metastasis incidence than same cells expressing mRas/mp53/wtErbB2. K5+/K19+ cells exhibit shorter overall tumor latency, and high metastatic potential than K5+/K19− cells, suggesting that these K19+ progenitors are more susceptible to oncogenesis and metastasis. Our results suggest that both genetic alterations and cell type of origin contribute to oncogenic phenotype of breast tumors. PMID:25940703
Cell-of-Origin DNA Methylation Signatures Are Maintained during Colorectal Carcinogenesis.

PubMed

Bormann, Felix; Rodríguez-Paredes, Manuel; Lasitschka, Felix; Edelmann, Dominic; Musch, Tanja; Benner, Axel; Bergman, Yehudit; Dieter, Sebastian M; Ball, Claudia R; Glimm, Hanno; Linhart, Heinz G; Lyko, Frank

2018-06-12

Colorectal adenomas are precursor lesions of colorectal cancers and represent clonal amplifications of single cells from colonic crypts. DNA methylation patterns specify cell-type identity during cellular differentiation and, therefore, provide opportunities for the molecular analysis of tumors. We have now analyzed DNA methylation patterns in colorectal adenomas and identified three biologically defined subclasses that describe different intestinal crypt differentiation stages. Importantly, colorectal carcinomas could be classified into the same methylation subtypes, reflecting their shared cell types of origin with adenomas. Further data analysis also revealed significantly reduced overall survival for one of the subtypes. Our results provide a concept for understanding the methylation patterns observed in colorectal cancer and provide opportunities for tumor subclassification and patient stratification. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
Ctip2-, Satb2-, Prox1-, and GAD65-Expressing Neurons in Rat Cultures: Preponderance of Single- and Double-Positive Cells, and Cell Type-Specific Expression of Neuron-Specific Gene Family Members, Nsg-1 (NEEP21) and Nsg-2 (P19).

PubMed

Digilio, Laura; Yap, Chan Choo; Winckler, Bettina

2015-01-01

The brain consists of many distinct neuronal cell types, but which cell types are present in widely used primary cultures of embryonic rodent brain is often not known. We characterized how abundantly four cell type markers (Ctip2, Satb2, Prox1, GAD65) were represented in cultured rat neurons, how easily neurons expressing different markers can be transfected with commonly used plasmids, and whether neuronal-enriched endosomal proteins Nsg-1 (NEEP21) and Nsg-2 (P19) are ubiquitously expressed in all types of cultured neurons. We found that cultured neurons stably maintain cell type identities that are reflective of cell types in vivo. This includes neurons maintaining simultaneous expression of two transcription factors, such as Ctip2+/Satb2+ or Prox1+/Ctip2+ double-positive cells, which have also been described in vivo. Secondly, we established the superior efficiency of CAG promoters for both Lipofectamine-mediated transfection as well as for electroporation. Thirdly, we discovered that Nsg-1 and Nsg-2 were not expressed equally in all neurons: whereas high levels of both Nsg-1 and Nsg-2 were found in Satb2-, Ctip2-, and GAD65-positive neurons, Prox1-positive neurons in hippocampal cultures expressed low levels of both. Our findings thus highlight the importance of identifying neuronal cell types for doing cell biology in cultured neurons: Keeping track of neuronal cell type might uncover effects in assays that might otherwise be masked by the mixture of responsive and non-responsive neurons in the dish.
Molecular and phenotypic analyses reveal the non-identity of the Phaeobacter gallaeciensis type strain deposits CIP 105210T and DSM 17395.

PubMed

Buddruhs, Nora; Pradella, Silke; Göker, Markus; Päuker, Orsola; Pukall, Rüdiger; Spröer, Cathrin; Schumann, Peter; Petersen, Jörn; Brinkhoff, Thorsten

2013-11-01

The marine genus Phaeobacter currently comprises six species, some of which were intensively studied mainly due to their ability to produce secondary metabolites. The type strain of the type species, Phaeobacter gallaeciensis BS107(T), has been deposited at several public culture collections worldwide. Based on differences in plasmid profiles, we detected that the alleged P. gallaeciensis type strains deposited at the Collection Institute Pasteur (CIP; Paris, France) as CIP 105210 and at the German Collection of Microorganisms and Cell Cultures (DSMZ; Braunschweig, Germany) as DSM 17395 are not identical. To determine the identity of these strains, we conducted DNA-DNA hybridization, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), 16S rRNA gene and internal transcribed spacer (ITS) sequence analyses, as well as physiological experiments. Based on the detailed 16S rRNA gene reanalysis we showed that strain CIP 105210 most likely corresponds to the original P. gallaeciensis type strain BS107(T). In contrast, the Phaeobacter strain DSM 17395 exhibits a much closer affiliation to Phaeobacter inhibens DSM 16374(T) ( = T5(T)) and should thus be allocated to this species. The detection of the dissimilarity of strains CIP 105210(T) and DSM 17395 will influence future comparative studies within the genus Phaeobacter.
Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

PubMed Central

Mizuno, Takako; Sridharan, Anusha; Du, Yina; Guo, Minzhe; Wikenheiser-Brokamp, Kathryn A.; Perl, Anne-Karina T.; Funari, Vincent A.; Gokey, Jason J.; Stripp, Barry R.; Whitsett, Jeffrey A.

2016-01-01

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease. PMID:27942595

Changes in Ca(2+) channel expression upon differentiation of SN56 cholinergic cells.

PubMed

Kushmerick, C; Romano-Silva, M A; Gomez, M V; Prado, M A

2001-10-19

The SN56 cell line, a fusion of septal neurons and neuroblastoma cells, has been used as a model for central cholinergic neurons. These cells show increased expression of cholinergic neurochemical features upon differentiation, but little is known about how differentiation affects their electrophysiological properties. We examined the changes in Ca(2+) channel expression that occur as these cells undergo morphological differentiation in response to serum withdrawal and exposure to dibutyryl-cAMP. Undifferentiated cells expressed a T-type current with biophysical and pharmacological properties similar, although not identical, to those reported for the current generated by the alpha(1H) (CaV3.2) Ca(2+) channel subunit. Differentiated cells expressed, in addition to this T-type current, high voltage activated currents which were inhibited 38% by the L-type channel antagonist nifedipine (5 microM), 37% by the N-type channel antagonist omega-conotoxin-GVIA (1 microM), and 15% by the P/Q-type channel antagonist omega-agatoxin-IVA (200 nM). Current resistant to these inhibitors accounted for 15% of the high voltage activated current in differentiated SN56 cells. Our data demonstrate that differentiation increases the expression of neuronal type voltage gated Ca(2+) channels in this cell line, and that the channels expressed are comparable to those reported for native basal forebrain cholinergic neurons. This cell line should thus provide a useful model system to study the relationship between calcium currents and cholinergic function and dysfunction.
[Induced pluripotent stem cells: a new paradigm to study human tissues].

PubMed

Sansac, Caroline; Assou, Said; Bouckenheimer, Julien; Lemaître, Jean-Marc; De Vos, John

2016-01-01

Induced pluripotent stem cells (iPSCs) are obtained by reprogramming differentiated cells through forced expression of four embryonic transcription factors. The discovery of this technology, able to transform a differentiated cell into a pluripotent cell, has profoundly shifted the paradigm of the concept of cell identity, since it is now possible to obtain in vitro any cell type from an initial sample of skin or blood cells from a healthy volunteer or patient. Applications of iPSCs are exceedingly large, and comprise the in vitro modeling of normal or pathological tissues, including for massive drug screening. They also open new therapeutic avenues in the field of regenerative medicine. © Société de Biologie, 2016.
The science of stem cell biobanking: investing in the future.

PubMed

Diaferia, Giuseppe R; Cardano, Marina; Cattaneo, Monica; Spinelli, Chiara C; Dessì, Sara S; DeBlasio, Pasquale; Biunno, Ida

2012-01-01

The use of human stem cells in biomedical research projects is increasing steadily and the number of cells that are being derived develops at a remarkable pace. However, stem cells around the world are vastly different in their provenance, programming, and potentials. Furthermore, knowledge on the actual number of cell types, their derivation, availability, and characteristics is rather sparse. Usually, "colleague-supply" avenues constantly furnish cells to laboratories around the world without ensuring their correct identity, characterization, and quality. These parameters are critical if the cells will be eventually used in toxicology studies and drug discovery. Here, we outline some basic principles in establishing a stem cell-specific bank. Copyright © 2011 Wiley Periodicals, Inc.
Wnt Signaling Specifies Anteroposterior Progenitor Zone Identity in the Drosophila Visual Center.

PubMed

Suzuki, Takumi; Trush, Olena; Yasugi, Tetsuo; Takayama, Rie; Sato, Makoto

2016-06-15

During brain development, various types of neuronal populations are produced from different progenitor pools to produce neuronal diversity that is sufficient to establish functional neuronal circuits. However, the molecular mechanisms that specify the identity of each progenitor pool remain obscure. Here, we show that Wnt signaling is essential for the specification of the identity of posterior progenitor pools in the Drosophila visual center. In the medulla, the largest component of the visual center, different types of neurons are produced from two progenitor pools: the outer proliferation center (OPC) and glial precursor cells (GPCs; also known as tips of the OPC). We found that OPC-type neurons are produced from the GPCs at the expense of GPC-type neurons when Wnt signaling is suppressed in the GPCs. In contrast, GPC-type neurons are ectopically induced when Wnt signaling is ectopically activated in the OPC. These results suggest that Wnt signaling is necessary and sufficient for the specification of the progenitor pool identity. We also found that Homothorax (Hth), which is temporally expressed in the OPC, is ectopically induced in the GPCs by suppression of Wnt signaling and that ectopic induction of Hth phenocopies the suppression of Wnt signaling in the GPCs. Thus, Wnt signaling is involved in regionalization of the fly visual center through the specification of the progenitor pool located posterior to the medulla by suppressing Hth expression. Brain consists of considerably diverse neurons of different origins. In mammalian brain, excitatory and inhibitory neurons derive from the dorsal and ventral telencephalon, respectively. Multiple progenitor pools also contribute to the neuronal diversity in fly brain. However, it has been unclear how differences between these progenitor pools are established. Here, we show that Wnt signaling, an evolutionarily conserved signaling, is involved in the process that establishes the differences between these progenitor pools. Because β-catenin signaling, which is under the control of Wnt ligands, specifies progenitor pool identity in the developing mammalian thalamus, Wnt signaling-mediated specification of progenitor pool identity may be conserved in insect and mammalian brains. Copyright © 2016 the authors 0270-6474/16/366503-11$15.00/0.
Proposal of a neutron transmutation doping facility for n-type spherical silicon solar cell at high-temperature engineering test reactor.

PubMed

Ho, Hai Quan; Honda, Yuki; Motoyama, Mizuki; Hamamoto, Shimpei; Ishii, Toshiaki; Ishitsuka, Etsuo

2018-05-01

The p-type spherical silicon solar cell is a candidate for future solar energy with low fabrication cost, however, its conversion efficiency is only about 10%. The conversion efficiency of a silicon solar cell can be increased by using n-type silicon semiconductor as a substrate. This study proposed a new method of neutron transmutation doping silicon (NTD-Si) for producing the n-type spherical solar cell, in which the Si-particles are irradiated directly instead of the cylinder Si-ingot as in the conventional NTD-Si. By using a 'screw', an identical resistivity could be achieved for the Si-particles without a complicated procedure as in the NTD with Si-ingot. Also, the reactivity and neutron flux swing could be kept to a minimum because of the continuous irradiation of the Si-particles. A high temperature engineering test reactor (HTTR), which is located in Japan, was used as a reference reactor in this study. Neutronic calculations showed that the HTTR has a capability to produce about 40t/EFPY of 10Ωcm resistivity Si-particles for fabrication of the n-type spherical solar cell. Copyright © 2018 Elsevier Ltd. All rights reserved.
Development of Extranodal NK/T-cell Lymphoma Nasal Type in Cerebrum Following Epstein-Barr Virus-positive Uveitis.

PubMed

Imai, Ayano; Takase, Hiroshi; Imadome, Ken-Ichi; Matsuda, Go; Ohnishi, Iichiro; Yamamoto, Kouhei; Kudo, Takumi; Tanaka, Yoji; Maehara, Taketoshi; Miura, Osamu; Arai, Ayako

2017-01-01

A 74-year-old woman developed bilateral uveitis with high Epstein-Barr virus (EBV) DNA load in the vitreous fluid without lymphoma cells. Four years after the onset, T2-weighted contrast-enhanced MRI revealed hyperintense lesions in the right occipital and parietal lobe. A biopsy resulted in the diagnosis of extranodal NK/T-cell lymphoma nasal type (ENKL). The repeat region of LMP1, an EBV gene, detected in the brain lesion was identical to that detected in the vitreous fluid. ENKL of the central nervous system is quite rare, and the pathogenesis has not been determined. The lymphoma in this case might have been closely associated with the EBV-positive uveitis.
Multiple scale model for cell migration in monolayers: Elastic mismatch between cells enhances motility

NASA Astrophysics Data System (ADS)

Palmieri, Benoit; Bresler, Yony; Wirtz, Denis; Grant, Martin

2015-07-01

We propose a multiscale model for monolayer of motile cells that comprise normal and cancer cells. In the model, the two types of cells have identical properties except for their elasticity; cancer cells are softer and normal cells are stiffer. The goal is to isolate the role of elasticity mismatch on the migration potential of cancer cells in the absence of other contributions that are present in real cells. The methodology is based on a phase-field description where each cell is modeled as a highly-deformable self-propelled droplet. We simulated two types of nearly confluent monolayers. One contains a single cancer cell in a layer of normal cells and the other contains normal cells only. The simulation results demonstrate that elasticity mismatch alone is sufficient to increase the motility of the cancer cell significantly. Further, the trajectory of the cancer cell is decorated by several speed “bursts” where the cancer cell quickly relaxes from a largely deformed shape and consequently increases its translational motion. The increased motility and the amplitude and frequency of the bursts are in qualitative agreement with recent experiments.
THE PURINERGIC NEUROTRANSMITTER REVISITED: A SINGLE SUBSTANCE OR MULTIPLE PLAYERS?

PubMed Central

Mutafova-Yambolieva, Violeta N.; Durnin, Leonie

2014-01-01

The past half century has witnessed tremendous advances in our understanding of extracellular purinergic signaling pathways. Purinergic neurotransmission, in particular, has emerged as a key contributor in the efficient control mechanisms in the nervous system. The identity of the purine neurotransmitter, however, remains controversial. Identifying it is difficult because purines are present in all cell types, have a large variety of cell sources, and are released via numerous pathways. Moreover, studies on purinergic neurotransmission have relied heavily on indirect measurements of integrated postjunctional responses that do not provide direct information for neurotransmitter identity. This paper discusses experimental support for adenosine 5′-triphosphate (ATP) as a neurotransmitter and recent evidence for possible contribution of other purines, in addition to or instead of ATP, in chemical neurotransmission in the peripheral, enteric and central nervous systems. Sites of release and action of purines in model systems such as vas deferens, blood vessels, urinary bladder and chromaffin cells are discussed. This is preceded by a brief discussion of studies demonstrating storage of purines in synaptic vesicles. We examine recent evidence for cell type targets (e.g., smooth muscle cells, interstitial cells, neurons and glia) for purine neurotransmitters in different systems. This is followed by brief discussion of mechanisms of terminating the action of purine neurotransmitters, including extracellular nucleotide hydrolysis and possible salvage and reuptake in the cell. The significance of direct neurotransmitter release measurements is highlighted. Possibilities for involvement of multiple purines (e.g., ATP, ADP, NAD+, ADP-ribose, adenosine, and diadenosine polyphosphates) in neurotransmission are considered throughout. PMID:24887688
A cysteine protease encoded by the baculovirus Bombyx mori nuclear polyhedrosis virus.

PubMed Central

Ohkawa, T; Majima, K; Maeda, S

1994-01-01

Sequence analysis of the BamHI F fragment of the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV) revealed an open reading frame whose deduced amino acid sequence had homology to those of cysteine proteases of the papain superfamily. The putative cysteine protease sequence (BmNPV-CP) was 323 amino acids long and showed 35% identity to a cysteine proteinase precursor from Trypanosoma brucei. Of 36 residues conserved among cathepsins B, H, L, and S and papain, 31 were identical in BmNPV-CP. In order to determine the activity and function of the putative cysteine protease, a BmNPV mutant (BmCysPD) was constructed by homologous recombination of the protease gene with a beta-galactosidase gene cassette. BmCysPD-infected BmN cell extracts were significantly reduced in acid protease activity compared with wild-type virus-infected cell extracts. The cysteine protease inhibitor E-64 [trans-epoxysuccinylleucylamido-(4-guanidino)butane] inhibited wild-type virus-expressed protease activity. Deletion of the cysteine protease gene had no significant effect on viral growth or polyhedron production in BmN cells, indicating that the cysteine protease was not essential for viral replication in vitro. However, B. mori larvae infected with BmCysPD showed symptoms different from those of wild-type BmNPV-infected larvae, e.g., less degradation of the body, including fat body cells, white body surface color due presumably to undegraded epidermal cells, and an increase in the number of polyhedra released into the hemolymph. This is the first report of (i) a virus-encoded protease with activity on general substrates and (ii) evidence that a virus-encoded protease may play a role in degradation of infected larvae to facilitate horizontal transmission of the virus. Images PMID:8083997
Automatic identification of informative regions with epigenomic changes associated to hematopoiesis

PubMed Central

Carrillo-de-Santa-Pau, Enrique; Pancaldi, Vera; Were, Felipe; Martin-Subero, Ignacio

2017-01-01

Abstract Hematopoiesis is one of the best characterized biological systems but the connection between chromatin changes and lineage differentiation is not yet well understood. We have developed a bioinformatic workflow to generate a chromatin space that allows to classify 42 human healthy blood epigenomes from the BLUEPRINT, NIH ROADMAP and ENCODE consortia by their cell type. This approach let us to distinguish different cells types based on their epigenomic profiles, thus recapitulating important aspects of human hematopoiesis. The analysis of the orthogonal dimension of the chromatin space identify 32,662 chromatin determinant regions (CDRs), genomic regions with different epigenetic characteristics between the cell types. Functional analysis revealed that these regions are linked with cell identities. The inclusion of leukemia epigenomes in the healthy hematological chromatin sample space gives us insights on the healthy cell types that are more epigenetically similar to the disease samples. Further analysis of tumoral epigenetic alterations in hematopoietic CDRs points to sets of genes that are tightly regulated in leukemic transformations and commonly mutated in other tumors. Our method provides an analytical approach to study the relationship between epigenomic changes and cell lineage differentiation. Method availability: https://github.com/david-juan/ChromDet. PMID:28934481
Distribution and photobiology of Symbiodinium types in different light environments for three colour morphs of the coral Madracis pharensis: is there more to it than total irradiance?

NASA Astrophysics Data System (ADS)

Frade, P. R.; Englebert, N.; Faria, J.; Visser, P. M.; Bak, R. P. M.

2008-12-01

The role of symbiont variation in the photobiology of reef corals was addressed by investigating the links among symbiont genetic diversity, function and ecological distribution in a single host species, Madracis pharensis. Symbiont distribution was studied for two depths (10 and 25 m), two different light habitats (exposed and shaded) and three host colour morphs (brown, purple and green). Two Symbiodinium genotypes were present, as defined by nuclear internal transcribed spacer 2 ribosomal DNA (ITS2-rDNA) variation. Symbiont distribution was depth- and colour morph-dependent. Type B15 occurred predominantly on the deeper reef and in green and purple colonies, while type B7 was present in shallow environments and brown colonies. Different light microhabitats at fixed depths had no effect on symbiont presence. This ecological distribution suggests that symbiont presence is potentially driven by light spectral niches. A reciprocal depth transplantation experiment indicated steady symbiont populations under environment change. Functional parameters such as pigment composition, chlorophyll a fluorescence and cell densities were measured for 25 m and included in multivariate analyses. Most functional variation was explained by two photobiological assemblages that relate to either symbiont identity or light microhabitat, suggesting adaptation and acclimation, respectively. Type B15 occurs with lower cell densities and larger sizes, higher cellular pigment concentrations and higher peridinin to chlorophyll a ratio than type B7. Type B7 relates to a larger xanthophyll-pool size. These unambiguous differences between symbionts can explain their distributional patterns, with type B15 being potentially more adapted to darker or deeper environments than B7. Symbiont cell size may play a central role in the adaptation of coral holobionts to the deeper reef. The existence of functional differences between B-types shows that the clade classification does not necessarily correspond to functional identity. This study supports the use of ITS2 as an ecological and functionally meaningful marker in Symbiodinium.
Compensatory branching morphogenesis of stalk cells in the Drosophila trachea.

PubMed

Francis, Deanne; Ghabrial, Amin S

2015-06-01

Tubes are essential for nutrient transport and gas exchange in multicellular eukaryotes, but how connections between different tube types are maintained over time is unknown. In the Drosophila tracheal system, mutations in oak gall (okg) and conjoined (cnj) confer identical defects, including late onset blockage near the terminal cell-stalk cell junction and the ectopic extension of autocellular, seamed tubes into the terminal cell. We determined that okg and cnj encode the E and G subunits of the vacuolar ATPase (vATPase) and showed that both the V0 and V1 domains are required for terminal cell morphogenesis. Remarkably, the ectopic seamed tubes running along vATPase-deficient terminal cells belonged to the neighboring stalk cells. All vATPase-deficient tracheal cells had reduced apical domains and terminal cells displayed mislocalized apical proteins. Consistent with recent reports that the mTOR and vATPase pathways intersect, we found that mTOR pathway mutants phenocopied okg and cnj. Furthermore, terminal cells depleted for the apical determinants Par6 or aPKC had identical ectopic seamed tube defects. We thus identify a novel mechanism of compensatory branching in which stalk cells extend autocellular tubes into neighboring terminal cells with undersized apical domains. This compensatory branching also occurs in response to injury, with damaged terminal cells being rapidly invaded by their stalk cell neighbor. © 2015. Published by The Company of Biologists Ltd.
Deep sequencing reveals cell-type-specific patterns of single-cell transcriptome variation.

PubMed

Dueck, Hannah; Khaladkar, Mugdha; Kim, Tae Kyung; Spaethling, Jennifer M; Francis, Chantal; Suresh, Sangita; Fisher, Stephen A; Seale, Patrick; Beck, Sheryl G; Bartfai, Tamas; Kuhn, Bernhard; Eberwine, James; Kim, Junhyong

2015-06-09

Differentiation of metazoan cells requires execution of different gene expression programs but recent single-cell transcriptome profiling has revealed considerable variation within cells of seeming identical phenotype. This brings into question the relationship between transcriptome states and cell phenotypes. Additionally, single-cell transcriptomics presents unique analysis challenges that need to be addressed to answer this question. We present high quality deep read-depth single-cell RNA sequencing for 91 cells from five mouse tissues and 18 cells from two rat tissues, along with 30 control samples of bulk RNA diluted to single-cell levels. We find that transcriptomes differ globally across tissues with regard to the number of genes expressed, the average expression patterns, and within-cell-type variation patterns. We develop methods to filter genes for reliable quantification and to calibrate biological variation. All cell types include genes with high variability in expression, in a tissue-specific manner. We also find evidence that single-cell variability of neuronal genes in mice is correlated with that in rats consistent with the hypothesis that levels of variation may be conserved. Single-cell RNA-sequencing data provide a unique view of transcriptome function; however, careful analysis is required in order to use single-cell RNA-sequencing measurements for this purpose. Technical variation must be considered in single-cell RNA-sequencing studies of expression variation. For a subset of genes, biological variability within each cell type appears to be regulated in order to perform dynamic functions, rather than solely molecular noise.
Regulation of the Arabidopsis root vascular initial population by LONESOME HIGHWAY

PubMed Central

Ohashi-Ito, Kyoko; Bergmann, Dominique C.

2011-01-01

Complex organisms consist of a multitude of cell types arranged in precise spatial relation to each other. Arabidopsis roots generally exhibit radial tissue organization; however, within a tissue layer, cells are not identical. Specific vascular cell types are arranged in diametrically opposed longitudinal files that maximize the distance between them and create a bilaterally symmetric (diarch) root. Mutations in the LONESOME HIGHWAY (LHW) gene eliminate bilateral symmetry and reduce the number of cells in the center of the root, resulting in roots with only single and xylem and phloem poles. LHW does not appear to be required for the creation of any specific cell type, but coordinately controls the number of all vascular cell types by regulating the size of the pool of cells from which they arise. We cloned LHW and found that it encodes a protein with weak sequence similarity to basic helix-loop-helix (bHLH) domain proteins. LHW is a transcriptional activator in vitro. In plants, LHW is nuclear localized and is expressed in the root meristems where we hypothesize it acts independently of other known root patterning genes to promote the production of stele cells, but may also indirectly feed into established regulatory networks for the maintenance of the root meristem. PMID:17626058
[Asymmetric effect of in-group social values on identity with common-identity and common-bond in-groups].

PubMed

Nakashima, Ken'ichiro; Isobe, Chikae; Toshihiko, Souma; Ura, Mitsuhiro

2013-06-01

Moderating effects of group type on the relationship between in-group social values and group identity were investigated. Previous research has indicated that values attached to the in-group, such as its status, privileges, and power, lead to increased group identity. However, these studies have not investigated the role of the type of in-groups on this effect. We conducted an experiment that manipulated the in-group type. In the common-identity type of in-group condition, formation of in- and out-groups on the basis of social categorization was established. In the common-bond type of in-group condition, interactions between the group members were conducted. Results indicated that in the former condition, the degree of in-group social values affected group identity; however, this effect was not found in the latter condition. These results suggest that social values of the in-group have an asymmetric effect on group identity, depending upon the in-group type as a common-identity or common-bond group.
Support of Unrelated Stem Cell Donor Searches by Donor Center-Initiated HLA Typing of Potentially Matching Donors

PubMed Central

Schmidt, Alexander H.; Solloch, Ute V.; Baier, Daniel; Grathwohl, Alois; Hofmann, Jan; Pingel, Julia; Stahr, Andrea; Ehninger, Gerhard

2011-01-01

Large registries of potential unrelated stem cell donors have been established in order to enable stem cell transplantation for patients without HLA-identical related donors. Donor search is complicated by the fact that the stored HLA information of many registered donors is incomplete. We carried out a project that was aimed to improve chances of patients with ongoing donor searches to find an HLA-matched unrelated donor. For that purpose, we carried out additional donor center-initiated HLA-DRB1 typing of donors who were only typed for the HLA loci A and B so far and were potential matches for patients in need of a stem cell transplant. In total, 8,861 donors were contacted for donor center-initiated HLA-DRB1 typing within 1,089 donor searches. 12 of these donors have donated stem cells so far, 8 thereof for their respective target patients. We conclude that chances of patients with ongoing donor searches to find an HLA-matched unrelated donor can indeed be improved by donor-center initiated typing that is carried out in addition to the standard donor search process. Our results also raise questions regarding the appropriate use of incompletely typed donors within unrelated donor searches. PMID:21625451
Mutation in the peb1A Locus of Campylobacter jejuni Reduces Interactions with Epithelial Cells and Intestinal Colonization of Mice

PubMed Central

Pei, Zhiheng; Burucoa, Christophe; Grignon, Bernadette; Baqar, Shahida; Huang, Xiao-Zhe; Kopecko, Dennis J.; Bourgeois, A. L.; Fauchere, Jean-Louis; Blaser, Martin J.

1998-01-01

Campylobacter jejuni is one of the leading causes of bacterial diarrhea throughout the world. We previously found that PEB1 is a homolog of cluster 3 binding proteins of bacterial ABC transporters and that a C. jejuni adhesin, cell-binding factor 1 (CBF1), if not identical to, contains PEB1. A single protein migrating at approximately 27 to 28 kDa was recognized by anti-CBF1 and anti-PEB1. To determine the role that the operon encoding PEB1 plays in C. jejuni adherence, peb1A, the gene encoding PEB1, was disrupted in strain 81-176 by insertion of a kanamycin resistance gene through homologous recombination. Inactivation of this operon completely abolished expression of CBF1, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. In comparison to the wild-type strain, the mutant strain showed 50- to 100-fold less adherence to and 15-fold less invasion of epithelial cells in culture. Mouse challenge studies showed that the rate and duration of intestinal colonization by the mutant were significantly lower and shorter than with the wild-type strain. In summary, PEB1 is identical to a previously identified cell-binding factor, CBF1, in C. jejuni, and the peb1A locus plays an important role in epithelial cell interactions and in intestinal colonization in a mouse model. PMID:9488379
Adoptive immunotherapy against kidney targets in dog-leukocyte antigen-identical mixed hematopoietic canine chimeras.

PubMed

Junghanss, Christian; Takatu, Alessandra; Little, Marie-Terese; Maciej Zaucha, J; Zellmer, Eustacia; Yunusov, Murad; Sale, George; Georges, George E; Storb, Rainer

2003-02-15

Stable mixed-donor-host-hematopoietic chimerism can serve as a platform for adoptive immunotherapy. Infusions of donor lymphocytes (DLI) sensitized against hematopoietic cells converted mixed hematopoietic into full-donor chimerism in dog-leukocyte antigen (DLA)-identical littermates. Whether sensitization against tissue of solid organs leads to organ-specific immunity that can be transferred by DLI was unknown and was investigated in these experiments using the kidney as target. DLA-identical recipients with established stable mixed-donor-host-hematopoietic chimerism were used. In five pairs, hematopoietic stem-cell transplant (HSCT) donors were sensitized by kidney transplantation from the respective chimeras. In a second group, five HSCT donors received vaccinations that were generated from kidney cells of the respective mixed chimeras. Twenty-eight days after sensitization, DLI were administered to the mixed-hematopoietic chimeras. All HSCT donors rejected their kidney grafts from their mixed-chimeric recipients within 22 to 45 days. DLI caused no sustained graft-versus-kidney effects in the mixed-chimeric recipients. However, DLI donors sensitized by kidney transplantation converted 4 of 5 mixed chimeras into virtually complete (>95%) donor-type chimeras, compared with 1 of 5 mixed chimeras given DLI by vaccination from sensitized donors. Although DLA-identical kidney grafts from mixed-hematopoietic chimeras were readily rejected by their HSCT donors, subsequent transfusions of sensitized-donor lymphocytes into mixed chimeras converted mixed to all-donor chimerism but failed to induce graft-versus-kidney effects. Vaccination strategies in lieu of kidney grafts failed to convert mixed chimerism.
Jacalin and peanut agglutinin (PNA) bindings in the taste bud cells of the rat: new reliable markers for type IV cells of the rat taste buds.

PubMed

Taniguchi, Ryo; Shi, Lei; Fujii, Masae; Ueda, Katsura; Honma, Shiho; Wakisaka, Satoshi

2005-12-01

Lectin histochemistry of Jacalin (Artocarpus integrifolia) and peanut agglutinin (PNA), specific lectins for galactosyl (beta-1, 3) N-acetylgalactosamine (galactosyl (beta-1, 3) GalNAc), was applied to the gustatory epithelium of the adult rat. In the ordinary lingual epithelium, Jacalin and PNA labeled the cell membrane from the basal to granular cell layer. They also bound membranes of rounded-cells at the basal portion of taste buds, but the number of PNA labeled cells was smaller than that of Jacalin labeled cells. There was no apparent difference in the binding patterns of Jacalin and PNA among the taste buds of the lingual papillae and those of the palatal epithelium. Occasionally, a few spindle-shaped cells were labeled with Jacalin, but not with PNA. Double labeling of Jacalin and alpha-gustducin, a specific marker for type II cells, revealed that Jacalin-labeled spindle-shaped taste cells were immunonegative for alpha-gustducin. Spindle-shaped cells expressing protein gene product 9.5 (PGP 9.5) immunoreactivity lacked Jacalin labeling. During the development of taste buds in circumvallate papillae, the binding pattern of Jacalin became almost identical from postnatal day 5. The present results indicate that rounded cells at the basal portion of the taste buds cells (type IV cells) bind to Jacalin and PNA, and these lectins are specific markers for type IV cells of the rat taste cells.
Collective school-type identity: predicting students' motivation beyond academic self-concept.

PubMed

Knigge, Michel; Hannover, Bettina

2011-06-01

In Germany, according to their prior achievement students are tracked into different types of secondary school that provide profoundly different options for their future educational careers. In this paper we suggest that as a result, school tracks clearly differ in their social status or reputation. This should translate into different collective school-type identities for their students, irrespective of the students' personal academic self-concepts. We examine the extent to which collective school-type identity systematically varies as a function of the school track students are enrolled in, and the extent to which students' collective school-type identity makes a unique contribution beyond academic self-concept and school track in predicting scholastic motivation. In two cross-sectional studies a measure of collective school-type identity is established and applied to explain motivational differences between two school tracks in Berlin. In Study 1 (N = 39 students) the content of the collective school-type identity is explored by means of an open format questionnaire. Based on these findings a structured instrument (semantic differential) to measure collective school-type identity is developed. In Study 2 (N = 1278 students) the assumed structure with four subscales (Stereotype Achievement, Stereotype Motivation, Stereotype Social, and Compensation) is proved with confirmatory factor analysis. This measure is used to compare the collective school-type identity across school tracks and predict motivational outcomes. Results show large differences in collective school-type identity between students of different school tracks. Furthermore, these differences can explain motivational differences between school tracks. Collective school-type identity has incremental predictive power for scholastic motivation, over and above the effects of academic self-concept and school track.

Keratin 17 null mice exhibit age- and strain-dependent alopecia

PubMed Central

McGowan, Kevin M.; Tong, Xuemei; Colucci-Guyon, Emma; Langa, Francina; Babinet, Charles; Coulombe, Pierre A.

2002-01-01

Onset of type I keratin 17 (K17) synthesis marks the adoption of an appendageal fate within embryonic ectoderm, and its expression persists in specific cell types within mature hair, glands, and nail. We report that K17 null mice develop severe alopecia during the first week postbirth, correlating with hair fragility, alterations in follicular histology, and apoptosis in matrix cells. These alterations are incompletely penetrant and normalize starting with the first postnatal cycle. Absence of a hair phenotype correlates with a genetic strain-dependent compensation by related keratins, including K16. These findings reveal a crucial role for K17 in the structural integrity of the first hair produced and the survival of hair-producing cells. Given that identical inherited mutations in this gene can cause either pachyonychia congenita or steatocystoma multiplex, the features of this mouse model suggest that this clinical heterogeneity arises from a cell type-specific, genetically determined compensation by related keratins. PMID:12050118
Reversal of β cell de-differentiation by a small molecule inhibitor of the TGFβ pathway

PubMed Central

Blum, Barak; Roose, Adam N; Barrandon, Ornella; Maehr, René; Arvanites, Anthony C; Davidow, Lance S; Davis, Jeffrey C; Peterson, Quinn P; Rubin, Lee L; Melton, Douglas A

2014-01-01

Dysfunction or death of pancreatic β cells underlies both types of diabetes. This functional decline begins with β cell stress and de-differentiation. Current drugs for type 2 diabetes (T2D) lower blood glucose levels but they do not directly alleviate β cell stress nor prevent, let alone reverse, β cell de-differentiation. We show here that Urocortin 3 (Ucn3), a marker for mature β cells, is down-regulated in the early stages of T2D in mice and when β cells are stressed in vitro. Using an insulin expression-coupled lineage tracer, with Ucn3 as a reporter for the mature β cell state, we screen for factors that reverse β cell de-differentiation. We find that a small molecule inhibitor of TGFβ receptor I (Alk5) protects cells from the loss of key β cell transcription factors and restores a mature β cell identity even after exposure to prolonged and severe diabetes. DOI: http://dx.doi.org/10.7554/eLife.02809.001 PMID:25233132
Fingerprint Recognition with Identical Twin Fingerprints

PubMed Central

Yang, Xin; Tian, Jie

2012-01-01

Fingerprint recognition with identical twins is a challenging task due to the closest genetics-based relationship existing in the identical twins. Several pioneers have analyzed the similarity between twins' fingerprints. In this work we continue to investigate the topic of the similarity of identical twin fingerprints. Our study was tested based on a large identical twin fingerprint database that contains 83 twin pairs, 4 fingers per individual and six impressions per finger: 3984 (83*2*4*6) images. Compared to the previous work, our contributions are summarized as follows: (1) Two state-of-the-art fingerprint identification methods: P071 and VeriFinger 6.1 were used, rather than one fingerprint identification method in previous studies. (2) Six impressions per finger were captured, rather than just one impression, which makes the genuine distribution of matching scores more realistic. (3) A larger sample (83 pairs) was collected. (4) A novel statistical analysis, which aims at showing the probability distribution of the fingerprint types for the corresponding fingers of identical twins which have same fingerprint type, has been conducted. (5) A novel analysis, which aims at showing which finger from identical twins has higher probability of having same fingerprint type, has been conducted. Our results showed that: (a) A state-of-the-art automatic fingerprint verification system can distinguish identical twins without drastic degradation in performance. (b) The chance that the fingerprints have the same type from identical twins is 0.7440, comparing to 0.3215 from non-identical twins. (c) For the corresponding fingers of identical twins which have same fingerprint type, the probability distribution of five major fingerprint types is similar to the probability distribution for all the fingers' fingerprint type. (d) For each of four fingers of identical twins, the probability of having same fingerprint type is similar. PMID:22558204
Fingerprint recognition with identical twin fingerprints.

PubMed

Tao, Xunqiang; Chen, Xinjian; Yang, Xin; Tian, Jie

2012-01-01

Fingerprint recognition with identical twins is a challenging task due to the closest genetics-based relationship existing in the identical twins. Several pioneers have analyzed the similarity between twins' fingerprints. In this work we continue to investigate the topic of the similarity of identical twin fingerprints. Our study was tested based on a large identical twin fingerprint database that contains 83 twin pairs, 4 fingers per individual and six impressions per finger: 3984 (83*2*4*6) images. Compared to the previous work, our contributions are summarized as follows: (1) Two state-of-the-art fingerprint identification methods: P071 and VeriFinger 6.1 were used, rather than one fingerprint identification method in previous studies. (2) Six impressions per finger were captured, rather than just one impression, which makes the genuine distribution of matching scores more realistic. (3) A larger sample (83 pairs) was collected. (4) A novel statistical analysis, which aims at showing the probability distribution of the fingerprint types for the corresponding fingers of identical twins which have same fingerprint type, has been conducted. (5) A novel analysis, which aims at showing which finger from identical twins has higher probability of having same fingerprint type, has been conducted. Our results showed that: (a) A state-of-the-art automatic fingerprint verification system can distinguish identical twins without drastic degradation in performance. (b) The chance that the fingerprints have the same type from identical twins is 0.7440, comparing to 0.3215 from non-identical twins. (c) For the corresponding fingers of identical twins which have same fingerprint type, the probability distribution of five major fingerprint types is similar to the probability distribution for all the fingers' fingerprint type. (d) For each of four fingers of identical twins, the probability of having same fingerprint type is similar.
Auxin physiology of the tomato mutant diageotropica

NASA Technical Reports Server (NTRS)

Daniel, S. G.; Rayle, D. L.; Cleland, R. E.

1989-01-01

The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.
Do FY antigens act as minor histocompatibility antigens in the graft-versus-host disease paradigm after human leukocyte antigen-identical sibling hematopoietic stem cell transplantation?

PubMed

Sellami, Mohamed Hichem; Chaabane, Manel; Kaabi, Houda; Torjemane, Lamia; Ladeb, Saloua; Ben Othmane, Tarek; Hmida, Slama

2012-03-01

FY antigens are candidate minor histocompatibility antigens relevant to renal allograft rejection, but no data have been reported about their role in graft-versus-host disease (GVHD) incidence after human leukocyte antigen (HLA)-identical siblings hematopoietic stem cell transplantation (HSCT). The aim of this study was to examine the effect of donor/recipient disparity at FY antigens on the incidence of GVHD in Tunisian patients receiving an HLA-identical HSCT. This work enrolled 105 Tunisian pairs of recipients and their HLA-identical sibling donors of HSCs. FY genotyping was performed with the polymerase chain reaction-sequence-specific primer method and donor/recipient disparity for these antigens was analyzed at two levels: incompatibility and nonidentity. The case-control analyses showed no significant correlation between FY disparity and the incidence of either acute or chronic GVHD. Sample size calculation showed that 572 cases and 1716 controls would be necessary to be able to detect a significant association with 80% power and two-sided type I error level of 5% (α=0.05). The lack of association in the studied cohort may be explained by the low immunogenicity of FY antigens in HSCT context, compared with other antigens such as HA-1 and CD31.
Discovering sparse transcription factor codes for cell states and state transitions during development

PubMed Central

Furchtgott, Leon A; Melton, Samuel; Menon, Vilas; Ramanathan, Sharad

2017-01-01

Computational analysis of gene expression to determine both the sequence of lineage choices made by multipotent cells and to identify the genes influencing these decisions is challenging. Here we discover a pattern in the expression levels of a sparse subset of genes among cell types in B- and T-cell developmental lineages that correlates with developmental topologies. We develop a statistical framework using this pattern to simultaneously infer lineage transitions and the genes that determine these relationships. We use this technique to reconstruct the early hematopoietic and intestinal developmental trees. We extend this framework to analyze single-cell RNA-seq data from early human cortical development, inferring a neocortical-hindbrain split in early progenitor cells and the key genes that could control this lineage decision. Our work allows us to simultaneously infer both the identity and lineage of cell types as well as a small set of key genes whose expression patterns reflect these relationships. DOI: http://dx.doi.org/10.7554/eLife.20488.001 PMID:28296636
Target-specific expression of presynaptic NMDA receptors in neocortical microcircuits.

PubMed

Buchanan, Katherine A; Blackman, Arne V; Moreau, Alexandre W; Elgar, Dale; Costa, Rui P; Lalanne, Txomin; Tudor Jones, Adam A; Oyrer, Julia; Sjöström, P Jesper

2012-08-09

Traditionally, NMDA receptors are located postsynaptically; yet, putatively presynaptic NMDA receptors (preNMDARs) have been reported. Although implicated in controlling synaptic plasticity, their function is not well understood and their expression patterns are debated. We demonstrate that, in layer 5 of developing mouse visual cortex, preNMDARs specifically control synaptic transmission at pyramidal cell inputs to other pyramidal cells and to Martinotti cells, while leaving those to basket cells unaffected. We also reveal a type of interneuron that mediates ascending inhibition. In agreement with synapse-specific expression, we find preNMDAR-mediated calcium signals in a subset of pyramidal cell terminals. A tuned network model predicts that preNMDARs specifically reroute information flow in local circuits during high-frequency firing, in particular by impacting frequency-dependent disynaptic inhibition mediated by Martinotti cells, a finding that we experimentally verify. We conclude that postsynaptic cell type determines presynaptic terminal molecular identity and that preNMDARs govern information processing in neocortical columns. Copyright © 2012 Elsevier Inc. All rights reserved.
Single-nucleus Hi-C of mammalian oocytes and zygotes.

PubMed

Gassler, Johanna; Flyamer, Ilya M; Tachibana, Kikuë

2018-01-01

The 3D folding of the genome is linked to essential nuclear processes including gene expression, DNA repair, and replication. Chromatin conformation capture assays such as Hi-C are providing unprecedented insights into higher-order chromatin structure. Bulk Hi-C of millions of cells enables detection of average chromatin features at high resolution but is challenging to apply to rare cell types. This chapter describes our recently developed single-nucleus Hi-C (snHi-C) approach for detection of chromatin contacts in single nuclei of murine oocytes and one-cell embryos (zygotes). The step-by-step protocol includes isolation of these cells, extraction of nuclei, fixation, restriction digestion, ligation, and whole genome amplification. Contacts obtained by snHi-C allow detection of chromatin features including loops, topologically associating domains, and compartments when averaged over the genome. The combination of snHi-C with other single-cell techniques in these and other rare cell types will likely provide a comprehensive picture of how chromatin architecture shapes cell identity. © 2018 Elsevier Inc. All rights reserved.
SBR-Blood: systems biology repository for hematopoietic cells.

PubMed

Lichtenberg, Jens; Heuston, Elisabeth F; Mishra, Tejaswini; Keller, Cheryl A; Hardison, Ross C; Bodine, David M

2016-01-04

Extensive research into hematopoiesis (the development of blood cells) over several decades has generated large sets of expression and epigenetic profiles in multiple human and mouse blood cell types. However, there is no single location to analyze how gene regulatory processes lead to different mature blood cells. We have developed a new database framework called hematopoietic Systems Biology Repository (SBR-Blood), available online at http://sbrblood.nhgri.nih.gov, which allows user-initiated analyses for cell type correlations or gene-specific behavior during differentiation using publicly available datasets for array- and sequencing-based platforms from mouse hematopoietic cells. SBR-Blood organizes information by both cell identity and by hematopoietic lineage. The validity and usability of SBR-Blood has been established through the reproduction of workflows relevant to expression data, DNA methylation, histone modifications and transcription factor occupancy profiles. Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.
Engineering kidney cells: reprogramming and directed differentiation to renal tissues.

PubMed

Kaminski, Michael M; Tosic, Jelena; Pichler, Roman; Arnold, Sebastian J; Lienkamp, Soeren S

2017-07-01

Growing knowledge of how cell identity is determined at the molecular level has enabled the generation of diverse tissue types, including renal cells from pluripotent or somatic cells. Recently, several in vitro protocols involving either directed differentiation or transcription-factor-based reprogramming to kidney cells have been established. Embryonic stem cells or induced pluripotent stem cells can be guided towards a kidney fate by exposing them to combinations of growth factors or small molecules. Here, renal development is recapitulated in vitro resulting in kidney cells or organoids that show striking similarities to mammalian embryonic nephrons. In addition, culture conditions are also defined that allow the expansion of renal progenitor cells in vitro. Another route towards the generation of kidney cells is direct reprogramming. Key transcription factors are used to directly impose renal cell identity on somatic cells, thus circumventing the pluripotent stage. This complementary approach to stem-cell-based differentiation has been demonstrated to generate renal tubule cells and nephron progenitors. In-vitro-generated renal cells offer new opportunities for modelling inherited and acquired renal diseases on a patient-specific genetic background. These cells represent a potential source for developing novel models for kidney diseases, drug screening and nephrotoxicity testing and might represent the first steps towards kidney cell replacement therapies. In this review, we summarize current approaches for the generation of renal cells in vitro and discuss the advantages of each approach and their potential applications.
The Relationship between Cell Phone Use and Identity Theft

ERIC Educational Resources Information Center

Saunders, Lewis O.

2014-01-01

The growth of mobile phone use has paralleled increased reports of identity theft. Identity theft can result in financial loss and threats to a victim's personal safety. Although trends in identity theft are well-known, less is known about individual cell phone users' attitudes toward identity theft and the extent to which they connect it to cell…
Constitutive expression of tert in thymocytes leads to increased incidence and dissemination of T-cell lymphoma in Lck-Tert mice.

PubMed

Canela, Andrés; Martín-Caballero, Juan; Flores, Juana M; Blasco, María A

2004-05-01

Here we describe a new mouse model with constitutive expression of the catalytic subunit of telomerase (Tert) targeted to thymocytes and peripheral T cells (Lck-Tert mice). Two independent Lck-Tert mouse lines showed higher incidences of spontaneous T-cell lymphoma than the corresponding age-matched wild-type controls, indicating that constitutive expression of Tert promotes lymphoma. Interestingly, T-cell lymphomas in Lck-Tert mice were more disseminated than those in wild-type controls and affected both lymphoid and nonlymphoid tissues, while nonlymphoid tissues were never affected with lymphoma in age-matched wild-type controls. Importantly, these roles of Tert constitutive expression in promoting tumor progression and dissemination were independent of the role of telomerase in telomere length maintenance, since telomere length distributions on a single-cell basis were identical in Lck-Tert and wild-type thymocytes. Finally, Tert constitutive expression did not interfere with telomere capping in Lck-Tert primary thymocytes, although it resulted in greater chromosomal instability upon gamma irradiation in Lck-Tert primary lymphocytes than in controls, suggesting that Tert overexpression may interfere with the cellular response to DNA damage.
Ultrastructural characterization of the hemocytes of Lasiodora sp. (Koch, 1850) (Araneae: Theraphosidae).

PubMed

Soares, Tatiana; Cavalcanti, Marília Gabriela Dos Santos; Ferreira, Felipe Roberto Borba; Cavalcanti, Maria do Socorro de Mendonça; Alves, Luiz Carlos; Brayner, Fábio André; Paiva, Patrícia Maria Guedes

2013-05-01

This paper is the first descriptive review of hemolymph cell types in the circulation of the tarantula spider Lasiodora sp. These animals are more long-lived than other arthropods, and may live for approximately twenty years. Such remarkable longevity may result from a highly successful immune system, which in turn is directly correlated with hemocyte function. Since the literature on the genus Lasiodora sp. is limited, the main goal of the present study was to identify the different cell types by optical and transmission microscope. Six hemocyte types were characterized and called prohemocyte, granulocyte type I, granulocyte type II, spherulocyte, oenocytoid and plasmatocyte. Prohemocytes presented a large nucleus, elongated granulocytes type I showed the nucleus with the same cell format, elliptical granulocytes type II showed the central nucleus of identical shape, spherulocytes exhibited the nucleus filling almost the whole cell, oval oenocytoids showed eccentric nucleus and less dense cytoplasm, and irregular plasmatocytes showed a nucleus and no granules in cytoplasm. These polymorphic granulocytes presented a round, elongated, elliptical, oval or irregular profile with large and varied numbers of granules, except for plasmatocytes, that were agranular. Different densities and different concentrations of these granules were found at the periphery of the cell. The possible reasons and implications of differences and similarities between arthropods hemocytes are discussed. It can be concluded that there are six cell types in Lasiodora sp. This study is of the first step in the elucidation of the role these cells play in the circulatory and immune system in spiders. Copyright © 2013 Elsevier Ltd. All rights reserved.
Whole-organism clone tracing using single-cell sequencing.

PubMed

Alemany, Anna; Florescu, Maria; Baron, Chloé S; Peterson-Maduro, Josi; van Oudenaarden, Alexander

2018-04-05

Embryonic development is a crucial period in the life of a multicellular organism, during which limited sets of embryonic progenitors produce all cells in the adult body. Determining which fate these progenitors acquire in adult tissues requires the simultaneous measurement of clonal history and cell identity at single-cell resolution, which has been a major challenge. Clonal history has traditionally been investigated by microscopically tracking cells during development, monitoring the heritable expression of genetically encoded fluorescent proteins and, more recently, using next-generation sequencing technologies that exploit somatic mutations, microsatellite instability, transposon tagging, viral barcoding, CRISPR-Cas9 genome editing and Cre-loxP recombination. Single-cell transcriptomics provides a powerful platform for unbiased cell-type classification. Here we present ScarTrace, a single-cell sequencing strategy that enables the simultaneous quantification of clonal history and cell type for thousands of cells obtained from different organs of the adult zebrafish. Using ScarTrace, we show that a small set of multipotent embryonic progenitors generate all haematopoietic cells in the kidney marrow, and that many progenitors produce specific cell types in the eyes and brain. In addition, we study when embryonic progenitors commit to the left or right eye. ScarTrace reveals that epidermal and mesenchymal cells in the caudal fin arise from the same progenitors, and that osteoblast-restricted precursors can produce mesenchymal cells during regeneration. Furthermore, we identify resident immune cells in the fin with a distinct clonal origin from other blood cell types. We envision that similar approaches will have major applications in other experimental systems, in which the matching of embryonic clonal origin to adult cell type will ultimately allow reconstruction of how the adult body is built from a single cell.
Medical identity theft: an emerging problem for informatics.

PubMed

Gillette, William; Patrick, Timothy B

2007-10-11

This poster reports a preliminary review of medical identity theft. Financial identity theft has received a great deal of media attention. Medical identity theft is a particular kind of identity theft that has received little attention. There are two main subtypes of medical identity theft. In the first type the stolen medical identity is used to receive medical services, and in the second type the stolen medical identity is used to commit healthcare fraud.
Microconnectomics of the pretectum and ventral thalamus in the chicken (Gallus gallus).

PubMed

Vega-Zuniga, Tomas; Marín, Gonzalo; González-Cabrera, Cristian; Planitscher, Eva; Hartmann, Anja; Marks, Vanessa; Mpodozis, Jorge; Luksch, Harald

2016-08-01

The avian pretectal and ventrothalamic nuclei, encompassing the griseum tectale (GT), n. lentiformis mesencephali (LM), and n. geniculatus lateralis pars ventralis (GLv), are prominent retinorecipient structures related to optic flow operations and visuomotor control. Hence, a close coordination of these neural circuits is to be expected. Yet the connectivity among these nuclei is poorly known. Here, using intracellular labeling and in situ hybridization, we investigated the detailed morphology, connectivity, and neurochemical identity of neurons in these nuclei. Two different cell types exist in the GT: one that generates an axonal projection to the optic tectum (TeO), LM, GLv, and n. intercalatus thalami (ICT), and a second population that only projects to the LM and GLv. In situ hybridization revealed that most neurons in the GT express the vesicular glutamate transporter (VGluT2) mRNA, indicating a glutamatergic identity. In the LM, three morphological cell types were defined, two of which project axons towards dorsal targets. The LM neurons showed strong VGluT2 expression. Finally, the cells located in the GLv project to the TeO, LM, GT, n. principalis precommisuralis (PPC), and ICT. All neurons in the GLv showed strong expression of the vesicular inhibitory amino acid transporter (VIAAT) mRNA, suggesting a GABAergic identity. Our results show that the pretectal and ventrothalamic nuclei are highly interconnected, especially by glutamatergic and GABAergic neurons from the GT and GLv, respectively. This complex morphology and connectivity might be required to organize orienting visuomotor behaviors and coordinate the specific optic flow patterns that they induce. J. Comp. Neurol. 524:2208-2229, 2016. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.
A Historical Perspective on the Identification of Cell Types in Pancreatic Islets of Langerhans by Staining and Histochemical Techniques

PubMed Central

2015-01-01

Before the middle of the previous century, cell types of the pancreatic islets of Langerhans were identified primarily on the basis of their color reactions with histological dyes. At that time, the chemical basis for the staining properties of islet cells in relation to the identity, chemistry and structure of their hormones was not fully understood. Nevertheless, the definitive islet cell types that secrete glucagon, insulin, and somatostatin (A, B, and D cells, respectively) could reliably be differentiated from each other with staining protocols that involved variations of one or more tinctorial techniques, such as the Mallory-Heidenhain azan trichrome, chromium hematoxylin and phloxine, aldehyde fuchsin, and silver impregnation methods, which were popularly used until supplanted by immunohistochemical techniques. Before antibody-based staining methods, the most bona fide histochemical techniques for the identification of islet B cells were based on the detection of sulfhydryl and disulfide groups of insulin. The application of the classical islet tinctorial staining methods for pathophysiological studies and physiological experiments was fundamental to our understanding of islet architecture and the physiological roles of A and B cells in glucose regulation and diabetes. PMID:26216133
Cell stimulus and lysis in a microfluidic device with segmented gas-liquid flow.

PubMed

El-Ali, Jamil; Gaudet, Suzanne; Günther, Axel; Sorger, Peter K; Jensen, Klavs F

2005-06-01

We describe a microfluidic device with rapid stimulus and lysis of mammalian cells for resolving fast transient responses in cell signaling networks. The device uses segmented gas-liquid flow to enhance mixing and has integrated thermoelectric heaters and coolers to control the temperature during cell stimulus and lysis. Potential negative effects of segmented flow on cell responses are investigated in three different cell types, with no morphological changes and no activation of the cell stress-sensitive mitogen activated protein kinases observed. Jurkat E6-1 cells are stimulated in the device using alpha-CD3, and the resulting activations of ERK and JNK are presented for different time points. Stimulation of cells performed on chip results in pathway activation identical to that of conventionally treated cells under the same conditions.
The founder-cell transcriptome in the Arabidopsis apetala1 cauliflower inflorescence meristem.

PubMed

Frerichs, Anneke; Thoma, Rahere; Abdallah, Ali Taleb; Frommolt, Peter; Werr, Wolfgang; Chandler, John William

2016-11-03

Although the pattern of lateral organ formation from apical meristems establishes species-specific plant architecture, the positional information that confers cell fate to cells as they transit to the meristem flanks where they differentiate, remains largely unknown. We have combined fluorescence-activated cell sorting and RNA-seq to characterise the cell-type-specific transcriptome at the earliest developmental time-point of lateral organ formation using DORNRÖSCHEN-LIKE::GFP to mark founder-cell populations at the periphery of the inflorescence meristem (IM) in apetala1 cauliflower double mutants, which overproliferate IMs. Within the lateral organ founder-cell population at the inflorescence meristem, floral primordium identity genes are upregulated and stem-cell identity markers are downregulated. Additional differentially expressed transcripts are involved in polarity generation and boundary formation, and in epigenetic and post-translational changes. However, only subtle transcriptional reprogramming within the global auxin network was observed. The transcriptional network of differentially expressed genes supports the hypothesis that lateral organ founder-cell specification involves the creation of polarity from the centre to the periphery of the IM and the establishment of a boundary from surrounding cells, consistent with bract initiation. However, contrary to the established paradigm that sites of auxin response maxima pre-pattern lateral organ initiation in the IM, auxin response might play a minor role in the earliest stages of lateral floral initiation.

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
The haemagglutination activity of equine herpesvirus type 1 glycoprotein C.

PubMed

Andoh, Kiyohiko; Hattori, Shiho; Mahmoud, Hassan Y A H; Takasugi, Maaya; Shimoda, Hiroshi; Bannai, Hiroshi; Tsujimura, Koji; Matsumura, Tomio; Kondo, Takashi; Kirisawa, Rikio; Mochizuki, Masami; Maeda, Ken

2015-01-02

Equine herpesvirus type 1 (EHV-1) has haemagglutination (HA) activity toward equine red blood cells (RBCs), but the identity of its haemagglutinin is unknown. To identify the haemagglutinin of EHV-1, the major glycoproteins of EHV-1 were expressed in 293T cells, and the cells or cell lysates were mixed with equine RBCs. The results showed that only EHV-1 glycoprotein C (gC)-producing cells adsorbed equine RBCs, and that the lysate of EHV-1 gC-expressing cells agglutinated equine RBCs. EHV-1 lacking gC did not show HA activity. HA activity was inhibited by monoclonal antibodies (MAbs) specific for gC, but not by antibodies directed against other glycoproteins. In addition, HA activity was not inhibited by the addition of heparin. These results indicate that EHV-1 gC can bind equine RBCs irrespective of heparin, in contrast to other herpesvirus gC proteins. Copyright © 2014 Elsevier B.V. All rights reserved.
Photodamage of the cells in culture sensitized with bilirubin

NASA Astrophysics Data System (ADS)

Kozlenkova, O. A.; Plavskaya, L. G.; Mikulich, A. V.; Leusenko, I. A.; Tretyakova, A. I.; Plavskii, V. Yu

2016-08-01

It has been shown that exposure to radiation of LED sources of light with an emission band maximum at about 465 and 520 nm having substantially identical damaging effects on animal cells in culture, that are in a logarithmic growth phase and preincubated with pigment. Photobiological effect is caused by photodynamic processes involving singlet oxygen generated by triplet excited sensitizer. Mono-exponential type dependence of cell survival on the energy dose indicates that it is bilirubin that acts as a sensitizer but not its photoproducts. The inclusion of bilirubin in the cells, where it is primarily localized in the mitochondria cells, it is accompanied by multiple amplification photochemical stability compared to pigment molecules bound with albumin
Cancer of the Esophagus and Esophagogastric Junction: An Eighth Edition Staging Primer

PubMed Central

Rice, Thomas W.; Ishwaran, Hemant; Ferguson, Mark K.; Blackstone, Eugene H.; Goldstraw, Peter

2017-01-01

This primer for eighth edition staging of esophageal and esophagogastric epithelial cancers presents separate classifications for the clinical (cTNM), pathologic (pTNM), and postneoadjuvant pathologic (ypTNM) stage groups, which are no longer shared. For pTNM, pT1 has been subcategorized as pT1a and pT1b for the subgrouping pStage I adenocarcinoma and squamous cell carcinoma. A new, simplified esophagus-specific regional lymph node map has been introduced. Undifferentiated histologic grade (G4) has been eliminated; additional analysis is required to expose histopathologic cell type. Location has been removed as a category for pT2N0M0 squamous cell cancer. The definition of the esophagogastric junction has been revised. ypTNM stage groups are identical for both histopathologic cell types, unlike those for cTNM and pTNM. PMID:27810391
Molecular cloning of NILE glycoprotein and evidence for its continued expression in mature rat CNS.

PubMed

Prince, J T; Alberti, L; Healy, P A; Nauman, S J; Stallcup, W B

1991-11-01

The NILE glycoprotein is a rat neuronal cell adhesion molecule which has been reported to be very similar in structure, function, and distribution to the mouse L1 glycoprotein. Here we report the complete nucleotide sequence of the NILE message (5,208 nucleotides) and the deduced amino acid sequence of the NILE polypeptide (1,257 amino acids). The predicted NILE protein is 96% identical to L1 at the amino acid level, confirming that the two molecules are homologues. The sequence information shows that NILE is a transmembrane molecule with an extensive ectodomain and a much smaller cytoplasmic domain. The extracellular portion of the molecule contains six immunoglobulin C-2 type domains followed by five fibronectin type III repeats. These two structural motifs are characteristic of several other cell adhesion molecules. The cytoplasmic tails of NILE and L1 are identical to each other and distinct from the cytoplasmic regions of all other cell adhesion molecules except Ng-CAM and neuroglian. Several possible sites for phosphorylation are present in the cytoplasmic tail of NILE. Antisera were produced against two NILE-beta-galactosidase fusion proteins containing distinct segments of the NILE polypeptide: the cytoplasmic domain and the segment containing fibronectin type III repeats. Immunoblots with these antisera and Northern blots with a NILE cDNA probe indicate that NILE continues to be expressed in most areas of the mature rat brain. This contradicts previous immunofluorescence data, which suggested that NILE was substantially down-regulated in maturing nerve fiber tracts. This raises the possibility that NILE could be masked in situ by interactions with other cell surface molecules.
Whole-Body Single-Cell Sequencing Reveals Transcriptional Domains in the Annelid Larval Body

PubMed Central

Achim, Kaia; Eling, Nils; Vergara, Hernando Martinez; Bertucci, Paola Yanina; Musser, Jacob; Vopalensky, Pavel; Brunet, Thibaut; Collier, Paul; Benes, Vladimir; Marioni, John C; Arendt, Detlev

2018-01-01

Abstract Animal bodies comprise diverse arrays of cells. To characterize cellular identities across an entire body, we have compared the transcriptomes of single cells randomly picked from dissociated whole larvae of the marine annelid Platynereis dumerilii. We identify five transcriptionally distinct groups of differentiated cells, each expressing a unique set of transcription factors and effector genes that implement cellular phenotypes. Spatial mapping of cells into a cellular expression atlas, and wholemount in situ hybridization of group-specific genes reveals spatially coherent transcriptional domains in the larval body, comprising, for example, apical sensory-neurosecretory cells versus neural/epidermal surface cells. These domains represent new, basic subdivisions of the annelid body based entirely on differential gene expression, and are composed of multiple, transcriptionally similar cell types. They do not represent clonal domains, as revealed by developmental lineage analysis. We propose that the transcriptional domains that subdivide the annelid larval body represent families of related cell types that have arisen by evolutionary diversification. Their possible evolutionary conservation makes them a promising tool for evo–devo research. PMID:29373712
Arabidopsis female gametophyte gene expression map reveals similarities between plant and animal gametes.

PubMed

Wuest, Samuel E; Vijverberg, Kitty; Schmidt, Anja; Weiss, Manuel; Gheyselinck, Jacqueline; Lohr, Miriam; Wellmer, Frank; Rahnenführer, Jörg; von Mering, Christian; Grossniklaus, Ueli

2010-03-23

The development of multicellular organisms is controlled by differential gene expression whereby cells adopt distinct fates. A spatially resolved view of gene expression allows the elucidation of transcriptional networks that are linked to cellular identity and function. The haploid female gametophyte of flowering plants is a highly reduced organism: at maturity, it often consists of as few as three cell types derived from a common precursor [1, 2]. However, because of its inaccessibility and small size, we know little about the molecular basis of cell specification and differentiation in the female gametophyte. Here we report expression profiles of all cell types in the mature Arabidopsis female gametophyte. Differentially expressed posttranscriptional regulatory modules and metabolic pathways characterize the distinct cell types. Several transcription factor families are overrepresented in the female gametophyte in comparison to other plant tissues, e.g., type I MADS domain, RWP-RK, and reproductive meristem transcription factors. PAZ/Piwi-domain encoding genes are upregulated in the egg, indicating a role of epigenetic regulation through small RNA pathways-a feature paralleled in the germline of animals [3]. A comparison of human and Arabidopsis egg cells for enrichment of functional groups identified several similarities that may represent a consequence of coevolution or ancestral gametic features. 2010 Elsevier Ltd. All rights reserved.
Molecular phylogeny, systematics, and revision of the type species of Lobomonas, L. francei (Volvocales, Chlorophyta) and closely related taxa.

PubMed

Sausen, Nicole; Malavasi, Veronica; Melkonian, Michael

2018-04-01

In the present study, three new strains of the rare volvocalean green alga Lobomonas were isolated from field-collected samples, one from Sardinia (Italy) and two from Argentina, and comparatively studied. The Sardinian and one of the Argentinian strains were identified as Lobomonas francei, the type species of the genus, whereas the second Argentinian strain corresponded to L. panduriformis. Two additional nominal species of Lobomonas from culture collections (L. rostrata and L. sphaerica) were included in the analysis and shown to be morphologically and molecularly identical to the L. francei strains. The presence, number, and shapes of cell wall lobes, the diagnostic criterion of Lobomonas, were shown to be highly variable depending on the chemical composition of the culture medium used. The analyses by SEM gave evidence that the cell wall lobes in Lobomonas originate at the junctions of adjacent cell wall plates by extrusion of gelatinous material. The four L. francei strains had identical nrRNA gene sequences and differed by only one or two substitutions in the ITS1 + ITS2 sequences. In the phylogenetic analyses, L. francei and L. panduriformis were sister taxa; however, another nominal Lobomonas species (L. monstruosa) did not belong to this genus. Lobomonas, together with taxa designated as Vitreochlamys, Tetraspora, and Paulschulzia, formed a monophyletic group that in the combined analyses was sister to the "Chlamydomonas/Volvox-clade." Based on these results, Lobomonas was revised, the diagnosis of the type species emended, a lectotype and an epitype designated, and several taxa synonymized with the type species. © 2017 Phycological Society of America.
Basal Cells Are a Multipotent Progenitor Capable of Renewing the Bronchial Epithelium

PubMed Central

Hong, Kyung U.; Reynolds, Susan D.; Watkins, Simon; Fuchs, Elaine; Stripp, Barry R.

2004-01-01

Commitment of the pulmonary epithelium to bronchial and bronchiolar airway lineages occurs during the transition from pseudoglandular to cannalicular phases of lung development, suggesting that regional differences exist with respect to the identity of stem and progenitor cells that contribute to epithelial maintenance in adulthood. We previously defined a critical role for Clara cell secretory protein-expressing (CE) cells in renewal of bronchiolar airway epithelium following injury. Even though CE cells are also the principal progenitor for maintenance of the bronchial airway epithelium, CE cell injury is resolved through a mechanism involving recruitment of a second progenitor cell population that we now identify as a GSI-B4 reactive, cytokeratin-14-expressing basal cell. These cells exhibit multipotent differentiation capacity as assessed by analysis of cellular phenotype within clones of LacZ-tagged cells. Clones were derived from K14-expressing cells tagged in a cell-type-specific fashion by ligand-regulable Cre recombinase-mediated genomic rearrangement of the ROSA26 recombination substrate allele. We conclude that basal cells represent an alternative multipotent progenitor cell population of bronchial airways and that progenitor cell selection is dictated by the type of airway injury. PMID:14742263
Regulation of asparagine-linked oligosaccharide processing. Oligosaccharide processing in Aedes albopictus mosquito cells.

PubMed

Hsieh, P; Robbins, P W

1984-02-25

We have examined the synthesis and processing of asparagine-linked oligosaccharides from Aedes albopictus C6/36 mosquito cells. These cells synthesized a glucose-containing lipid-linked oligosaccharide with properties identical to that of Glc3Man9GlcNAc2-PP-dolichol. Results of brief pulse label experiments with [3H]mannose were consistent with the transfer of Glc3Man9GlcNAc2 to protein followed by the rapid removal of glucose residues. Pulse-chase experiments established that further processing of oligosaccharides in C6/36 cells resulted in the removal of up to six alpha-linked mannose residues yielding Man3GlcNAc2 whose structure is identical to that of the trimannosyl "core" of N-linked oligosaccharides of vertebrate cells and yeast. Complex-type oligosaccharides were not observed in C6/36 cells. When Sindbis virus was grown in mosquito cells, Man3GlcNAc2 glycans were preferentially located at the two glycosylation sites which were previously shown to have complex glycans in virus grown in vertebrate cells. These Man3GlcNAc2 structures are the most extensively processed oligosaccharides in A. albopictus, and as such, are analogous to the complex glycans of vertebrate cells. We suggest that determinants of oligosaccharide processing which reside in the polypeptide are universally recognized despite evolutionary divergence of the oligosaccharide-processing pathway between insects and vertebrates.
Receptive Field Vectors of Genetically-Identified Retinal Ganglion Cells Reveal Cell-Type-Dependent Visual Functions

PubMed Central

Katz, Matthew L.; Viney, Tim J.; Nikolic, Konstantin

2016-01-01

Sensory stimuli are encoded by diverse kinds of neurons but the identities of the recorded neurons that are studied are often unknown. We explored in detail the firing patterns of eight previously defined genetically-identified retinal ganglion cell (RGC) types from a single transgenic mouse line. We first introduce a new technique of deriving receptive field vectors (RFVs) which utilises a modified form of mutual information (“Quadratic Mutual Information”). We analysed the firing patterns of RGCs during presentation of short duration (~10 second) complex visual scenes (natural movies). We probed the high dimensional space formed by the visual input for a much smaller dimensional subspace of RFVs that give the most information about the response of each cell. The new technique is very efficient and fast and the derivation of novel types of RFVs formed by the natural scene visual input was possible even with limited numbers of spikes per cell. This approach enabled us to estimate the 'visual memory' of each cell type and the corresponding receptive field area by calculating Mutual Information as a function of the number of frames and radius. Finally, we made predictions of biologically relevant functions based on the RFVs of each cell type. RGC class analysis was complemented with results for the cells’ response to simple visual input in the form of black and white spot stimulation, and their classification on several key physiological metrics. Thus RFVs lead to predictions of biological roles based on limited data and facilitate analysis of sensory-evoked spiking data from defined cell types. PMID:26845435
Genome-Wide Analysis Reveals the Unique Stem Cell Identity of Human Amniocytes

PubMed Central

Maguire, Colin T.; Demarest, Bradley L.; Hill, Jonathon T.; Palmer, James D.; Brothman, Arthur R.; Yost, H. Joseph; Condic, Maureen L.

2013-01-01

Human amniotic fluid contains cells that potentially have important stem cell characteristics, yet the programs controlling their developmental potency are unclear. Here, we provide evidence that amniocytes derived from multiple patients are marked by heterogeneity and variability in expression levels of pluripotency markers. Clonal analysis from multiple patients indicates that amniocytes have large pools of self-renewing cells that have an inherent property to give rise to a distinct amniocyte phenotype with a heterogeneity of pluripotent markers. Significant to their therapeutic potential, genome-wide profiles are distinct at different gestational ages and times in culture, but do not differ between genders. Based on hierarchical clustering and differential expression analyses of the entire transcriptome, amniocytes express canonical regulators associated with pluripotency and stem cell repression. Their profiles are distinct from human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and newborn foreskin fibroblasts. Amniocytes have a complex molecular signature, coexpressing trophoblastic, ectodermal, mesodermal, and endodermal cell-type-specific regulators. In contrast to the current view of the ground state of stem cells, ESCs and iPSCs also express high levels of a wide range of cell-type-specific regulators. The coexpression of multilineage differentiation markers combined with the strong expression of a subset of ES cell repressors in amniocytes suggests that these cells have a distinct phenotype that is unlike any other known cell-type or lineage. PMID:23326421
Cellular interaction of a layer-by-layer based drug delivery system depending on material properties and cell types.

PubMed

Brueckner, Mandy; Jankuhn, Steffen; Jülke, Eva-Maria; Reibetanz, Uta

2018-01-01

Drug delivery systems (DDS) and their interaction with cells are a controversial topic in the development of therapeutic concepts and approaches. On one hand, DDS are very useful for protected and targeted transport of defined dosages of active agents. On the other hand, their physicochemical properties such as material, size, shape, charge, or stiffness have a huge impact on cellular uptake and intracellular processing. Additionally, even identical DDS can undergo a completely diverse interaction with different cell types. However, quite often in in vitro DDS/cell interaction experiments, those aspects are not considered and DDS and cells are randomly chosen. Hence, our investigations provide an insight into layer-by-layer designed microcarriers with modifications of only some of the most important parameters (surface charge, stiffness, and applied microcarrier/cell ratio) and their influence on cellular uptake and viability. We also considered the interaction of these differently equipped DDS with several cell types and investigated professional phagocytes (neutrophil granulocytes; macrophages) as well as non-professional phagocytes (epithelial cells) under comparable conditions. We found that even small modifications such as layer-by-layer (LbL)-microcarriers with positive or negative surface charge, or LbL-microcarriers with solid core or as hollow capsules but equipped with the same surface properties, show significant differences in interaction and viability, and several cell types react very differently to the offered DDS. As a consequence, the properties of the DDS have to be carefully chosen with respect to the addressed cell type with the aim to efficiently transport a desired agent.
Selection of stable reference genes for quantitative rt-PCR comparisons of mouse embryonic and extra-embryonic stem cells.

PubMed

Veazey, Kylee J; Golding, Michael C

2011-01-01

Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined.Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively.Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified mRNAs, offers a reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo.
Single cell visualization of transcription kinetics variance of highly mobile identical genes using 3D nanoimaging

PubMed Central

Annibale, Paolo; Gratton, Enrico

2015-01-01

Multi-cell biochemical assays and single cell fluorescence measurements revealed that the elongation rate of Polymerase II (PolII) in eukaryotes varies largely across different cell types and genes. However, there is not yet a consensus whether intrinsic factors such as the position, local mobility or the engagement by an active molecular mechanism of a genetic locus could be the determinants of the observed heterogeneity. Here by employing high-speed 3D fluorescence nanoimaging techniques we resolve and track at the single cell level multiple, distinct regions of mRNA synthesis within the model system of a large transgene array. We demonstrate that these regions are active transcription sites that release mRNA molecules in the nucleoplasm. Using fluctuation spectroscopy and the phasor analysis approach we were able to extract the local PolII elongation rate at each site as a function of time. We measured a four-fold variation in the average elongation between identical copies of the same gene measured simultaneously within the same cell, demonstrating a correlation between local transcription kinetics and the movement of the transcription site. Together these observations demonstrate that local factors, such as chromatin local mobility and the microenvironment of the transcription site, are an important source of transcription kinetics variability. PMID:25788248
Combined Transcriptome and Proteome Analysis Identifies Pathways and Markers Associated with the Establishment of Rapeseed Microspore-Derived Embryo Development1[W

PubMed Central

Joosen, Ronny; Cordewener, Jan; Supena, Ence Darmo Jaya; Vorst, Oscar; Lammers, Michiel; Maliepaard, Chris; Zeilmaker, Tieme; Miki, Brian; America, Twan; Custers, Jan; Boutilier, Kim

2007-01-01

Microspore-derived embryo (MDE) cultures are used as a model system to study plant cell totipotency and as an in vitro system to study embryo development. We characterized and compared the transcriptome and proteome of rapeseed (Brassica napus) MDEs from the few-celled stage to the globular/heart stage using two MDE culture systems: conventional cultures in which MDEs initially develop as unorganized clusters that usually lack a suspensor, and a novel suspensor-bearing embryo culture system in which the embryo proper originates from the distal cell of a suspensor-like structure and undergoes the same ordered cell divisions as the zygotic embryo. Improved histodifferentiation of suspensor-bearing MDEs suggests a new role for the suspensor in driving embryo cell identity and patterning. An MDE culture cDNA array and two-dimensional gel electrophoresis and protein sequencing were used to compile global and specific expression profiles for the two types of MDE cultures. Analysis of the identities of 220 candidate embryo markers, as well as the identities of 32 sequenced embryo up-regulated protein spots, indicate general roles for protein synthesis, glycolysis, and ascorbate metabolism in the establishment of MDE development. A collection of 135 robust markers for the transition to MDE development was identified, a number of which may be coregulated at the gene and protein expression level. Comparison of the expression profiles of preglobular-stage conventional MDEs and suspensor-bearing MDEs identified genes whose differential expression may reflect improved histodifferentiation of suspensor-bearing embryos. This collection of early embryo-expressed genes and proteins serves as a starting point for future marker development and gene function studies aimed at understanding the molecular regulation of cell totipotency and early embryo development in plants. PMID:17384159
T-cell epitope analysis using subtracted expression libraries (TEASEL): application to a 38-kDA autoantigen recognized by T cells from an insulin-dependent diabetic patient.

PubMed Central

Neophytou, P I; Roep, B O; Arden, S D; Muir, E M; Duinkerken, G; Kallan, A; de Vries, R R; Hutton, J C

1996-01-01

Studies on circulating T cells and antibodies in newly diagnosed type 1 diabetic patients and rodent models of autoimmune diabetes suggest that beta-cell membrane proteins of 38 kDa may be important molecular targets of autoimmune attack. Biochemical approaches to the isolation and identification of the 38-kDa autoantigen have been hampered by the restricted availability of islet tissue and the low abundance of the protein. A procedure of epitope analysis for CD4+ T cells using subtracted expression libraries (TEASEL) was developed and used to clone a 70-amino acid pancreatic beta-cell peptide incorporating an epitope recognized by a 38-kDa-reactive CD4+ T-cell clone (1C6) isolated from a human diabetic patient. The minimal epitope was mapped to a 10-amino acid synthetic peptide containing a DR1 consensus binding motif. Data base searches did not reveal the identity of the protein, though a weak homology to the bacterial superantigens SEA (Streptococcus pyogenes exotoxin A) and SEB (Staphylococcus aureus enterotoxin B) (23% identity) was evident. The TEASEL procedure might be used to identify epitopes of other autoantigens recognized by CD4+ T cells in diabetes as well as be more generally applicable to the study low-abundance autoantigens in other tissue-specific autoimmune diseases. PMID:8700877
Creutzfeldt-Jakob disease with severe involvement of cerebral white matter and cerebellum.

PubMed

Berciano, J; Berciano, M T; Polo, J M; Figols, J; Ciudad, J; Lafarga, M

1990-01-01

We describe a patient with Creutzfeldt-Jakob disease (CJD) of the ataxic and panencephalopathic type. Postmortem examination revealed the characteristic lesions of CJD in the grey matter and profound white matter involvement was seen with immunocytochemical techniques. Ultrastructural white matter lesions were identical to those described in experimentally transmitted CJD. There was marked loss of cerebellar granule cells with virtual disappearance of parallel fibres, but Purkinje cells were only slightly reduced. Electron microscopic studies revealed extensive degenerative changes including cytoplasmic vacuoles in both cell types. Silver methods disclosed massive impregnation of white matter and striking abnormalities of Purkinje cells consisting of hypertrophy and flattening of thick dendritic branches, reduction in the number of terminal branchlets, segmentary loss of spines and polymorphic spines. These findings show the extensive involvement of all three cerebellar cortical layers and the reactive plasticity of Purkinje cells to deafferentiation. They favour the hypothesis that demyelination represents a primary lesion of the white matter.
HOX and TALE signatures specify human stromal stem cell populations from different sources.

PubMed

Picchi, Jacopo; Trombi, Luisa; Spugnesi, Laura; Barachini, Serena; Maroni, Giorgia; Brodano, Giovanni Barbanti; Boriani, Stefano; Valtieri, Mauro; Petrini, Mario; Magli, Maria Cristina

2013-04-01

Human stromal stem cell populations reside in different tissues and anatomical sites, however a critical question related to their efficient use in regenerative medicine is whether they exhibit equivalent biological properties. Here, we compared cellular and molecular characteristics of stromal stem cells derived from the bone marrow, at different body sites (iliac crest, sternum, and vertebrae) and other tissues (dental pulp and colon). In particular, we investigated whether homeobox genes of the HOX and TALE subfamilies might provide suitable markers to identify distinct stromal cell populations, as HOX proteins control cell positional identity and, together with their co-factors TALE, are involved in orchestrating differentiation of adult tissues. Our results show that stromal populations from different sources, although immunophenotypically similar, display distinct HOX and TALE signatures, as well as different growth and differentiation abilities. Stromal stem cells from different tissues are characterized by specific HOX profiles, differing in the number and type of active genes, as well as in their level of expression. Conversely, bone marrow-derived cell populations can be essentially distinguished for the expression levels of specific HOX members, strongly suggesting that quantitative differences in HOX activity may be crucial. Taken together, our data indicate that the HOX and TALE profiles provide positional, embryological and hierarchical identity of human stromal stem cells. Furthermore, our data suggest that cell populations derived from different body sites may not represent equivalent cell sources for cell-based therapeutical strategies for regeneration and repair of specific tissues. Copyright © 2012 Wiley Periodicals, Inc.
Clinicopathological features of growth hormone-producing pituitary adenomas: difference among various types defined by cytokeratin distribution pattern including a transitional form.

PubMed

Obari, Abdulkader; Sano, Toshiaki; Ohyama, Kenichi; Kudo, Eiji; Qian, Zhi Rong; Yoneda, Akiko; Rayhan, Nasim; Mustafizur Rahman, Muhammad; Yamada, Shozo

2008-01-01

Pituitary adenomas producing almost exclusively growth hormones (GH) have been ultrastructurally classified into two distinct types: densely granulated somatotroph (DG) adenomas and sparsely granulated (SG) adenomas. Fibrous body (FB), an intracytoplasmic globular aggregation of cytokeratin (CK) filaments, is a hallmark of SG adenomas. Under light microscope, FB could be identified by CK immunohistochemistry as a dot-pattern immunoreaction versus a perinuclear pattern for cells without FB. However, it has been noted that numerous adenomas contain mixed populations of the two patterns. To clarify clinicopathological characteristics of the adenomas with mixed populations ("intermediate type" adenomas) and to confirm clinicopathological differences between strictly defined DG-type and SG-type adenomas, we performed this study on 104 GH cell adenomas. Having segregated "intermediate-type" adenomas (26 cases), we found significant differences between typical DG-type (47 cases) and SG-type adenomas (31 cases); SG-type adenomas had younger ages (44 vs. 50), higher frequency of macroadenomas (86% vs. 58%), invasiveness (65% vs. 38%), advanced grades (3 or 4) in Knosp's classification (50% vs. 24%), and weaker immunoreaction for GH, beta-TSH, alpha-subunit, E-cadherin, and beta-catenin. Clinicopathological characteristics of "intermediate-type" adenomas were identical to those of DG-type adenomas. These findings confirm that SG-type adenoma is a distinct section of GH cell adenomas with special properties and biological behavior, and suggest that intermediate-phenotype adenomas are enrolled in DG-type adenomas. Special properties and biological behavior of SG-type adenomas may appear after the majority of tumor cells possess a fully developed fibrous body.

Nanoparticle growth and surface chemistry changes in cell-conditioned culture medium.

PubMed

Kendall, Michaela; Hodges, Nikolas J; Whitwell, Harry; Tyrrell, Jess; Cangul, Hakan

2015-02-05

When biomolecules attach to engineered nanoparticle (ENP) surfaces, they confer the particles with a new biological identity. Physical format may also radically alter, changing ENP stability and agglomeration state within seconds. In order to measure which biomolecules are associated with early ENP growth, we studied ENPs in conditioned medium from A549 cell culture, using dynamic light scattering (DLS) and linear trap quadrupole electron transfer dissociation mass spectrometry. Two types of 100 nm polystyrene particles (one uncoated and one with an amine functionalized surface) were used to measure the influence of surface type. In identically prepared conditioned medium, agglomeration was visible in all samples after 1 h, but was variable, indicating inter-sample variability in secretion rates and extracellular medium conditions. In samples conditioned for 1 h or more, ENP agglomeration rates varied significantly. Agglomerate size measured by DLS was well correlated with surface sequestered peptide number for uncoated but not for amine coated polystyrene ENPs. Amine-coated ENPs grew much faster and into larger agglomerates associated with fewer sequestered peptides, but including significant sequestered lactose dehydrogenase. We conclude that interference with extracellular peptide balance and oxidoreductase activity via sequestration is worthy of further study, as increased oxidative stress via this new mechanism may be important for cell toxicity. © 2014 The Author(s) Published by the Royal Society. All rights reserved.
Bursting synchronization dynamics of pancreatic β-cells with electrical and chemical coupling.

PubMed

Meng, Pan; Wang, Qingyun; Lu, Qishao

2013-06-01

Based on bifurcation analysis, the synchronization behaviors of two identical pancreatic β-cells connected by electrical and chemical coupling are investigated, respectively. Various firing patterns are produced in coupled cells when a single cell exhibits tonic spiking or square-wave bursting individually, irrespectively of what the cells are connected by electrical or chemical coupling. On the one hand, cells can burst synchronously for both weak electrical and chemical coupling when an isolated cell exhibits tonic spiking itself. In particular, for electrically coupled cells, under the variation of the coupling strength there exist complex transition processes of synchronous firing patterns such as "fold/limit cycle" type of bursting, then anti-phase continuous spiking, followed by the "fold/torus" type of bursting, and finally in-phase tonic spiking. On the other hand, it is shown that when the individual cell exhibits square-wave bursting, suitable coupling strength can make the electrically coupled system generate "fold/Hopf" bursting via "fold/fold" hysteresis loop; whereas, the chemically coupled cells generate "fold/subHopf" bursting. Especially, chemically coupled bursters can exhibit inverse period-adding bursting sequence. Fast-slow dynamics analysis is applied to explore the generation mechanism of these bursting oscillations. The above analysis of bursting types and the transition may provide us with better insight into understanding the role of coupling in the dynamic behaviors of pancreatic β-cells.
Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

PubMed

Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

2016-01-01

The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling.
Two cell-counting factors regulate the aggregate size of the cellular slime mold Dictyostelium discoideum.

PubMed

Okuwa, T; Katayama, T; Takano, A; Kodaira, K; Yasukawa, H

2001-12-01

Countin, a cell-counting factor in Dictyostelium discoideum, is considered to limit the maximum size of the multicellular structure, because a countin null strain forms a huge fruiting body compared to that of the wild-type. A novel gene, countin2, that is highly homologous to countin (40% identity in amino acid sequence) was identified in the D. discoideum genome. The countin2 null strain formed a 1.7-fold higher number of the aggregates, resulting in smaller fruiting bodies compared with those of wild-type cells. Thus, the Countin2 protein is thought to limit the minimum size of the multicellular structure. The size and number of aggregates formed by a mixture of countin null and countin2 null strains were the same as those of the wild-type. These findings demonstrate that a combination of Countin and Countin2 proteins determines the appropriate size of the multicellular structure of D. discoideum.
Regulation of mammalian cell differentiation by long non-coding RNAs

PubMed Central

Hu, Wenqian; Alvarez-Dominguez, Juan R; Lodish, Harvey F

2012-01-01

Differentiation of specialized cell types from stem and progenitor cells is tightly regulated at several levels, both during development and during somatic tissue homeostasis. Many long non-coding RNAs have been recognized as an additional layer of regulation in the specification of cellular identities; these non-coding species can modulate gene-expression programmes in various biological contexts through diverse mechanisms at the transcriptional, translational or messenger RNA stability levels. Here, we summarize findings that implicate long non-coding RNAs in the control of mammalian cell differentiation. We focus on several representative differentiation systems and discuss how specific long non-coding RNAs contribute to the regulation of mammalian development. PMID:23070366
Integument pattern formation involves genetic and epigenetic controls: feather arrays simulated by digital hormone models

PubMed Central

Jiang, Ting-Xin; Widelitz, Randall B.; Shen, Wei-Min; Will, Peter; Wu, Da-Yu; Lin, Chih-Min; Jung, Han-Sung; Chuong, Cheng-Ming

2015-01-01

Pattern formation is a fundamental morphogenetic process. Models based on genetic and epigenetic control have been proposed but remain controversial. Here we use feather morphogenesis for further evaluation. Adhesion molecules and/or signaling molecules were first expressed homogenously in feather tracts (restrictive mode, appear earlier) or directly in bud or inter-bud regions (de novo mode, appear later). They either activate or inhibit bud formation, but paradoxically co-localize in the bud. Using feather bud reconstitution, we showed that completely dissociated cells can reform periodic patterns without reference to previous positional codes. The patterning process has the characteristics of being self-organizing, dynamic and plastic. The final pattern is an equilibrium state reached by competition, and the number and size of buds can be altered based on cell number and activator/inhibitor ratio, respectively. We developed a Digital Hormone Model which consists of (1) competent cells without identity that move randomly in a space, (2) extracellular signaling hormones which diffuse by a reaction-diffusion mechanism and activate or inhibit cell adhesion, and (3) cells which respond with topological stochastic actions manifested as changes in cell adhesion. Based on probability, the results are cell clusters arranged in dots or stripes. Thus genetic control provides combinational molecular information which defines the properties of the cells but not the final pattern. Epigenetic control governs interactions among cells and their environment based on physical-chemical rules (such as those described in the Digital Hormone Model). Complex integument patterning is the sum of these two components of control and that is why integument patterns are usually similar but non-identical. These principles may be shared by other pattern formation processes such as barb ridge formation, fingerprints, pigmentation patterning, etc. The Digital Hormone Model can also be applied to swarming robot navigation, reaching intelligent automata and representing a self-re-configurable type of control rather than a follow-the-instruction type of control. PMID:15272377
Direct transdifferentiation in the vertebrate retina.

PubMed

Opas, M; Dziak, E

1998-03-01

Transdifferentiation is the process by which differentiated cells alter their identity to become other, distinct cell types. The conversion of neural retina into lens epithelium is one of the most spectacular examples of transdifferentiation. We show that the redirection of cell fate from neural retina to lens and subsequent transdifferentiation is independent of cell replication as it occurs in growth-arrested cell populations. Using DNA ratiometry of individual cells in these cultures we show that, indeed, individual amitotic cells do transdifferentiate. Hence, choice of fate in transdifferentiating cells does not rely on a "community effect" but instead can be categorized as a For lack of overt lens progenitors, and most importantly, for its mitotic independence, we conclude that lens colony formation in vitro does occur by direct transdifferentiation and not by clonal proliferation of progenitor cells.
Metabolic and morphological differences between rapidly proliferating cancerous and normal breast epithelial cells.

PubMed

Meadows, Adam L; Kong, Becky; Berdichevsky, Marina; Roy, Siddhartha; Rosiva, Rosiva; Blanch, Harvey W; Clark, Douglas S

2008-01-01

The metabolic and morphological characteristics of two human epithelial breast cell populations--MCF7 cells, a cancerous cell line, and 48R human mammary epithelial cells (48R HMECs), a noncancerous, finite lifespan cell strain--were compared at identical growth rates. Both cell types were induced to grow rapidly in nutrient-rich media containing 13C-labeled glucose, and the isotopic enrichment of cellular metabolites was quantified to calculate metabolic fluxes in key pathways. Despite their similar growth rates, the cells exhibited distinctly different metabolic and morphological profiles. MCF7 cells have an 80% smaller exposed surface area and contain 26% less protein per cell than the 48R cells. Surprisingly, rapidly proliferating 48R cells exhibited a 225% higher per-cell glucose consumption rate, a 250% higher per-cell lactate production rate, and a nearly identical per-cell glutamine consumption rate relative to the cancer cell line. However, when fluxes were considered on the basis of exposed area, the cancer cells were observed to have higher glucose, lactate, and glutamine fluxes, demonstrating superior transport capabilities per unit area of cell membrane. MCF7 cells also consumed amino acids at rates much higher than are generally required for protein synthesis, whereas 48R cells generally did not. Pentose phosphate pathway activity was higher in MCF7 cells, and the flux of glutamine to glutamate was less reversible. Energy efficiency was significantly higher in MCF7 cells, as a result of a combination of their smaller size and greater reliance on the TCA cycle than the 48R cells. These observations support evolutionary models of cancer cell metabolism and suggest targets for metabolic drugs in metastatic breast cancers.
Relation of Disability Type and Career Thoughts to Vocational Identity

ERIC Educational Resources Information Center

Yanchak, Kristen V.; Lease, Suzanne H.; Strauser, David R.

2005-01-01

This study compared dysfunctional career thoughts and perceptions of vocational identity for individuals with different types of disabilities and examined whether the relation of career thoughts to vocational identity was moderated by type of disability. Ninety adults with cognitive and physical impairments were administered the Career Thoughts…
bHLH-O proteins balance the self-renewal and differentiation of Drosophila neural stem cells by regulating Earmuff expression.

PubMed

Li, Xiaosu; Chen, Rui; Zhu, Sijun

2017-11-15

Balancing self-renewal and differentiation of stem cells requires differential expression of self-renewing factors in two daughter cells generated from the asymmetric division of the stem cells. In Drosophila type II neural stem cell (or neuroblast, NB) lineages, the expression of the basic helix-loop-helix-Orange (bHLH-O) family proteins, including Deadpan (Dpn) and E(spl) proteins, is required for maintaining the self-renewal and identity of type II NBs, whereas the absence of these self-renewing factors is essential for the differentiation of intermediate neural progenitors (INPs) generated from type II NBs. Here, we demonstrate that Dpn maintains type II NBs by suppressing the expression of Earmuff (Erm). We provide evidence that Dpn and E(spl) proteins suppress Erm by directly binding to C-sites and N-boxes in the cis-regulatory region of erm. Conversely, the absence of bHLH-O proteins in INPs allows activation of erm and Erm-mediated maturation of INPs. Our results further suggest that Pointed P1 (PntP1) mediates the dedifferentiation of INPs resulting from the loss of Erm or overexpression of Dpn or E(spl) proteins. Taken together, these findings reveal mechanisms underlying the regulation of the maintenance of type II NBs and differentiation of INPs through the differential expression of bHLH-O family proteins. Copyright © 2017 Elsevier Inc. All rights reserved.
Flexible method for monitoring fuel cell voltage

DOEpatents

Mowery, Kenneth D.; Ripley, Eugene V.

2002-01-01

A method for equalizing the measured voltage of each cluster in a fuel cell stack wherein at least one of the clusters has a different number of cells than the identical number of cells in the remaining clusters by creating a pseudo voltage for the different cell numbered cluster. The average cell voltage of the all of the cells in the fuel cell stack is calculated and multiplied by a constant equal to the difference in the number of cells in the identical cell clusters and the number of cells in the different numbered cell cluster. The resultant product is added to the actual voltage measured across the different numbered cell cluster to create a pseudo voltage which is equivalent in cell number to the number of cells in the other identical numbered cell clusters.
Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets

PubMed Central

Macosko, Evan Z.; Basu, Anindita; Satija, Rahul; Nemesh, James; Shekhar, Karthik; Goldman, Melissa; Tirosh, Itay; Bialas, Allison R.; Kamitaki, Nolan; Martersteck, Emily M.; Trombetta, John J.; Weitz, David A.; Sanes, Joshua R.; Shalek, Alex K.; Regev, Aviv; McCarroll, Steven A.

2015-01-01

Summary Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-Seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. Drop-Seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts’ cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-Seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. PMID:26000488
Autoubiquitination of feline E3 ubiquitin ligase BCA2.

PubMed

Wang, Weiran; Qu, Meng; Wang, Jiawen; Zhang, Xin; Zhang, Haihong; Wu, Jiaxin; Yu, Bin; Wu, Hui; Kong, Wei; Yu, Xianghui

2018-01-05

BCA2/RNF115/Rabring7 is a RING type E3 ubiquitin ligase that is overexpressed in human breast tumors and is important for regulating breast cancer cell migration. In the present investigation, feline BCA2 (fBCA2) was identified and characterized. Compared with its human counterpart, the fBCA2 cDNA was confirmed to be 918 base pairs in length showing 92.6% consensus and identity positions, encoding a protein of 305 amino acids with 96.7% consensus and 93.1% identity positions. The fBCA2 protein contains a RING domain at the C-terminus, which was found to be essential for its autoubiquitination. Copyright © 2017. Published by Elsevier B.V.
A computational model of the development of separate representations of facial identity and expression in the primate visual system.

PubMed

Tromans, James Matthew; Harris, Mitchell; Stringer, Simon Maitland

2011-01-01

Experimental studies have provided evidence that the visual processing areas of the primate brain represent facial identity and facial expression within different subpopulations of neurons. For example, in non-human primates there is evidence that cells within the inferior temporal gyrus (TE) respond primarily to facial identity, while cells within the superior temporal sulcus (STS) respond to facial expression. More recently, it has been found that the orbitofrontal cortex (OFC) of non-human primates contains some cells that respond exclusively to changes in facial identity, while other cells respond exclusively to facial expression. How might the primate visual system develop physically separate representations of facial identity and expression given that the visual system is always exposed to simultaneous combinations of facial identity and expression during learning? In this paper, a biologically plausible neural network model, VisNet, of the ventral visual pathway is trained on a set of carefully-designed cartoon faces with different identities and expressions. The VisNet model architecture is composed of a hierarchical series of four Self-Organising Maps (SOMs), with associative learning in the feedforward synaptic connections between successive layers. During learning, the network develops separate clusters of cells that respond exclusively to either facial identity or facial expression. We interpret the performance of the network in terms of the learning properties of SOMs, which are able to exploit the statistical indendependence between facial identity and expression.
Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis.

PubMed

Chapman, Mark A; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David; Lieber, Richard L

2017-02-01

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173-183, 2009; Kjaer M. Physiol Rev 84: 649-98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins-fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. Copyright © 2017 the American Physiological Society.
Three distinct cell populations express extracellular matrix proteins and increase in number during skeletal muscle fibrosis

PubMed Central

Chapman, Mark A.; Mukund, Kavitha; Subramaniam, Shankar; Brenner, David

2017-01-01

Tissue extracellular matrix (ECM) provides structural support and creates unique environments for resident cells (Bateman JF, Boot-Handford RP, Lamandé SR. Nat Rev Genet 10: 173–183, 2009; Kjaer M. Physiol Rev 84: 649–98, 2004). However, the identities of cells responsible for creating specific ECM components have not been determined. In striated muscle, the identity of these cells becomes important in disease when ECM changes result in fibrosis and subsequent increased tissue stiffness and dysfunction. Here we describe a novel approach to isolate and identify cells that maintain the ECM in both healthy and fibrotic muscle. Using a collagen I reporter mouse, we show that there are three distinct cell populations that express collagen I in both healthy and fibrotic skeletal muscle. Interestingly, the number of collagen I-expressing cells in all three cell populations increases proportionally in fibrotic muscle, indicating that all cell types participate in the fibrosis process. Furthermore, while some profibrotic ECM and ECM-associated genes are significantly upregulated in fibrotic muscle, the fibrillar collagen gene expression profile is not qualitatively altered. This suggests that muscle fibrosis in this model results from an increased number of collagen I-expressing cells and not the initiation of a specific fibrotic collagen gene expression program. Finally, in fibrotic muscle, we show that these collagen I-expressing cell populations differentially express distinct ECM proteins—fibroblasts express the fibrillar components of ECM, fibro/adipogenic progenitors cells differentially express basal laminar proteins, and skeletal muscle progenitor cells differentially express genes important for the satellite cell. PMID:27881411
Transitions from mono- to co- to tri-culture uniquely affect gene expression in breast cancer, stromal, and immune compartments.

PubMed

Regier, Mary C; Maccoux, Lindsey J; Weinberger, Emma M; Regehr, Keil J; Berry, Scott M; Beebe, David J; Alarid, Elaine T

2016-08-01

Heterotypic interactions in cancer microenvironments play important roles in disease initiation, progression, and spread. Co-culture is the predominant approach used in dissecting paracrine interactions between tumor and stromal cells, but functional results from simple co-cultures frequently fail to correlate to in vivo conditions. Though complex heterotypic in vitro models have improved functional relevance, there is little systematic knowledge of how multi-culture parameters influence this recapitulation. We therefore have employed a more iterative approach to investigate the influence of increasing model complexity; increased heterotypic complexity specifically. Here we describe how the compartmentalized and microscale elements of our multi-culture device allowed us to obtain gene expression data from one cell type at a time in a heterotypic culture where cells communicated through paracrine interactions. With our device we generated a large dataset comprised of cell type specific gene-expression patterns for cultures of increasing complexity (three cell types in mono-, co-, or tri-culture) not readily accessible in other systems. Principal component analysis indicated that gene expression was changed in co-culture but was often more strongly altered in tri-culture as compared to mono-culture. Our analysis revealed that cell type identity and the complexity around it (mono-, co-, or tri-culture) influence gene regulation. We also observed evidence of complementary regulation between cell types in the same heterotypic culture. Here we demonstrate the utility of our platform in providing insight into how tumor and stromal cells respond to microenvironments of varying complexities highlighting the expanding importance of heterotypic cultures that go beyond conventional co-culture.
Redox regulation of stress signals: possible roles of dendritic stellate TRX producer cells (DST cell types).

PubMed

Yodoi, Junji; Nakamura, Hajime; Masutani, Hiroshi

2002-01-01

Thioredoxin (TRX) is a 12 kDa protein with redox-active dithiol (Cys-Gly-Pro-Cys) in the active site. TRX is induced by a variety of stresses including viral infection and inflammation. The promoter sequences of the TRX gene contain a series of stress-responsive elements including ORE, ARE, XRE, CRE and SP-1. TRX promotes DNA binding of transcription factors such as NF-kappaB, AP-1 and p53. TRX interacts with target proteins modulating the activity of those proteins. We have identified TRX binding protein-2 (TBP-2), which was identical to vitamin D3 up-regulated protein 1 (VDUP1). Potential action of TBP-2/VDUP1 as a redox-sensitive tumor suppressor will be discussed. There is accumulating evidence for the involvement of TRX in the protection against infectious and inflammatory disorders. We will discuss the role of TRX-dependent redox regulation of the host defense mechanism, in particular its relation to the emerging concept of constitutive and/or inducible TRX on special cell types with dendritic and stellate morphology in the immune, endocrine and nervous systems, which we provisionally designate as dendritic stellate TRX producer cells (DST cell types).
Cellular interaction of a layer-by-layer based drug delivery system depending on material properties and cell types

PubMed Central

Brueckner, Mandy; Jankuhn, Steffen; Jülke, Eva-Maria; Reibetanz, Uta

2018-01-01

Background Drug delivery systems (DDS) and their interaction with cells are a controversial topic in the development of therapeutic concepts and approaches. On one hand, DDS are very useful for protected and targeted transport of defined dosages of active agents. On the other hand, their physicochemical properties such as material, size, shape, charge, or stiffness have a huge impact on cellular uptake and intracellular processing. Additionally, even identical DDS can undergo a completely diverse interaction with different cell types. However, quite often in in vitro DDS/cell interaction experiments, those aspects are not considered and DDS and cells are randomly chosen. Methods and results Hence, our investigations provide an insight into layer-by-layer designed microcarriers with modifications of only some of the most important parameters (surface charge, stiffness, and applied microcarrier/cell ratio) and their influence on cellular uptake and viability. We also considered the interaction of these differently equipped DDS with several cell types and investigated professional phagocytes (neutrophil granulocytes; macrophages) as well as non-professional phagocytes (epithelial cells) under comparable conditions. We found that even small modifications such as layer-by-layer (LbL)-microcarriers with positive or negative surface charge, or LbL-microcarriers with solid core or as hollow capsules but equipped with the same surface properties, show significant differences in interaction and viability, and several cell types react very differently to the offered DDS. Conclusion As a consequence, the properties of the DDS have to be carefully chosen with respect to the addressed cell type with the aim to efficiently transport a desired agent. PMID:29670351
Differential tumor biology effects of double-initiation in a mouse skin chemical carcinogenesis model comparing wild type versus protein kinase Cepsilon overexpression mice.

PubMed

Li, Yafan; Wheeler, Deric L; Ananthaswamy, Honnavara N; Verma, Ajit K; Oberley, Terry D

2007-12-01

Our previous studies showed that protein kinase Cepsilon (PKCepsilon) verexpression in mouse skin resulted in metastatic squamous cell carcinoma (SCC) elicited by single 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in the absence of preceding papilloma formation as is typically observed in wild type mice. The present study demonstrates that double-DMBA initiation modulates tumor incidence, multiplicity, and latency period in both wild type and PKCepsilon overexpression transgenic (PKCepsilon-Tg) mice. After 17 weeks (wks) of tumor promotion, a reduction in papilloma multiplicity was observed in double- versus single-DMBA initiated wild type mice. Papilloma multiplicity was inversely correlated with cell death indices of interfollicular keratinocytes, indicating decreased papilloma formation was caused by increased cell death and suggesting the origin of papillomas is in interfollicular epidermis. Double-initiated PKCepsilon-Tg mice had accelerated carcinoma formation and cancer incidence in comparison to single-initiated PKCepsilon-Tg mice. Morphologic analysis of mouse skin following double initiation and tumor promotion showed a similar if not identical series of events to those previously observed following single initiation and tumor promotion: putative preneoplastic cells were observed arising from hyperplastic hair follicles (HFs) with subsequent cancer cell infiltration into the dermis. Single-initiated PKCepsilon-Tg mice exhibited increased mitosis in epidermal cells of HFs during tumor promotion.

C-Terminal Region of EBNA-2 Determines the Superior Transforming Ability of Type 1 Epstein-Barr Virus by Enhanced Gene Regulation of LMP-1 and CXCR7

PubMed Central

Cancian, Laila; Bosshard, Rachel; Lucchesi, Walter; Karstegl, Claudio Elgueta; Farrell, Paul J.

2011-01-01

Type 1 Epstein-Barr virus (EBV) strains immortalize B lymphocytes in vitro much more efficiently than type 2 EBV, a difference previously mapped to the EBNA-2 locus. Here we demonstrate that the greater transforming activity of type 1 EBV correlates with a stronger and more rapid induction of the viral oncogene LMP-1 and the cell gene CXCR7 (which are both required for proliferation of EBV-LCLs) during infection of primary B cells with recombinant viruses. Surprisingly, although the major sequence differences between type 1 and type 2 EBNA-2 lie in N-terminal parts of the protein, the superior ability of type 1 EBNA-2 to induce proliferation of EBV-infected lymphoblasts is mostly determined by the C-terminus of EBNA-2. Substitution of the C-terminus of type 1 EBNA-2 into the type 2 protein is sufficient to confer a type 1 growth phenotype and type 1 expression levels of LMP-1 and CXCR7 in an EREB2.5 cell growth assay. Within this region, the RG, CR7 and TAD domains are the minimum type 1 sequences required. Sequencing the C-terminus of EBNA-2 from additional EBV isolates showed high sequence identity within type 1 isolates or within type 2 isolates, indicating that the functional differences mapped are typical of EBV type sequences. The results indicate that the C-terminus of EBNA-2 accounts for the greater ability of type 1 EBV to promote B cell proliferation, through mechanisms that include higher induction of genes (LMP-1 and CXCR7) required for proliferation and survival of EBV-LCLs. PMID:21857817
G to A substitution in 5{prime} donor splice site of introns 18 and 48 of COL1A1 gene of type I collagen results in different splicing alternatives in osteogenesis imperfecta type I cell strains

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

Willing, M.; Deschenes, S.

We have identified a G to A substitution in the 5{prime} donor splice site of intron 18 of one COL1A1 allele in two unrelated families with osteogenesis imperfecta (OI) type I. A third OI type I family has a G to A substitution at the identical position in intron 48 of one COL1A1 allele. Both mutations abolish normal splicing and lead to reduced steady-state levels of mRNA from the mutant COL1A1 allele. The intron 18 mutation leads to both exon 18 skipping in the mRNA and to utilization of a single alternative splice site near the 3{prime} end of exonmore » 18. The latter results in deletion of the last 8 nucleotides of exon 18 from the mRNA, a shift in the translational reading-frame, and the creation of a premature termination codon in exon 19. Of the potential alternative 5{prime} splice sites in exon 18 and intron 18, the one utilized has a surrounding nucleotide sequence which most closely resembles that of the natural splice site. Although a G to A mutation was detected at the identical position in intron 48 of one COL1A1 allele in another OI type I family, nine complex alternative splicing patterns were identified by sequence analysis of cDNA clones derived from fibroblast mRNA from this cell strain. All result in partial or complete skipping of exon 48, with in-frame deletions of portions of exons 47 and/or 49. The different patterns of RNA splicing were not explained by their sequence homology with naturally occuring 5{prime} splice sites, but rather by recombination between highly homologous exon sequences, suggesting that we may not have identified the major splicing alternative(s) in this cell strain. Both G to A mutations result in decreased production of type I collagen, the common biochemical correlate of OI type I.« less
Antiviral Type I and Type III Interferon Responses in the Central Nervous System

PubMed Central

Sorgeloos, Frédéric; Kreit, Marguerite; Hermant, Pascale; Lardinois, Cécile; Michiels, Thomas

2013-01-01

The central nervous system (CNS) harbors highly differentiated cells, such as neurons that are essential to coordinate the functions of complex organisms. This organ is partly protected by the blood-brain barrier (BBB) from toxic substances and pathogens carried in the bloodstream. Yet, neurotropic viruses can reach the CNS either by crossing the BBB after viremia, or by exploiting motile infected cells as Trojan horses, or by using axonal transport. Type I and type III interferons (IFNs) are cytokines that are critical to control early steps of viral infections. Deficiencies in the IFN pathway have been associated with fatal viral encephalitis both in humans and mice. Therefore, the IFN system provides an essential protection of the CNS against viral infections. Yet, basal activity of the IFN system appears to be low within the CNS, likely owing to the toxicity of IFN to this organ. Moreover, after viral infection, neurons and oligodendrocytes were reported to be relatively poor IFN producers and appear to keep some susceptibility to neurotropic viruses, even in the presence of IFN. This review addresses some trends and recent developments concerning the role of type I and type III IFNs in: i) preventing neuroinvasion and infection of CNS cells; ii) the identity of IFN-producing cells in the CNS; iii) the antiviral activity of ISGs; and iv) the activity of viral proteins of neurotropic viruses that target the IFN pathway. PMID:23503326
Antiviral type I and type III interferon responses in the central nervous system.

PubMed

Sorgeloos, Frédéric; Kreit, Marguerite; Hermant, Pascale; Lardinois, Cécile; Michiels, Thomas

2013-03-15

The central nervous system (CNS) harbors highly differentiated cells, such as neurons that are essential to coordinate the functions of complex organisms. This organ is partly protected by the blood-brain barrier (BBB) from toxic substances and pathogens carried in the bloodstream. Yet, neurotropic viruses can reach the CNS either by crossing the BBB after viremia, or by exploiting motile infected cells as Trojan horses, or by using axonal transport. Type I and type III interferons (IFNs) are cytokines that are critical to control early steps of viral infections. Deficiencies in the IFN pathway have been associated with fatal viral encephalitis both in humans and mice. Therefore, the IFN system provides an essential protection of the CNS against viral infections. Yet, basal activity of the IFN system appears to be low within the CNS, likely owing to the toxicity of IFN to this organ. Moreover, after viral infection, neurons and oligodendrocytes were reported to be relatively poor IFN producers and appear to keep some susceptibility to neurotropic viruses, even in the presence of IFN. This review addresses some trends and recent developments concerning the role of type I and type III IFNs in: i) preventing neuroinvasion and infection of CNS cells; ii) the identity of IFN-producing cells in the CNS; iii) the antiviral activity of ISGs; and iv) the activity of viral proteins of neurotropic viruses that target the IFN pathway.
UFO in the Arabidopsis inflorescence apex is required for floral-meristem identity and bract suppression.

PubMed

Hepworth, Shelley R; Klenz, Jennifer E; Haughn, George W

2006-03-01

The UNUSUAL FLORAL ORGANS (UFO) gene of Arabidopsis encodes an F-box protein required for the determination of floral-organ and floral-meristem identity. Mutation of UFO leads to dramatic changes in floral-organ type which are well-characterized whereas inflorescence defects are more subtle and less understood. These defects include an increase in the number of secondary inflorescences, nodes that alternate between forming flowers and secondary inflorescences, and nodes in which a single flower is subtended by a bract. Here, we show how inflorescence defects correlate with the abnormal development of floral primordia and establish a temporal requirement for UFO in this process. At the inflorescence apex of ufo mutants, newly formed primordia are initially bract-like. Expression of the floral-meristem identity genes LFY and AP1 are confined to a relatively small adaxial region of these primordia with expression of the bract-identity marker FIL observed in cells that comprise the balance of the primordia. Proliferation of cells in the adaxial region of these early primordia is delayed by several nodes such that primordia appear "chimeric" at several nodes, having visible floral and bract components. However, by late stage 2 of floral development, growth of the bract generally ceases and is overtaken by development of the floral primordium. This abnormal pattern of floral meristem development is not rescued by expression of UFO from the AP1 promoter, indicating that UFO is required prior to AP1 activation for normal development of floral primordia. We propose that UFO and LFY are jointly required in the inflorescence meristem to both promote floral meristem development and inhibit, in a non-cell autonomous manner, growth of the bract.
ELECTRON MICROSCOPY OF HELA CELLS INFECTED WITH ADENOVIRUSES

PubMed Central

Harford, Carl G.; Hamlin, Alice; Parker, Esther; van Ravenswaay, Theodore

1956-01-01

HeLa cells were infected with adenoviruses (types 1–4) and sectioned for electron microscopy after intervals of 20 to 48 hours. Clusters of virus-like particles were found within the nuclei of infected cultures but not in those of uninfected controls. The particles were often arranged in rows as if in crystalline formation. Maximal diameter of particles was approximately 65 mµ, and internal bodies were demonstrated. Lesions of infected cells included target-like structures of the nuclear membrane, large nuclear vacuoles (type 2), and increased numbers of large irregular electron-dense granules in the cytoplasm 48 hours after infection. Examination of infected cultures by light microscopy, using the Feulgen reaction, showed intranuclear inclusion bodies and a cytopathogenic effect consisting of clumping of cells without pyknosis of nuclei. A lipide stain showed numerous cytoplasmic granules that were not identical with the large, irregular, electron-dense granules of the cytoplasm. Practically all the cells showed the viral cytopathogenic effect, but only a minority of cells were found to contain virus-like particles or intranuclear inclusion bodies. PMID:13357696
The impact of family status on gender identity and on sex-typing of household tasks in Israel.

PubMed

Kulik, Liat

2005-06-01

The author examined differences in sex-typing of household tasks (adult gender roles and children's chores) and differences in gender identity among adult Israelis. The author compared 2 groups of participants: single people without children (single-family participants; n = 62) and married people with children (full-family participants; n = 62). Regarding sex-typing of household tasks and direct assessments of masculine and feminine identity, there were no differences between single-family participants and full-family participants. However, family status affected self-assessments of gender identity that were based on cultural definitions of masculine and feminine attributes. Furthermore, correlations between direct assessments of gender identity and sex-typing of household tasks differed according to family status.
Evolution of pathogenicity and sexual reproduction in eight Candida genomes

PubMed Central

Butler, Geraldine; Rasmussen, Matthew D.; Lin, Michael F.; Santos, Manuel A.S.; Sakthikumar, Sharadha; Munro, Carol A.; Rheinbay, Esther; Grabherr, Manfred; Forche, Anja; Reedy, Jennifer L.; Agrafioti, Ino; Arnaud, Martha B.; Bates, Steven; Brown, Alistair J.P.; Brunke, Sascha; Costanzo, Maria C.; Fitzpatrick, David A.; de Groot, Piet W. J.; Harris, David; Hoyer, Lois L.; Hube, Bernhard; Klis, Frans M.; Kodira, Chinnappa; Lennard, Nicola; Logue, Mary E.; Martin, Ronny; Neiman, Aaron M.; Nikolaou, Elissavet; Quail, Michael A.; Quinn, Janet; Santos, Maria C.; Schmitzberger, Florian F.; Sherlock, Gavin; Shah, Prachi; Silverstein, Kevin; Skrzypek, Marek S.; Soll, David; Staggs, Rodney; Stansfield, Ian; Stumpf, Michael P H; Sudbery, Peter E.; Thyagarajan, Srikantha; Zeng, Qiandong; Berman, Judith; Berriman, Matthew; Heitman, Joseph; Gow, Neil A. R.; Lorenz, Michael C.; Birren, Bruce W.; Kellis, Manolis; Cuomo, Christina A.

2009-01-01

Candida species are the most common cause of opportunistic fungal infection worldwide. We report the genome sequences of six Candida species and compare these and related pathogens and nonpathogens. There are significant expansions of cell wall, secreted, and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the Mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/alpha2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine to serine genetic code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the C. albicans gene catalog, identifying many new genes. PMID:19465905
Cell Identity Switching Regulated by Retinoic Acid Signaling Maintains Homogeneous Segments in the Hindbrain.

PubMed

Addison, Megan; Xu, Qiling; Cayuso, Jordi; Wilkinson, David G

2018-06-04

The patterning of tissues to form subdivisions with distinct and homogeneous regional identity is potentially disrupted by cell intermingling. Transplantation studies suggest that homogeneous segmental identity in the hindbrain is maintained by identity switching of cells that intermingle into another segment. We show that switching occurs during normal development and is mediated by feedback between segment identity and the retinoic acid degrading enzymes, cyp26b1 and cyp26c1. egr2, which specifies the segmental identity of rhombomeres r3 and r5, underlies the lower expression level of cyp26b1 and cyp26c1 in r3 and r5 compared with r2, r4, and r6. Consequently, r3 or r5 cells that intermingle into adjacent segments encounter cells with higher cyp26b1/c1 expression, which we find is required for downregulation of egr2b expression. Furthermore, egr2b expression is regulated in r2, r4, and r6 by non-autonomous mechanisms that depend upon the number of neighbors that express egr2b. These findings reveal that a community regulation of retinoid signaling maintains homogeneous segmental identity. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
Bilinear identities for an extended B-type Kadomtsev-Petviashvili hierarchy

NASA Astrophysics Data System (ADS)

Lin, Runliang; Cao, Tiancheng; Liu, Xiaojun; Zeng, Yunbo

2016-03-01

We construct bilinear identities for wave functions of an extended B-type Kadomtsev-Petviashvili (BKP) hierarchy containing two types of (2+1)-dimensional Sawada-Kotera equations with a self-consistent source. Introducing an auxiliary variable corresponding to the extended flow for the BKP hierarchy, we find the τ -function and bilinear identities for this extended BKP hierarchy. The bilinear identities generate all the Hirota bilinear equations for the zero-curvature forms of this extended BKP hierarchy. As examples, we obtain the Hirota bilinear equations for the two types of (2+1)-dimensional Sawada-Kotera equations in explicit form.
Detailed molecular analyses of the hexon loop-1 and fibers of fowl aviadenoviruses reveal new insights into the antigenic relationship and confirm that specific genotypes are involved in field outbreaks of inclusion body hepatitis.

PubMed

Schachner, Anna; Marek, Ana; Grafl, Beatrice; Hess, Michael

2016-04-15

Forty-eight fowl aviadenoviruses (FAdVs) isolated from recent IBH outbreaks across Europe were investigated, by utilizing for the first time the two major adenoviral antigenic domains, hexon loop-1 and fiber, for compound molecular characterization of IBH-associated FAdVs. Successful target gene amplification, following virus isolation in cell culture or from FTA-card samples, demonstrated presence of FAdVs in all cases indicative for IBH. Based on hexon loop-1 analysis, 31 European field isolates exhibited highest nucleotide identity (>97.2%) to reference strains FAdV-2 or -11 representing FAdV-D, while 16 and one European isolates shared >96.0% nucleotide identity with FAdV-8a and -8b, or FAdV-7, the prototype strains representing FAdV-E. These results extend recognition of specific FAdV-D and FAdV-E affiliate genotypes as causative agents of IBH to the European continent. In all isolates, species specificity determined by fiber gene analysis correlated with hexon-based typing. A threshold of 72.0% intraspecies nucleotide identity between fibers from investigated prototype and field strains corresponded with demarcation criteria proposed for hexon, suggesting fiber-based analysis as a complementary tool for molecular FAdV typing. A limited number of strains exhibited inconsistencies between hexon and fiber subclustering, indicating potential constraints for single-gene based typing of those FAdVs. Within FAdV-D, field isolate fibers shared a high degree of nucleotide (>96.7%) and aa (>95.8%) identity, while FAdV-E field isolate fibers displayed greater nucleotide divergence of up to 22.6%, resulting in lower aa identities of >81.7%. Furthermore, comparison with FAdVs from IBH outbreaks outside Europe revealed close genetic relationship in the fiber, independent of the strains' geographic origin. Copyright © 2016 Elsevier B.V. All rights reserved.
Assessing similarity to primary tissue and cortical layer identity in induced pluripotent stem cell-derived cortical neurons through single-cell transcriptomics

PubMed Central

Handel, Adam E.; Chintawar, Satyan; Lalic, Tatjana; Whiteley, Emma; Vowles, Jane; Giustacchini, Alice; Argoud, Karene; Sopp, Paul; Nakanishi, Mahito; Bowden, Rory; Cowley, Sally; Newey, Sarah; Akerman, Colin; Ponting, Chris P.; Cader, M. Zameel

2016-01-01

Induced pluripotent stem cell (iPSC)-derived cortical neurons potentially present a powerful new model to understand corticogenesis and neurological disease. Previous work has established that differentiation protocols can produce cortical neurons, but little has been done to characterize these at cellular resolution. In particular, it is unclear to what extent in vitro two-dimensional, relatively disordered culture conditions recapitulate the development of in vivo cortical layer identity. Single-cell multiplex reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to interrogate the expression of genes previously implicated in cortical layer or phenotypic identity in individual cells. Totally, 93.6% of single cells derived from iPSCs expressed genes indicative of neuronal identity. High proportions of single neurons derived from iPSCs expressed glutamatergic receptors and synaptic genes. And, 68.4% of iPSC-derived neurons expressing at least one layer marker could be assigned to a laminar identity using canonical cortical layer marker genes. We compared single-cell RNA-seq of our iPSC-derived neurons to available single-cell RNA-seq data from human fetal and adult brain and found that iPSC-derived cortical neurons closely resembled primary fetal brain cells. Unexpectedly, a subpopulation of iPSC-derived neurons co-expressed canonical fetal deep and upper cortical layer markers. However, this appeared to be concordant with data from primary cells. Our results therefore provide reassurance that iPSC-derived cortical neurons are highly similar to primary cortical neurons at the level of single cells but suggest that current layer markers, although effective, may not be able to disambiguate cortical layer identity in all cells. PMID:26740550
Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes and progenitors

PubMed Central

Villani, Alexandra-Chloé; Satija, Rahul; Reynolds, Gary; Sarkizova, Siranush; Shekhar, Karthik; Fletcher, James; Griesbeck, Morgane; Butler, Andrew; Zheng, Shiwei; Lazo, Suzan; Jardine, Laura; Dixon, David; Stephenson, Emily; Nilsson, Emil; Grundberg, Ida; McDonald, David; Filby, Andrew; Li, Weibo; De Jager, Philip L.; Rozenblatt-Rosen, Orit; Lane, Andrew A.; Haniffa, Muzlifah; Regev, Aviv; Hacohen, Nir

2017-01-01

Dendritic cells (DCs) and monocytes play a central role in pathogen sensing, phagocytosis and antigen presentation and consist of multiple specialized subtypes. However, their identities and interrelationships are not fully understood. Using unbiased single-cell RNA sequencing (RNA-seq) of ~2400 cells, we identified six human DCs and four monocyte subtypes in human blood. Our study reveals: a new DC subset that shares properties with plasmacytoid DCs (pDCs) but potently activates T cells, thus redefining pDCs; a new subdivision within the CD1C+ subset of DCs; the relationship between blastic plasmacytoid DC neoplasia cells and healthy DCs; and circulating progenitor of conventional DCs (cDCs). Our revised taxonomy will enable more accurate functional and developmental analyses as well as immune monitoring in health and disease. PMID:28428369
The murine ufo receptor: molecular cloning, chromosomal localization and in situ expression analysis.

PubMed

Faust, M; Ebensperger, C; Schulz, A S; Schleithoff, L; Hameister, H; Bartram, C R; Janssen, J W

1992-07-01

We have cloned the mouse homologue of the ufo oncogene. It encodes a novel tyrosine kinase receptor characterized by a unique extracellular domain containing two immunoglobulin-like and two fibronectin type III repeats. Comparison of the predicted ufo amino acid sequences of mouse and man revealed an overall identity of 87.6%. The ufo locus maps to mouse chromosome 7A3-B1 and thereby extends the known conserved linkage group between mouse chromosome 7 and human chromosome 19. RNA in situ hybridization analysis established the onset of specific ufo expression in the late embryogenesis at day 12.5 post coitum (p.c.) and localized ufo transcription to distinct substructures of a broad spectrum of developing tissues (e.g. subepidermal cells of the skin, mesenchymal cells of the periosteum). In adult animals ufo is expressed in cells forming organ capsules as well as in connective tissue structures. ufo may function as a signal transducer between specific cell types of mesodermal origin.
Integrins are required for tissue organization and restriction of neurogenesis in regenerating planarians

PubMed Central

Seebeck, Florian; März, Martin; Meyer, Anna-Wiebke; Reuter, Hanna; Vogg, Matthias C.; Stehling, Martin; Mildner, Karina; Zeuschner, Dagmar; Rabert, Franziska

2017-01-01

Tissue regeneration depends on proliferative cells and on cues that regulate cell division, differentiation, patterning and the restriction of these processes once regeneration is complete. In planarians, flatworms with high regenerative potential, muscle cells express some of these instructive cues. Here, we show that members of the integrin family of adhesion molecules are required for the integrity of regenerating tissues, including the musculature. Remarkably, in regenerating β1-integrin RNAi planarians, we detected increased numbers of mitotic cells and progenitor cell types, as well as a reduced ability of stem cells and lineage-restricted progenitor cells to accumulate at wound sites. These animals also formed ectopic spheroid structures of neural identity in regenerating heads. Interestingly, those polarized assemblies comprised a variety of neural cells and underwent continuous growth. Our study indicates that integrin-mediated cell adhesion is required for the regenerative formation of organized tissues and for restricting neurogenesis during planarian regeneration. PMID:28137894
ODE constrained mixture modelling: a method for unraveling subpopulation structures and dynamics.

PubMed

Hasenauer, Jan; Hasenauer, Christine; Hucho, Tim; Theis, Fabian J

2014-07-01

Functional cell-to-cell variability is ubiquitous in multicellular organisms as well as bacterial populations. Even genetically identical cells of the same cell type can respond differently to identical stimuli. Methods have been developed to analyse heterogeneous populations, e.g., mixture models and stochastic population models. The available methods are, however, either incapable of simultaneously analysing different experimental conditions or are computationally demanding and difficult to apply. Furthermore, they do not account for biological information available in the literature. To overcome disadvantages of existing methods, we combine mixture models and ordinary differential equation (ODE) models. The ODE models provide a mechanistic description of the underlying processes while mixture models provide an easy way to capture variability. In a simulation study, we show that the class of ODE constrained mixture models can unravel the subpopulation structure and determine the sources of cell-to-cell variability. In addition, the method provides reliable estimates for kinetic rates and subpopulation characteristics. We use ODE constrained mixture modelling to study NGF-induced Erk1/2 phosphorylation in primary sensory neurones, a process relevant in inflammatory and neuropathic pain. We propose a mechanistic pathway model for this process and reconstructed static and dynamical subpopulation characteristics across experimental conditions. We validate the model predictions experimentally, which verifies the capabilities of ODE constrained mixture models. These results illustrate that ODE constrained mixture models can reveal novel mechanistic insights and possess a high sensitivity.
Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells.

PubMed

Milenkovic, Andrea; Brandl, Caroline; Milenkovic, Vladimir M; Jendryke, Thomas; Sirianant, Lalida; Wanitchakool, Potchanart; Zimmermann, Stephanie; Reiff, Charlotte M; Horling, Franziska; Schrewe, Heinrich; Schreiber, Rainer; Kunzelmann, Karl; Wetzel, Christian H; Weber, Bernhard H F

2015-05-19

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1(-/-)) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1(-/-) mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex--that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies.
Molecular analysis of neocortical layer structure in the ferret

PubMed Central

Rowell, Joanna J.; Mallik, Atul K.; Dugas-Ford, Jennifer; Ragsdale, Clifton W.

2010-01-01

Molecular markers that distinguish specific layers of rodent neocortex are increasingly employed to study cortical development and the physiology of cortical circuits. The extent to which these markers represent general features of neocortical cell type identity across mammals is, however, unknown. To assess the conservation of layer markers more broadly, we isolated orthologs for fifteen layer-enriched genes in the ferret, a carnivore with a large, gyrencephalic brain, and analyzed their patterns of neocortical gene expression. Our major findings are: (1) Many but not all layer markers tested show similar patterns of layer-specific gene expression between mouse and ferret cortex, supporting the view that layer-specific cell type identity is conserved at a molecular level across mammalian superorders; (2) Our panel of deep layer markers (ER81/ETV1, SULF2, PCP4, FEZF2/ZNF312, CACNA1H, KCNN2/SK2, SYT6, FOXP2, CTGF) provides molecular evidence that the specific stratifications of layer 5 and 6 into 5a, 5b, 6a and 6b are also conserved between rodents and carnivores. (3) Variations in layer-specific gene expression are more pronounced across areas of ferret cortex than between homologous areas of mouse and ferret cortex; (4) This variation of area gene expression was clearest with the superficial layer markers studied (SERPINE2, MDGA1, CUX1, UNC5D, RORB/NR1F2, EAG2/KCNH5). Most dramatically, the layer 4 markers RORB and EAG2 disclosed a molecular sublamination to ferret visual cortex and demonstrated a molecular dissociation among the so-called agranular areas of the neocortex. Our findings establish molecular markers as a powerful complement to cytoarchitecture for neocortical layer and cell-type comparisons across mammals. PMID:20575059
Molecular analysis of neocortical layer structure in the ferret.

PubMed

Rowell, Joanna J; Mallik, Atul K; Dugas-Ford, Jennifer; Ragsdale, Clifton W

2010-08-15

Molecular markers that distinguish specific layers of rodent neocortex are increasingly employed to study cortical development and the physiology of cortical circuits. The extent to which these markers represent general features of neocortical cell type identity across mammals, however, is unknown. To assess the conservation of layer markers more broadly, we isolated orthologs for 15 layer-enriched genes in the ferret, a carnivore with a large, gyrencephalic brain, and analyzed their patterns of neocortical gene expression. Our major findings are: 1) Many but not all layer markers tested show similar patterns of layer-specific gene expression between mouse and ferret cortex, supporting the view that layer-specific cell type identity is conserved at a molecular level across mammalian superorders; 2) Our panel of deep layer markers (ER81/ETV1, SULF2, PCP4, FEZF2/ZNF312, CACNA1H, KCNN2/SK2, SYT6, FOXP2, CTGF) provides molecular evidence that the specific stratifications of layers 5 and 6 into 5a, 5b, 6a, and 6b are also conserved between rodents and carnivores; 3) Variations in layer-specific gene expression are more pronounced across areas of ferret cortex than between homologous areas of mouse and ferret cortex; 4) This variation of area gene expression was clearest with the superficial layer markers studied (SERPINE2, MDGA1, CUX1, UNC5D, RORB/NR1F2, EAG2/KCNH5). Most dramatically, the layer 4 markers RORB and EAG2 disclosed a molecular sublamination to ferret visual cortex and demonstrated a molecular dissociation among the so-called agranular areas of the neocortex. Our findings establish molecular markers as a powerful complement to cytoarchitecture for neocortical layer and cell-type comparisons across mammals. (c) 2010 Wiley-Liss, Inc.
Norepinephrine is coreleased with serotonin in mouse taste buds.

PubMed

Huang, Yijen A; Maruyama, Yutaka; Roper, Stephen D

2008-12-03

ATP and serotonin (5-HT) are neurotransmitters secreted from taste bud receptor (type II) and presynaptic (type III) cells, respectively. Norepinephrine (NE) has also been proposed to be a neurotransmitter or paracrine hormone in taste buds. Yet, to date, the specific stimulus for NE release in taste buds is not well understood, and the identity of the taste cells that secrete NE is not known. Chinese hamster ovary cells were transfected with alpha(1A) adrenoceptors and loaded with fura-2 ("biosensors") to detect NE secreted from isolated mouse taste buds and taste cells. Biosensors responded to low concentrations of NE (>or=10 nm) with a reliable fura-2 signal. NE biosensors did not respond to stimulation with KCl or taste compounds. However, we recorded robust responses from NE biosensors when they were positioned against mouse circumvallate taste buds and the taste buds were stimulated with KCl (50 mm) or a mixture of taste compounds (cycloheximide, 10 microm; saccharin, 2 mm; denatonium, 1 mm; SC45647, 100 microm). NE biosensor responses evoked by stimulating taste buds were reversibly blocked by prazosin, an alpha(1A) receptor antagonist. Together, these findings indicate that taste bud cells secrete NE when they are stimulated. We isolated individual taste bud cells to identify the origin of NE release. NE was secreted only from presynaptic (type III) taste cells and not receptor (type II) cells. Stimulus-evoked NE release depended on Ca(2+) in the bathing medium. Using dual biosensors (sensitive to 5-HT and NE), we found all presynaptic cells secrete 5-HT and 33% corelease NE with 5-HT.

An experimentally validated network of nine haematopoietic transcription factors reveals mechanisms of cell state stability

PubMed Central

Schütte, Judith; Wang, Huange; Antoniou, Stella; Jarratt, Andrew; Wilson, Nicola K; Riepsaame, Joey; Calero-Nieto, Fernando J; Moignard, Victoria; Basilico, Silvia; Kinston, Sarah J; Hannah, Rebecca L; Chan, Mun Chiang; Nürnberg, Sylvia T; Ouwehand, Willem H; Bonzanni, Nicola; de Bruijn, Marella FTR; Göttgens, Berthold

2016-01-01

Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes. DOI: http://dx.doi.org/10.7554/eLife.11469.001 PMID:26901438
REMEDIAL APPLICATIONS OF EXOSOMES IN CANCER, INFECTIONS AND DIABETES.

PubMed

Wang, Qianhuai; Ding, Xiaoqing; Zhen, Fei; Ma, Jianwei; Meng, Fanxing

2017-03-01

Different cell types under normal and diseased states constantly secrete numerous membrane vesicles including exosomes into extracellular space wkich can be isolated from various biological fluids and cell culture supernatants. Exosomal diameter ranges between 40-100 nm. In current research, exosomes are being exploited as biomarkers for pathological diagnosis and potential remedy against various disease conditions such as infections and autoimmune disorders. In cancer immunotherapy, exosomes have promisingly been employed due to the identical immunogenic antigens of exosomes produced by neoplastic cells and the originating tissues. Antigen-specific T-cell activation and immunomodulatory activity is observed to be performed by exosomes isolated from tumor and dendritic cells (DCs). However, more research is still required to uncover the realistic uses of exosomes, particularly as drug delivery tool in autoimmune diseases, cancers and diabetes.
Similarly Torn, Differentially Shorn? The Experience and Management of Conflict between Multiple Roles, Relationships, and Social Categories

PubMed Central

Jones, Janelle M.; Hynie, Michaela

2017-01-01

In three studies we examined the experience and management of conflict between different types of multiple identities. Participants described a conflict between pairs of role, relational, or social identities before rating the experience (i.e., magnitude, stress, and growth) and management of conflict on a newly developed scale assessing four strategies: reconciliation, where identities are integrated, realignment, where one identity is chosen over another, retreat, where both identities are avoided, and reflection, where fit (with others, situation) determines identity selection. In general, the types of identities mattered for conflict management but not its experience: Magnitude and growth did not differ, however, stress was greater for role identity conflicts (Study 3 only) and participants endorsed the use of more realignment for role conflicts (Study 2) and more retreat for relational conflicts (Study 3) relative to other types of identity conflicts. Furthermore, findings suggested that the perceived flexibility of identities, not their importance or valence, were associated with realignment and retreat for roles and with retreat for relationships. Experiencing conflicts between multiple identities leaves people similarly torn, but multiple roles and relationships may be differentially shorn to manage conflict. PMID:29051744
Similarly Torn, Differentially Shorn? The Experience and Management of Conflict between Multiple Roles, Relationships, and Social Categories.

PubMed

Jones, Janelle M; Hynie, Michaela

2017-01-01

In three studies we examined the experience and management of conflict between different types of multiple identities. Participants described a conflict between pairs of role, relational, or social identities before rating the experience (i.e., magnitude, stress, and growth) and management of conflict on a newly developed scale assessing four strategies: reconciliation , where identities are integrated, realignment , where one identity is chosen over another, retreat , where both identities are avoided, and reflection , where fit (with others, situation) determines identity selection. In general, the types of identities mattered for conflict management but not its experience: Magnitude and growth did not differ, however, stress was greater for role identity conflicts ( Study 3 only ) and participants endorsed the use of more realignment for role conflicts ( Study 2 ) and more retreat for relational conflicts ( Study 3 ) relative to other types of identity conflicts. Furthermore, findings suggested that the perceived flexibility of identities, not their importance or valence, were associated with realignment and retreat for roles and with retreat for relationships. Experiencing conflicts between multiple identities leaves people similarly torn, but multiple roles and relationships may be differentially shorn to manage conflict.
Adeno-Associated Virus Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles of Human Diploid Fibroblasts Analyzed by Third-Generation PacBio DNA Sequencing

PubMed Central

Hüser, Daniela; Gogol-Döring, Andreas; Chen, Wei

2014-01-01

ABSTRACT Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats. IMPORTANCE Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will have impact on vector targeting to be considered during gene therapy. PMID:25031342
Gender Identity and Adjustment: Understanding the Impact of Individual and Normative Differences in Sex Typing

PubMed Central

Lurye, Leah E.; Zosuls, Kristina M.; Ruble, Diane N.

2009-01-01

The relationship among gender identity, sex typing, and adjustment has attracted the attention of social and developmental psychologists for many years. However, they have explored this issue with different assumptions and different approaches. Generally the approaches differ regarding whether sex typing is considered adaptive versus maladaptive, measured as an individual or normative difference, and whether gender identity is regarded as a unidimensional or multidimensional construct. In this chapter, we consider both perspectives and suggest that the developmental timing and degree of sex typing, as well as the multidimensionality of gender identity, be considered when examining their relationship to adjustment. PMID:18521861
Gender identity and adjustment: understanding the impact of individual and normative differences in sex typing.

PubMed

Lurye, Leah E; Zosuls, Kristina M; Ruble, Diane N

2008-01-01

The relationship among gender identity, sex typing, and adjustment has attracted the attention of social and developmental psychologists for many years. However, they have explored this issue with different assumptions and different approaches. Generally the approaches differ regarding whether sex typing is considered adaptive versus maladaptive, measured as an individual or normative difference, and whether gender identity is regarded as a unidimensional or multidimensional construct. In this chapter, we consider both perspectives and suggest that the developmental timing and degree of sex typing, as well as the multidimensionality of gender identity, be considered when examining their relationship to adjustment.
Conceptual Challenges of the Systemic Approach in Understanding Cell Differentiation.

PubMed

Paldi, Andras

2018-01-01

The cells of a multicellular organism are derived from a single zygote and genetically identical. Yet, they are phenotypically very different. This difference is the result of a process commonly called cell differentiation. How the phenotypic diversity emerges during ontogenesis or regeneration is a central and intensely studied but still unresolved issue in biology. Cell biology is facing conceptual challenges that are frequently confused with methodological difficulties. How to define a cell type? What stability or change means in the context of cell differentiation and how to deal with the ubiquitous molecular variations seen in the living cells? What are the driving forces of the change? We propose to reframe the problem of cell differentiation in a systemic way by incorporating different theoretical approaches. The new conceptual framework is able to capture the insights made at different levels of cellular organization and considered previously as contradictory. It also provides a formal strategy for further experimental studies.
CD34+ Testicular Stromal Cells Support Long-Term Expansion of Embryonic and Adult Stem and Progenitor Cells

PubMed Central

Kim, Jiyeon; Seandel, Marco; Falciatori, Ilaria; Wen, Duancheng; Rafii, Shahin

2010-01-01

Stem cells reside in specialized microenvironments created by supporting stromal cells that orchestrate self-renewal and lineage-specific differentiation. However, the precise identity of the cellular and molecular pathways that support self-renewal of stem cells is not known. For example, long-term culture of prototypical stem cells, such as adult spermatogonial stem and progenitor cells (SPCs), in vitro has been impeded by the lack of an optimal stromal cell line that initiates and sustains proliferation of these cells. Indeed, current methods, including the use of mouse embryonic fibroblasts (MEFs), have not been efficient and have generally led to inconsistent results. Here, we report the establishment of a novel CD34-positive cell line, referred to as JK1, derived from mouse testicular stromal cells that not only facilitated long-term SPC culture but also allowed faithful generation of SPCs and multipotent stem cells. SPCs generated on JK1 maintained key features of germ line stem cells, including expression of PLZF, DAZL, and GCNA. Furthermore, these feeders also promoted the long-term cultivation of other types of primitive cells including multi-potent adult spermatogonial-derived stem cells, pluripotent murine embryonic stem cells, and embryonic germ cells derived from primordial germ cells. Stem cells could be passaged serially and still maintained expression of characteristic markers such as OCT4 and NANOG in vitro, as well as the ability to generate all three germ layers in vivo. These results indicate that the JK1 cell line is capable of promoting long-term culture of primitive cells. As such, this cell line allows for identification of stromal-derived factors that support long-term proliferation of various types of stem cells and constitutes a convenient alternative to other types of feeder layers. PMID:18669907
Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans

PubMed Central

Sun, C-L; Kim, E; Crowder, C M

2014-01-01

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2–3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death. PMID:24317200
Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans.

PubMed

Sun, C-L; Kim, E; Crowder, C M

2014-04-01

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2-3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death.
Transitions from mono- to co- to tri-culture uniquely affect gene expression in breast cancer, stromal, and immune compartments

PubMed Central

Weinberger, Emma M.; Regehr, Keil J.; Berry, Scott M.; Beebe, David J.; Alarid, Elaine T.

2016-01-01

Heterotypic interactions in cancer microenvironments play important roles in disease initiation, progression, and spread. Co-culture is the predominant approach used in dissecting paracrine interactions between tumor and stromal cells, but functional results from simple co-cultures frequently fail to correlate to in vivo conditions. Though complex heterotypic in vitro models have improved functional relevance, there is little systematic knowledge of how multi-culture parameters influence this recapitulation. We therefore have employed a more iterative approach to investigate the influence of increasing model complexity; increased heterotypic complexity specifically. Here we describe how the compartmentalized and microscale elements of our multi-culture device allowed us to obtain gene expression data from one cell type at a time in a heterotypic culture where cells communicated through paracrine interactions. With our device we generated a large dataset comprised of cell type specific gene-expression patterns for cultures of increasing complexity (three cell types in mono-, co-, or tri-culture) not readily accessible in other systems. Principal component analysis indicated that gene expression was changed in co-culture but was often more strongly altered in tri-culture as compared to mono-culture. Our analysis revealed that cell type identity and the complexity around it (mono-, co-, or tri-culture) influence gene regulation. We also observed evidence of complementary regulation between cell types in the same heterotypic culture. Here we demonstrate the utility of our platform in providing insight into how tumor and stromal cells respond to microenvironments of varying complexities highlighting the expanding importance of heterotypic cultures that go beyond conventional co-culture. PMID:27432323
Building strong bones: molecular regulation of the osteoblast lineage.

PubMed

Long, Fanxin

2011-12-22

The past 15 years have witnessed tremendous progress in the molecular understanding of osteoblasts, the main bone-forming cells in the vertebrate skeleton. In particular, all of the major developmental signals (including WNT and Notch signalling), along with an increasing number of transcription factors (such as RUNX2 and osterix), have been shown to regulate the differentiation and/or function of osteoblasts. As evidence indicates that osteoblasts may also regulate the behaviour of other cell types, a clear understanding of the molecular identity and regulation of osteoblasts is important beyond the field of bone biology.
Prognostic significance of NOD2/CARD15 variants in HLA-identical sibling hematopoietic stem cell transplantation: effect on long-term outcome is confirmed in 2 independent cohorts and may be modulated by the type of gastrointestinal decontamination.

PubMed

Holler, Ernst; Rogler, Gerhard; Brenmoehl, Julia; Hahn, Joachim; Herfarth, Hans; Greinix, Hildegard; Dickinson, Anne M; Socié, Gerard; Wolff, Daniel; Fischer, Gottfried; Jackson, Graham; Rocha, Vanderson; Steiner, Beate; Eissner, Guenther; Marienhagen, Jeorg; Schoelmerich, Juergen; Andreesen, Reinhard

2006-05-15

To assess the role of NOD2/CARD15 variants on the long-term outcome of allogeneic stem cell transplantation in a genetically homogeneous group, we extended our previous study (cohort I, n = 78) and typed DNA for NOD2/CARD15 single nucleotide polymorphisms (SNPs) from an additional 225 recipients and their HLA-identical sibling donors (cohort II) treated at four other European centers. Results of genotyping were compared with clinical outcome. The strong association of NOD2/CARD15 variants with transplantation-related mortality (TRM) was confirmed in univariate and multivariate analysis; TRM increased from 20% in cohort I/22% in cohort II in recipient/donor pairs without any NOD2/CARD15 variants to 47% in cohort I/32% in cohort II in the presence of one variant in either donor or recipient and further to 57% in cohort I/74% in cohort II in the presence of 2 or more variants (P < .002 in both cohorts). NOD2/CARD15 SNPs were not associated with relapse rate but had a strong impact on overall survival. In an analysis of center effects, the type of gastrointestinal decontamination was the only factor interfering with the prognostic significance of NOD2/CARD15 SNPs. Our data further support an interaction between gastrointestinal defense mechanisms, activation of the innate immune system, and specific transplant-related complications.
Symbiotic Cell Differentiation and Cooperative Growth in Multicellular Aggregates

PubMed Central

Yamagishi, Jumpei F; Saito, Nen; Kaneko, Kunihiko

2016-01-01

As cells grow and divide under a given environment, they become crowded and resources are limited, as seen in bacterial biofilms and multicellular aggregates. These cells often show strong interactions through exchanging chemicals, as evident in quorum sensing, to achieve mutualism and division of labor. Here, to achieve stable division of labor, three characteristics are required. First, isogenous cells differentiate into several types. Second, this aggregate of distinct cell types shows better growth than that of isolated cells without interaction and differentiation, by achieving division of labor. Third, this cell aggregate is robust with respect to the number distribution of differentiated cell types. Indeed, theoretical studies have thus far considered how such cooperation is achieved when the ability of cell differentiation is presumed. Here, we address how cells acquire the ability of cell differentiation and division of labor simultaneously, which is also connected with the robustness of a cell society. For this purpose, we developed a dynamical-systems model of cells consisting of chemical components with intracellular catalytic reaction dynamics. The reactions convert external nutrients into internal components for cellular growth, and the divided cells interact through chemical diffusion. We found that cells sharing an identical catalytic network spontaneously differentiate via induction from cell-cell interactions, and then achieve division of labor, enabling a higher growth rate than that in the unicellular case. This symbiotic differentiation emerged for a class of reaction networks under the condition of nutrient limitation and strong cell-cell interactions. Then, robustness in the cell type distribution was achieved, while instability of collective growth could emerge even among the cooperative cells when the internal reserves of products were dominant. The present mechanism is simple and general as a natural consequence of interacting cells with limited resources, and is consistent with the observed behaviors and forms of several aggregates of unicellular organisms. PMID:27749898
Cytotoxicity of cadmium-free quantum dots and their use in cell bioimaging.

PubMed

Soenen, Stefaan J; Manshian, Bella B; Aubert, Tangi; Himmelreich, Uwe; Demeester, Jo; De Smedt, Stefaan C; Hens, Zeger; Braeckmans, Kevin

2014-06-16

The use of quantum dots (QDots) as bright and photostable probes for long-term fluorescence imaging is gaining more interest. Thus far, (pre)clinical use of QDots remains limited, which is primarily caused by the potential toxicity of QDots. Most QDots consist of Cd2+ ions, which are known to cause high levels of toxicity. In order to overcome this problem, several strategies have been tested, such as the generation of cadmium-free QDots. In the present study, two types of cadmium-free QDots, composed of ZnSe/ZnS (QDotZnSe) and InP/ZnS (QDotInP), were studied with respect to their cytotoxicity and cellular uptake in a variety of cell types. A multiparametric cytotoxicity approach is used, where the QDots are studied with respect to cell viability, oxidative stress, cell morphology, stem cell differentiation, and neurite outgrowth. The data reveal slight differences in uptake levels for both types of QDots (maximal for QDotZnSe), but clear differences in cytotoxicity and cell functionality effects exist, with highest toxicity for QDotZnSe. Differences between cell types and between both types of QDots can be explained by the intrinsic sensitivity of certain cell types and chemical composition of the QDots. At concentrations at which no toxic effects can be observed, the functionality of the QDots for fluorescence cell visualization is evaluated, revealing that the higher brightness of QDotZnSe overcomes most of the toxicity issues compared to that of QDotInP. Comparing the results obtained with common Cd2+-containing QDots tested under identical conditions, the importance of particle functionality is demonstrated, revealing that cadmium-free QDots tested in this study are not significantly better than Cd2+-containing QDots for long-term cell imaging and that more work needs to be performed in optimizing the brightness and surface chemistry of cadmium-free QDots for them to replace currently used Cd2+-containing QDots.
View-tolerant face recognition and Hebbian learning imply mirror-symmetric neural tuning to head orientation

PubMed Central

Leibo, Joel Z.; Liao, Qianli; Freiwald, Winrich A.; Anselmi, Fabio; Poggio, Tomaso

2017-01-01

SUMMARY The primate brain contains a hierarchy of visual areas, dubbed the ventral stream, which rapidly computes object representations that are both specific for object identity and robust against identity-preserving transformations like depth-rotations [1, 2]. Current computational models of object recognition, including recent deep learning networks, generate these properties through a hierarchy of alternating selectivity-increasing filtering and tolerance-increasing pooling operations, similar to simple-complex cells operations [3, 4, 5, 6]. Here we prove that a class of hierarchical architectures and a broad set of biologically plausible learning rules generate approximate invariance to identity-preserving transformations at the top level of the processing hierarchy. However, all past models tested failed to reproduce the most salient property of an intermediate representation of a three-level face-processing hierarchy in the brain: mirror-symmetric tuning to head orientation [7]. Here we demonstrate that one specific biologically-plausible Hebb-type learning rule generates mirror-symmetric tuning to bilaterally symmetric stimuli like faces at intermediate levels of the architecture and show why it does so. Thus the tuning properties of individual cells inside the visual stream appear to result from group properties of the stimuli they encode and to reflect the learning rules that sculpted the information-processing system within which they reside. PMID:27916522
Specific effects of surface carboxyl groups on anionic polystyrene particles in their interactions with mesenchymal stem cells

NASA Astrophysics Data System (ADS)

Jiang, Xiue; Musyanovych, Anna; Röcker, Carlheinz; Landfester, Katharina; Mailänder, Volker; Nienhaus, G. Ulrich

2011-05-01

Nanoparticle uptake by living cells is governed by chemical interactions between functional groups on the nanoparticle as well as the receptors on cell surfaces. Here we have investigated the uptake of anionic polystyrene (PS) nanoparticles of ~100 nm diameter by mesenchymal stem cells (MSCs) using spinning-disk confocal optical microscopy combined with a quantitative analysis of the fluorescence images. Two types of anionic PS nanoparticles with essentially identical sizes and ζ-potentials were employed in this study, carboxyl-functionalized nanoparticles (CPS) and plain PS nanoparticles, both coated with anionic detergent for stabilization. CPS nanoparticles were observed to internalize more rapidly and accumulate to a much higher level than plain PS nanoparticles. The relative importance of different uptake mechanisms for the two types of nanoparticles was investigated by using specific inhibitors. CPS nanoparticles were internalized mainly via the clathrin-mediated mechanism, whereas plain PS nanoparticles mainly utilized the macropinocytosis pathway. The pronounced difference in the internalization behavior of CPS and plain PS nanoparticles points to a specific interaction of the carboxyl group with receptors on the cell surface.
Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci

PubMed Central

Philippe, Claude; Vargas-Landin, Dulce B; Doucet, Aurélien J; van Essen, Dominic; Vera-Otarola, Jorge; Kuciak, Monika; Corbin, Antoine; Nigumann, Pilvi; Cristofari, Gaël

2016-01-01

LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants. DOI: http://dx.doi.org/10.7554/eLife.13926.001 PMID:27016617
A molecular scheme for improved characterization of human embryonic stem cell lines

PubMed Central

Josephson, Richard; Sykes, Gregory; Liu, Ying; Ording, Carol; Xu, Weining; Zeng, Xianmin; Shin, Soojung; Loring, Jeanne; Maitra, Anirban; Rao, Mahendra S; Auerbach, Jonathan M

2006-01-01

Background Human embryonic stem cells (hESC) offer a renewable source of a wide range of cell types for use in research and cell-based therapies to treat disease. Inspection of protein markers provides important information about the current state of the cells and data for subsequent manipulations. However, hESC must be routinely analyzed at the genomic level to guard against deleterious changes during extensive propagation, expansion, and manipulation in vitro. Results We found that short tandem repeat (STR) analysis, human leukocyte antigen (HLA) typing, single nucleotide polymorphism (SNP) genomic analysis, mitochondrial DNA sequencing, and gene expression analysis by microarray can be used to fully describe any hESC culture in terms of its identity, stability, and undifferentiated state. Conclusion Here we describe, using molecular biology alone, a comprehensive characterization of 17 different hESC lines. The use of amplified nucleic acids means that for the first time full characterization of hESC lines can be performed with little time investment and a minimum of material. The information thus gained will facilitate comparison of lines and replication of results between laboratories. PMID:16919167

Potassium recycling pathways in the human cochlea.

PubMed

Weber, P C; Cunningham, C D; Schulte, B A

2001-07-01

Potential pathways for recycling potassium (K+) used in the maintenance of inner ear electrochemical gradients have been elucidated in animal models. However, little is known about K+ transport in the human cochlea. This study was designed to characterize putative K+ recycling pathways in the human ear and to determine whether observations from animal models can be extrapolated to humans. A prospective laboratory study using an immunohistochemical approach to analyze the distribution of key ion transport mediators in the human cochlea. Human temporal bones were fixed in situ within 1 to 6 hours of death and subsequently harvested at autopsy. Decalcification was accomplished with the aid of microwaving. Immunohistochemical staining was then performed to define the presence and cell type-specific distribution of Na,K-ATPase, sodium-potassium-chloride cotransporter (NKCC), and carbonic anhydrase (CA) in the inner ear. Staining patterns visualized in the human cochlea closely paralleled those seen in other species. Anti-Na,K-ATPase stained strongly the basolateral plasma membrane of strial marginal cells and nerve endings underlying hair cells. This antibody also localized Na,K-ATPase to type II, type IV, and type V fibrocytes in the spiral ligament and in limbal fibrocytes. NKCC was present in the basolateral membrane of strial marginal cells as well as in type II, type V, and limbal fibrocytes. Immunoreactive carbonic anhydrase was present in type I and type III fibrocytes and in epithelial cells lining Reissner's membrane and the spiral prominence. The distribution of several major ion transport proteins in the human cochlea is similar but not identical to that described in various rodent models. These results support the presence of a complex system for recycling and regulating K+ homeostasis in the human cochlea, similar to that described in other mammalian species.
Vascular endothelial cells express isoforms of protein kinase A inhibitor.

PubMed

Lum, Hazel; Hao, Zengping; Gayle, Dave; Kumar, Priyadarsini; Patterson, Carolyn E; Uhler, Michael D

2002-01-01

The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs). RT-PCR using specific primers for human PKI alpha, human PKI gamma, and mouse PKI beta sequences detected PKI alpha and PKI gamma mRNA in all three cell types. Sequencing and BLAST analysis indicated that forward and reverse DNA strands for PKI alpha and PKI gamma were of >96% identity with database sequences. RNase protection assays showed protection of the 542 nucleotides in HBMEC and HPAEC PKI alpha mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKI gamma mRNA. Western blot analysis indicated that PKI gamma protein was detected in all three cell types, whereas PKI alpha was found in HBMECs. In summary, endothelial cells from three different vascular beds express PKI alpha and PKI gamma, which may be physiologically important in endothelial barrier function.
Sex-typed personality traits and gender identity as predictors of young adults' career interests.

PubMed

Dinella, Lisa M; Fulcher, Megan; Weisgram, Erica S

2014-04-01

Gender segregation of careers is still prominent in the U.S. workforce. The current study was designed to investigate the role of sex-typed personality traits and gender identity in predicting emerging adults' interests in sex-typed careers. Participants included 586 university students (185 males, 401 females). Participants reported their sex-typed personality traits (masculine and feminine traits), gender identities (gender typicality, contentment, felt pressure to conform, and intergroup bias), and interests in sex-typed careers. Results indicated both sex-typed personality traits and gender identity were important predictors of young adults' career interests, but in varying degrees and differentially for men and women. Men's sex-typed personality traits and gender typicality were predictive of their masculine career interests even more so when the interaction of their masculine traits and gender typicality were considered. When gender typicality and sex-typed personality traits were considered simultaneously, gender typicality was negatively related to men's feminine career interests and gender typicality was the only significant predictor of men's feminine career interests. For women, sex-typed personality traits and gender typicality were predictive of their sex-typed career interests. The level of pressure they felt to conform to their gender also positively predicted interest in feminine careers. The interaction of sex-typed personality traits and gender typicality did not predict women's career interests more than when these variables were considered as main effects. Results of the multidimensional assessment of gender identity confirmed that various dimensions of gender identity played different roles in predicting career interests and gender typicality was the strongest predictor of career interests.
Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue

PubMed Central

Yee, Karen K.; Li, Yan; Redding, Kevin M.; Iwatsuki, Ken; Margolskee, Robert F.; Jiang, Peihua

2013-01-01

Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine-rich repeat-containing G-protein coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5-expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5-expressing cells in posterior tongue are a subset of K14-positive epithelial cells. Lineage-tracing experiments using an inducible Cre knock-in allele in combination with Rosa26-LacZ and Rosa26-tdTomato reporter strains showed that Lgr5-expressing cells gave rise to taste cells, perigemmal cells, along with self-renewing cells at the bottom of trench areas at the base of circumvallate and foliate papillae. Moreover, using subtype-specific taste markers, we found that Lgr5-expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue. PMID:23377989
Lgr5-EGFP marks taste bud stem/progenitor cells in posterior tongue.

PubMed

Yee, Karen K; Li, Yan; Redding, Kevin M; Iwatsuki, Ken; Margolskee, Robert F; Jiang, Peihua

2013-05-01

Until recently, reliable markers for adult stem cells have been lacking for many regenerative mammalian tissues. Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5) has been identified as a marker for adult stem cells in intestine, stomach, and hair follicle; Lgr5-expressing cells give rise to all types of cells in these tissues. Taste epithelium also regenerates constantly, yet the identity of adult taste stem cells remains elusive. In this study, we found that Lgr5 is strongly expressed in cells at the bottom of trench areas at the base of circumvallate (CV) and foliate taste papillae and weakly expressed in the basal area of taste buds and that Lgr5-expressing cells in posterior tongue are a subset of K14-positive epithelial cells. Lineage-tracing experiments using an inducible Cre knockin allele in combination with Rosa26-LacZ and Rosa26-tdTomato reporter strains showed that Lgr5-expressing cells gave rise to taste cells, perigemmal cells, along with self-renewing cells at the bottom of trench areas at the base of CV and foliate papillae. Moreover, using subtype-specific taste markers, we found that Lgr5-expressing cell progeny include all three major types of adult taste cells. Our results indicate that Lgr5 may mark adult taste stem or progenitor cells in the posterior portion of the tongue. Copyright © 2013 AlphaMed Press.
G protein γ (Gγ) subtype dependent targeting of GRK2 to M3 receptor by Gβγ.

PubMed

Samaradivakara, Saroopa; Kankanamge, Dinesh; Senarath, Kanishka; Ratnayake, Kasun; Karunarathne, Ajith

2018-06-11

Interactions of cytosolic G protein coupled receptor kinase 2 (GRK2) with activated G protein coupled receptors (GPCRs) induce receptor phosphorylation and desensitization. GRK2 is recruited to active M3-muscarinic receptors (M3R) with the participation of the receptor, Gαq and Gβγ. Since we have shown that signaling efficacy of Gβγ is governed by its Gγ subtype identity, the present study examined whether recruitment of GRK2 to M3R is also Gγ subtype dependent. To capture the dynamics of GRK2-recruitment concurrently with GPCR-G protein activation, we employed live cell confocal imaging and a novel assay based on Gβγ translocation. Data show that M3R activation-induced GRK2 recruitment is Gγ subtype dependent in which Gβγ dimers with low PM-affinity Gγ9 exhibited a two-fold higher GRK2-recruitment compared to high PM affinity Gγ3 expressing cells. Since 12-mammalian Gγ types exhibit a cell and tissue specific expressions and the PM-affinity of a Gγ is linked to its subtype identity, our results indicate a mechanism by which Gγ profile of a cell controls GRK2 signaling and GPCR desensitization. Copyright © 2018 Elsevier Inc. All rights reserved.
Emerging pulmonary vasculature lacks fate specification.

PubMed

Schwarz, Margaret A; Caldwell, Lauren; Cafasso, Danielle; Zheng, Haihua

2009-01-01

Lung morphogenesis requires precise coordination between branching morphogenesis and vascularization to generate distal airways capable of supporting respiration at the cell-cell interface. The specific origins and types of blood vessels that initially form in the lung, however, remain obscure. Herein, we definitively show that during the early phases of lung development [i.e., embryonic day (E) 11.5], functional vessels, replete with blood flow, are restricted to the mesenchyme, distal to the epithelium. However, by day E14.5, and in response to epithelial-derived VEGF signals, functional vessels extend from the mesenchyme to the epithelial interface. Moreover, these vessels reside adjacent to multipotent mesenchymal stromal cells that likely play a regulatory role in this process. As well as and distinct from the systemic vasculature, immunostaining for EphrinB2 and EphB4 revealed that arterial and venous identity is not distinguishable in emergent pulmonary vasculature. Collectively, this study provides evidence that lung vascularization initially originates in the mesenchyme, distal to the epithelium, and that arterial-venous specification does not exist in the early lung. At a mechanistic level, we show that basilar epithelial VEGF prompts endothelial cells to move toward the epithelium where they undergo morphogenesis during the proliferative, canalicular stage. Thus our findings challenge existing notions of vascular origin and identity during development.
Arachidonic Acid-Induced Expression of the Organic Solute and Steroid Transporter-beta (Ost-beta) in a Cartilaginous Fish Cell Line

PubMed Central

Hwang, Jae-Ho; Parton, Angela; Czechanski, Anne; Ballatori, Nazzareno; Barnes, David

2008-01-01

The organic solute and steroid transporter (OST/Ost) is a unique membrane transport protein heterodimer composed of subunits designated alpha and beta, that transports conjugated steroids and prostaglandin E2 across the plasma membrane. Ost was first identified in the liver of the cartilaginous fish Leucoraja erinacea, the little skate, and subsequently was found in many other species, including humans and rodents. The present study describes the isolation of a new cell line, LEE-1, derived from an early embryo of L. erinacea, and characterizes the expression of Ost in these cells. The mRNA size and amino acid sequence of Ost-beta in LEE-1 was identical to that previously reported for Ost-beta from skate liver, and the primary structure was identical to that of the spiny dogfish shark (Squalus acanthias) with the exception of a single amino acid. Ost-beta was found both on the plasma membrane and intracellularly in LEE-1 cells, consistent with its localization in other cell types. Interestingly, arachidonic acid, the precursor to eiconsanoids, strongly induced Ost-beta expression in LEE-1 cells and a lipid mixture containing arachidonic acid also induced Ost-alpha. Overall, the present study describes the isolation of a novel marine cell line, and shows that this cell line expresses relatively high levels of Ost when cultured in the presence of arachidonic acid. Although the function of this transport protein in embryo-derived cells is unknown, it may play a role in the disposition of eicosanoids or steroid-derived molecules. PMID:18407792
Integrative Analysis of Transcription Factor Combinatorial Interactions Using a Bayesian Tensor Factorization Approach

PubMed Central

Ye, Yusen; Gao, Lin; Zhang, Shihua

2017-01-01

Transcription factors play a key role in transcriptional regulation of genes and determination of cellular identity through combinatorial interactions. However, current studies about combinatorial regulation is deficient due to lack of experimental data in the same cellular environment and extensive existence of data noise. Here, we adopt a Bayesian CANDECOMP/PARAFAC (CP) factorization approach (BCPF) to integrate multiple datasets in a network paradigm for determining precise TF interaction landscapes. In our first application, we apply BCPF to integrate three networks built based on diverse datasets of multiple cell lines from ENCODE respectively to predict a global and precise TF interaction network. This network gives 38 novel TF interactions with distinct biological functions. In our second application, we apply BCPF to seven types of cell type TF regulatory networks and predict seven cell lineage TF interaction networks, respectively. By further exploring the dynamics and modularity of them, we find cell lineage-specific hub TFs participate in cell type or lineage-specific regulation by interacting with non-specific TFs. Furthermore, we illustrate the biological function of hub TFs by taking those of cancer lineage and blood lineage as examples. Taken together, our integrative analysis can reveal more precise and extensive description about human TF combinatorial interactions. PMID:29033978
Integrative Analysis of Transcription Factor Combinatorial Interactions Using a Bayesian Tensor Factorization Approach.

PubMed

Ye, Yusen; Gao, Lin; Zhang, Shihua

2017-01-01

Transcription factors play a key role in transcriptional regulation of genes and determination of cellular identity through combinatorial interactions. However, current studies about combinatorial regulation is deficient due to lack of experimental data in the same cellular environment and extensive existence of data noise. Here, we adopt a Bayesian CANDECOMP/PARAFAC (CP) factorization approach (BCPF) to integrate multiple datasets in a network paradigm for determining precise TF interaction landscapes. In our first application, we apply BCPF to integrate three networks built based on diverse datasets of multiple cell lines from ENCODE respectively to predict a global and precise TF interaction network. This network gives 38 novel TF interactions with distinct biological functions. In our second application, we apply BCPF to seven types of cell type TF regulatory networks and predict seven cell lineage TF interaction networks, respectively. By further exploring the dynamics and modularity of them, we find cell lineage-specific hub TFs participate in cell type or lineage-specific regulation by interacting with non-specific TFs. Furthermore, we illustrate the biological function of hub TFs by taking those of cancer lineage and blood lineage as examples. Taken together, our integrative analysis can reveal more precise and extensive description about human TF combinatorial interactions.
Apheresis product identification in the transplant center: development of point-of-care protocols for extended blood typing of stem cell apheresis products.

PubMed

Cummerow, C; Schwind, P; Spicher, M; Spohn, G; Geisen, C; Seifried, E; Bönig, H

2012-06-01

Transfusion of the 'wrong' stem cell product would almost inevitably be lethal, yet assays to confirm the contents of the product bag, except by checking labels and paperwork, are lacking. To increase the likelihood that a product mix-up would be detected in the transplant center, we developed a simple protocol for extended blood typing and hence, for confirmation of donor/product identity, on a tube segment. Apheresis samples were applied, directly or after erythrocyte enrichment, to commercially available blood typing assays, including lateral flow cards and gel agglutination cards. Without sample modification, low hematocrit and high leukocyte count obviated definitive blood typing. Using the most simple erythrocyte enrichment protocol, that is, centrifugation, reliable blood group analysis became possible with either assay. Other, more cumbersome pre-analytical protocols were also successful but provided no advantage. The preferred method was validated on 100 samples; ABD was correctly identified in 100% of cases. Of the other Rh Ags, all except two 'small e', in both cases in heterozygous individuals, were detected; there were no false positives. A simple, inexpensive point-of-care assay for extended blood typing of apheresis products is available, which can reduce the fatal risk of administering the wrong stem cell product.
Two Genera of Magnetococci with Bean-like Morphology from Intertidal Sediments of the Yellow Sea, China

PubMed Central

Zhang, Wen-Yan; Zhou, Ke; Pan, Hong-Miao; Yue, Hai-Dong; Jiang, Ming

2012-01-01

Magnetotactic bacteria have the unique capacity of being able to swim along geomagnetic field lines. They are Gram-negative bacteria with diverse morphologies and variable phylogenetic relatedness. Here, we describe a group of uncultivated marine magnetococci collected from intertidal sediments of Huiquan Bay in the Yellow Sea. They were coccoid-ovoid in morphology, with an average size of 2.8 ± 0.3 μm by 2.0 ± 0.2 μm. Differential interference contrast microscopy, fluorescence microscopy, and transmission electron microscopy revealed that each cell was apparently composed of two hemispheres. The cells synthesized iron oxide-type magnetosomes that clustered on one side of the cell at the interface between the two hemispheres. In some cells two chains of magnetosomes were observed across the interface. Each cell had two bundles of flagella enveloped in a sheath and displayed north-seeking helical motion. Two 16S rRNA gene sequences having 91.8% identity were obtained, and their authenticity was confirmed by fluorescence in situ hybridization. Phylogenetic analysis revealed that the magnetococci are affiliated with the Alphaproteobacteria and are most closely related to two uncultured magnetococci with sequence identities of 92.7% and 92.4%, respectively. Because they display a >7% sequence divergence to all bacteria reported, the bean-like magnetococci may represent two novel genera. PMID:22660708
Purification and biochemical heterogeneity of the mammalian SWI-SNF complex.

PubMed Central

Wang, W; Côté, J; Xue, Y; Zhou, S; Khavari, P A; Biggar, S R; Muchardt, C; Kalpana, G V; Goff, S P; Yaniv, M; Workman, J L; Crabtree, G R

1996-01-01

We have purified distinct complexes of nine to 12 proteins [referred to as BRG1-associated factors (BAFs)] from several mammalian cell lines using an antibody to the SWI2-SNF2 homolog BRG1. Microsequencing revealed that the 47 kDa BAF is identical to INI1. Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene. A group of BAF47-associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co-purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI-SNF complex. Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4-VP16 to a nucleosomal template similar to the yeast SWI-SNF complex. Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition. The two SWI-SNF2 homologs, BRG1 and hbrm, were found in separate complexes. Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI-SNF complex may be tailored to the needs of a differentiated cell type. Images PMID:8895581
A Combination of Ontogeny and CNS Environment Establishes Microglial Identity.

PubMed

Bennett, F Chris; Bennett, Mariko L; Yaqoob, Fazeela; Mulinyawe, Sara B; Grant, Gerald A; Hayden Gephart, Melanie; Plowey, Edward D; Barres, Ben A

2018-05-22

Microglia, the brain's resident macrophages, are dynamic CNS custodians with surprising origins in the extra-embryonic yolk sac. The consequences of their distinct ontogeny are unknown but critical to understanding and treating brain diseases. We created a brain macrophage transplantation system to disentangle how environment and ontogeny specify microglial identity. We find that donor cells extensively engraft in the CNS of microglia-deficient mice, and even after exposure to a cell culture environment, microglia fully regain their identity when returned to the CNS. Though transplanted macrophages from multiple tissues can express microglial genes in the brain, only those of yolk-sac origin fully attain microglial identity. Transplanted macrophages of inappropriate origin, including primary human cells in a humanized host, express disease-associated genes and specific ontogeny markers. Through brain macrophage transplantation, we discover new principles of microglial identity that have broad applications to the study of disease and development of myeloid cell therapies. Copyright © 2018 Elsevier Inc. All rights reserved.
Political, religious and occupational identities in context: placing identity status paradigm in context.

PubMed

Solomontos-Kountouri, Olga; Hurry, Jane

2008-04-01

This study critically contrasts global identity with domain-specific identities (political, religious and occupational) and considers context and gender as integral parts of identity. In a cross-sectional survey, 1038 Greek Cypriot adolescents (449 boys and 589 girls, mean age 16.8) from the three different types of secondary schools (state, state technical and private) and from different SES completed part of the Extended Objective Measure of Ego Identity Status-2 (EOMEIS-2). The macro-context of Greek Cypriot society is used to understand the role of context in adolescents' identities. Results showed that Greek Cypriot young people were not in the same statuses across their global, political, religious and occupational identities. This heterogeneity in the status of global identity and of each identity domain is partially explained by differences in gender, type of school and SES (socio-economic status). The fact that identity status is found to be reactive to context suggests that developmental stage models of identity status should place greater emphasis on context.
Mammary Cancer and Activation of Transposable Elements

DTIC Science & Technology

2015-03-01

EGFP). No other cell type in the mammary fat pad was observed to express EGFP. Wholemount and FACS analyses of mammary fat pads after involution from...were sacrificed for PI-MEC isolation in groups of up to 4 control or cancer-prone uniparous or triparous females. Both 4 th mammary fat pads were...to unknown reason (n=46), and smaller numbers of animals with various conditions (malocclusion, head tilt , dystocia, respiratory complaints, identity
Influence of Adolescent Social Cliques on Vocational Identity.

ERIC Educational Resources Information Center

Johnson, John A.; Cheek, Jonathan M.

While Holland's (1973) theory of personality types and vocational identity is widely used, the theory does not specify the developmental antecedents of the six personality types. To examine the relationship between membership in adolescent social cliques and vocational identity in early adulthood, four groups of college students (N=192)…
The SU(r)2 string functions as q-diagrams

NASA Astrophysics Data System (ADS)

Genish, Arel; Gepner, Doron

2016-06-01

A generalized Roger Ramanujan (GRR) type expression for the characters of A-type parafermions has been a long standing puzzle dating back to conjectures made regarding some of the characters in the 90s. Not long ago we have put forward such GRR type identities describing any of the level two ADE-type generalized parafermions characters at any rank. These characters are the string functions of simply laced Lie algebras at level two, as such, they are also of mathematical interest. In our last joint paper we presented the complete derivation for the D-type generalized parafermions characters identities. Here we generalize our previous discussion and prove the GRR type expressions for the characters of A-type generalized parafermions. To prove the A-type GRR conjecture we study further the q-diagrams, introduced in our last joint paper, and examine the diagrammatic interpretations of known identities among them Slater identities for the characters of the first minimal model, which is the Ising model, and the Bailey lemma.
Plastids and gravitropic sensing

NASA Technical Reports Server (NTRS)

Sack, F. D.

1997-01-01

Data and theories about the identity of the mass that acts in gravitropic sensing are reviewed. Gravity sensing may have evolved several times in plants and algae in processes such as gravitropism of organs and tip-growing cells, gravimorphism, gravitaxis, and the regulation of cytoplasmic streaming in internodal cells of Chara. In the latter and in gravitaxis, the mass of the entire cell may function in sensing. But gravitropic sensing appears to rely upon the mass of amyloplasts that sediment since (i) the location of cells with sedimentation is highly regulated, (ii) such cells contain other morphological specializations favoring sedimentation, (iii) sedimentation always correlates with gravitropic competence in wild-type plants, (iv) magnetophoretic movement of rootcap amyloplasts mimics gravitropism, and (v) starchless and intermediate starch mutants show reduced gravitropic sensitivity. The simplest interpretation of these data is that gravitropic sensing is plastid-based.
The tailless Ortholog nhr-67 Regulates Patterning of Gene Expression and Morphogenesis in the C. elegans Vulva

PubMed Central

Fernandes, Jolene S; Sternberg, Paul W

2007-01-01

Regulation of spatio-temporal gene expression in diverse cell and tissue types is a critical aspect of development. Progression through Caenorhabditis elegans vulval development leads to the generation of seven distinct vulval cell types (vulA, vulB1, vulB2, vulC, vulD, vulE, and vulF), each with its own unique gene expression profile. The mechanisms that establish the precise spatial patterning of these mature cell types are largely unknown. Dissection of the gene regulatory networks involved in vulval patterning and differentiation would help us understand how cells generate a spatially defined pattern of cell fates during organogenesis. We disrupted the activity of 508 transcription factors via RNAi and assayed the expression of ceh-2, a marker for vulB fate during the L4 stage. From this screen, we identified the tailless ortholog nhr-67 as a novel regulator of gene expression in multiple vulval cell types. We find that one way in which nhr-67 maintains cell identity is by restricting inappropriate cell fusion events in specific vulval cells, namely vulE and vulF. nhr-67 exhibits a dynamic expression pattern in the vulval cells and interacts with three other transcriptional regulators cog-1 (Nkx6.1/6.2), lin-11 (LIM), and egl-38 (Pax2/5/8) to generate the composite expression patterns of their downstream targets. We provide evidence that egl-38 regulates gene expression in vulB1, vulC, vulD, vulE, as well as vulF cells. We demonstrate that the pairwise interactions between these regulatory genes are complex and vary among the seven cell types. We also discovered a striking regulatory circuit that affects a subset of the vulval lineages: cog-1 and nhr-67 inhibit both one another and themselves. We postulate that the differential levels and combinatorial patterns of lin-11, cog-1, and nhr-67 expression are a part of a regulatory code for the mature vulval cell types. PMID:17465684

SIRT6 knockout cells resist apoptosis initiation but not progression: a computational method to evaluate the progression of apoptosis.

PubMed

Domanskyi, Sergii; Nicholatos, Justin W; Schilling, Joshua E; Privman, Vladimir; Libert, Sergiy

2017-11-01

Apoptosis is essential for numerous processes, such as development, resistance to infections, and suppression of tumorigenesis. Here, we investigate the influence of the nutrient sensing and longevity-assuring enzyme SIRT6 on the dynamics of apoptosis triggered by serum starvation. Specifically, we characterize the progression of apoptosis in wild type and SIRT6 deficient mouse embryonic fibroblasts using time-lapse flow cytometry and computational modelling based on rate-equations and cell distribution analysis. We find that SIRT6 deficient cells resist apoptosis by delaying its initiation. Interestingly, once apoptosis is initiated, the rate of its progression is higher in SIRT6 null cells compared to identically cultured wild type cells. However, SIRT6 null cells succumb to apoptosis more slowly, not only in response to nutrient deprivation but also in response to other stresses. Our data suggest that SIRT6 plays a role in several distinct steps of apoptosis. Overall, we demonstrate the utility of our computational model to describe stages of apoptosis progression and the integrity of the cellular membrane. Such measurements will be useful in a broad range of biological applications.
Broad H3K4me3 is associated with increased transcription elongation and enhancer activity at tumor suppressor genes

PubMed Central

Chen, Kaifu; Chen, Zhong; Wu, Dayong; Zhang, Lili; Lin, Xueqiu; Su, Jianzhong; Rodriguez, Benjamin; Xi, Yuanxin; Xia, Zheng; Chen, Xi; Shi, Xiaobing; Wang, Qianben; Li, Wei

2016-01-01

Tumor suppressors are mostly defined by inactivating mutations in tumors, yet little is known about their epigenetic features in normal cells. Through integrative analysis of 1,134 genome-wide epigenetic profiles, mutations from >8,200 tumor-normal pairs, and our experimental data from clinical samples, we discovered broad H3K4me3 (wider than 4 kb) as the first epigenetic signature for tumor suppressors in normal cells. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity together leading to exceptionally high gene expression, and is distinct from other broad epigenetic features, such as super-enhancers. Broad H3K4me3 conserved across normal cells may represent pan-cancer tumor suppressors, such as P53 and PTEN, whereas cell-type-specific broad H3K4me3 may indicate cell-identity genes and cell-type-specific tumor suppressors. Furthermore, widespread shortening of broad H3K4me3 in cancers is associated with repression of tumor suppressors. Together, the broad H3K4me3 epigenetic signature provides mutation-independent information for the discovery and characterization of novel tumor suppressors. PMID:26301496
Heat Transport Enhancement of Turbulent Thermal Convection by Inserted Channels

NASA Astrophysics Data System (ADS)

Xia, Ke-Qing; Zhang, Lu

2017-11-01

We report an experimental study on the heat transport properties of turbulent Rayleigh Benard Convection (RBC) in a rectangular cell with two types of 3D-printed structures inserted inside. The first one splits the original rectangular cell into 60 identical sub cells whose aspect ratio is 1:1:10 (length, width, height). The second one splits the cell into 30 sub cells, each with a 1:2:10 aspect ratio and a baffle in the center. We find that for large Rayleigh numbers (Ra), the Nusselt numbers (Nu) of both structures increase compared with that of the empty rectangular cell. An enhancement in Nu as much as 20% is found for the second type of insertion at Rayleigh number 2 ×109 . Moreover, the Nu-Ra scaling shows a transition with both geometries. The particle image velocimetry (PIV) measurement within a single sub unit indicates that the transition may be related to the laminar to turbulent transition in flow field. Direct numerical simulations (DNS) confirm the experimental results. Our results demonstrate the potential in using insertions to enhance passive heat transfer. This work was supported by the Research Grants Council (RGC) of HKSAR (Nos. CUHK404513 and CUHK14301115).
Emergence and patterning of the five cell types of the Zea mays anther locule

PubMed Central

Kelliher, Timothy; Walbot, Virginia

2011-01-01

One fundamental difference between plants and animals is the existence of a germ-line in animals and its absence in plants. In flowering plants the sexual organs (stamens and carpels) are composed almost entirely of somatic cells, a small subset of which switch to meiosis, however, the mechanism of meiotic cell fate acquisition is a long-standing botanical mystery. In the maize (Zea mays) anther microsporangium the somatic tissues consist of four concentric cell layers which surround and support reproductive cells as they progress through meiosis and pollen maturation. Male sterility, defined as the absence of viable pollen, is a common phenotype in flowering plants, and many male sterile mutants have defects in somatic and reproductive cell fate acquisition. However, without a robust model of anther cell fate acquisition based on careful observation of wild type anther ontogeny, interpretation of cell fate mutants is limited. To address this, the pattern of cell proliferation, expansion, and differentiation was tracked in three dimensions over thirty days of wild type (W23) anther development, using anthers stained with propidium iodide (PI) and/or 5-ethynyl-2′-deoxyuridine (EdU) (S-phase label) and imaged by confocal microscopy. The pervading lineage model of anther development claims that new cell layers are generated by coordinated, oriented cell divisions in transient precursor cell types. In reconstructing anther cell division patterns, however, we can only confirm this for the origin of the middle layer (ml) and tapetum, while young anther development appears more complex. We find that each anther cell type undergoes a burst of cell division after specification with a characteristic pattern of both cell expansion and division. Comparisons between two inbreds lines and between ab- and adaxial anther florets indicated near identity: anther development is highly canalized and synchronized. Three classical models of plant organ development are tested and ruled out; however, local clustering of developmental events was identified for several processes, including the first evidence for a direct relationship between the development of ml and tapetal cells. We speculate that small groups of ml and tapetum cells function as a developmental unit dedicated to the development of a single pollen grain. PMID:21070762
Design and interpretation of cell trajectory assays

PubMed Central

Bowden, Lucie G.; Simpson, Matthew J.; Baker, Ruth E.

2013-01-01

Cell trajectory data are often reported in the experimental cell biology literature to distinguish between different types of cell migration. Unfortunately, there is no accepted protocol for designing or interpreting such experiments and this makes it difficult to quantitatively compare different published datasets and to understand how changes in experimental design influence our ability to interpret different experiments. Here, we use an individual-based mathematical model to simulate the key features of a cell trajectory experiment. This shows that our ability to correctly interpret trajectory data is extremely sensitive to the geometry and timing of the experiment, the degree of motility bias and the number of experimental replicates. We show that cell trajectory experiments produce data that are most reliable when the experiment is performed in a quasi-one-dimensional geometry with a large number of identically prepared experiments conducted over a relatively short time-interval rather than a few trajectories recorded over particularly long time-intervals. PMID:23985736
ODE Constrained Mixture Modelling: A Method for Unraveling Subpopulation Structures and Dynamics

PubMed Central

Hasenauer, Jan; Hasenauer, Christine; Hucho, Tim; Theis, Fabian J.

2014-01-01

Functional cell-to-cell variability is ubiquitous in multicellular organisms as well as bacterial populations. Even genetically identical cells of the same cell type can respond differently to identical stimuli. Methods have been developed to analyse heterogeneous populations, e.g., mixture models and stochastic population models. The available methods are, however, either incapable of simultaneously analysing different experimental conditions or are computationally demanding and difficult to apply. Furthermore, they do not account for biological information available in the literature. To overcome disadvantages of existing methods, we combine mixture models and ordinary differential equation (ODE) models. The ODE models provide a mechanistic description of the underlying processes while mixture models provide an easy way to capture variability. In a simulation study, we show that the class of ODE constrained mixture models can unravel the subpopulation structure and determine the sources of cell-to-cell variability. In addition, the method provides reliable estimates for kinetic rates and subpopulation characteristics. We use ODE constrained mixture modelling to study NGF-induced Erk1/2 phosphorylation in primary sensory neurones, a process relevant in inflammatory and neuropathic pain. We propose a mechanistic pathway model for this process and reconstructed static and dynamical subpopulation characteristics across experimental conditions. We validate the model predictions experimentally, which verifies the capabilities of ODE constrained mixture models. These results illustrate that ODE constrained mixture models can reveal novel mechanistic insights and possess a high sensitivity. PMID:24992156
Cellular origin of bladder neoplasia and tissue dynamics of its progression to invasive carcinoma

PubMed Central

Shin, Kunyoo; Lim, Agnes; Odegaard, Justin I.; Honeycutt, Jared D.; Kawano, Sally; Hsieh, Michael H.; Beachy, Philip A.

2014-01-01

Understanding how malignancies arise within normal tissues requires identification of the cancer cell of origin and knowledge of the cellular and tissue dynamics of tumor progression. Here we examine bladder cancer in a chemical carcinogenesis model that mimics muscle-invasive human bladder cancer. With no prior bias regarding genetic pathways or cell types, we prospectively mark or ablate cells to show that muscle-invasive bladder carcinomas arise exclusively from Sonic hedgehog (Shh)-expressing stem cells in basal urothelium. These carcinomas arise clonally from a single cell whose progeny aggressively colonize a major portion of the urothelium to generate a lesion with histological features identical to human carcinoma-in-situ. Shh-expressing basal cells within this precursor lesion become tumor-initiating cells, although Shh expression is lost in subsequent carcinomas. We thus find that invasive carcinoma is initiated from basal urothelial stem cells but that tumor cell phenotype can diverge significantly from that of the cancer cell-of-origin. PMID:24747439
Infection of Polarized MDCK Cells with Herpes Simplex Virus 1: Two Asymmetrically Distributed Cell Receptors Interact with Different Viral Proteins

NASA Astrophysics Data System (ADS)

Sears, Amy E.; McGwire, Bradford S.; Roizman, Bernard

1991-06-01

Herpes simplex virus 1 attaches to at least two cell surface receptors. In polarized epithelial (Madin-Darby canine kidney; MDCK) cells one receptor is located in the apical surface and attachment to the cells requires the presence of glycoprotein C in the virus. The second receptor is located in the basal surface and does not require the presence of glycoprotein C. Exposure of MDCK cells at either the apical or basal surface to wild-type virus yields plaques and viral products whereas infection by a glycoprotein C-negative mutant yields identical results only after exposure of MDCK cells to virus at the basal surface. Multiple receptors for viral entry into cells expand the host range of the virus. The observation that glycoprotein C-negative mutants are infectious in many nonpolarized cell lines suggests that cells in culture may express more than one receptor and explains why genes that specify the viral proteins that recognize redundant receptors, like glycoprotein C, are expendable.
Social Media Social Comparison of Ability (but not Opinion) Predicts Lower Identity Clarity: Identity Processing Style as a Mediator.

PubMed

Yang, Chia-Chen; Holden, Sean M; Carter, Mollie D K

2018-01-11

Social comparison on social media has received increasing attention, but most research has focused on one type of social comparison and its psycho-emotional implications. Little is known about how different types of social comparison influence youth's identity development. Drawing on the theories of identity processing styles and social comparison, we examined how two different forms of social comparison on social media related to three identity processing styles, which in turn predicted youth's global self-esteem and identity clarity. We surveyed 219 college freshmen (M age = 18.29; 74% female) once in the Fall and once in the Spring. Social comparison of ability on social media was related to concurrent diffuse-avoidant identity processing style, which predicted lower identity clarity months later. In contrast, social comparison of opinion on social media did not influence college freshmen's global self-esteem and identity clarity through identity processing styles. The findings clarified the implications of online social comparison for youth's identity development.
The C. elegans Tailless/TLX transcription factor nhr-67 controls neuronal identity and left/right asymmetric fate diversification.

PubMed

Sarin, Sumeet; Antonio, Celia; Tursun, Baris; Hobert, Oliver

2009-09-01

An understanding of the molecular mechanisms of cell fate determination in the nervous system requires the elucidation of transcriptional regulatory programs that ultimately control neuron-type-specific gene expression profiles. We show here that the C. elegans Tailless/TLX-type, orphan nuclear receptor NHR-67 acts at several distinct steps to determine the identity and subsequent left/right (L/R) asymmetric subtype diversification of a class of gustatory neurons, the ASE neurons. nhr-67 controls several broad aspects of sensory neuron development and, in addition, triggers the expression of a sensory neuron-type-specific selector gene, che-1, which encodes a zinc-finger transcription factor. Subsequent to its induction of overall ASE fate, nhr-67 diversifies the fate of the two ASE neurons ASEL and ASER across the L/R axis by promoting ASER and inhibiting ASEL fate. This function is achieved through direct expression activation by nhr-67 of the Nkx6-type homeobox gene cog-1, an inducer of ASER fate, that is inhibited in ASEL through the miRNA lsy-6. Besides controlling bilateral and asymmetric aspects of ASE development, nhr-67 is also required for many other neurons of diverse lineage history and function to appropriately differentiate, illustrating the broad and diverse use of this type of transcription factor in neuronal development.
The role of the SCRAMBLED receptor-like kinase in patterning the Arabidopsis root epidermis.

PubMed

Kwak, Su-Hwan; Schiefelbein, John

2007-02-01

Cell-type patterning in the Arabidopsis root epidermis is achieved by a network of transcription factors and influenced by a position-dependent mechanism. The SCRAMBLED receptor-like kinase is required for the normal pattern to arise, but its precise role is not understood. Here we describe genetic and molecular studies to define the spatial and temporal role of SCM in epidermal patterning and its relationship to the transcriptional network. Our results suggest that SCM helps unspecified epidermal cells interpret their position in relation to the underlying cortical cells and establish distinct cell identities. Furthermore, SCM loss-of-function and overexpression analyses suggest that SCM influences cell fate through its negative transcriptional regulation of the WEREWOLF MYB gene in epidermal cells at the H position. We also find that SCM function is specifically required for patterning the post-embryonic root epidermis and not for the analogous epidermal cell-type patterning during embryogenesis or hypocotyl development. In addition, we show that two closely related SCM-like genes in Arabidopsis (SRF1 and SRF3) are not required alone or together with SCM for proper epidermal patterning. These findings help define the developmental and mechanistic role of SCM and suggest a new model for its action in root epidermal cell patterning.
Accelerated and real-time geosynchronous life cycling test performance of nickel-hydrogen batteries

NASA Technical Reports Server (NTRS)

Green, R. S.

1985-01-01

RCA Astro-Electronics currently has four nickel-hydrogen storage battery modules (11 cells each) on test in simulated geosynchronous life cycle regimes. These battery modules are of identical design to those used on the GSTAR (GTE Satellite Corp.) and Spacenet (GTE Spacenet Corp.) communications satellites. The batteries are being tested using an automated test station equipped with computer-controlled environmental chambers and recording equipment. The two battery types, 30 ampere-hours and 40 ampere-hours (GSTAR and Spacenet, respectively), are being electrically cycled using identical 44-day eclipse sequences at 5 C and vary with respect to depth of discharge, recharge ratio, duration of accumulated suntime, and the use of a reconditioning sequence. The test parameters are outlined and the preliminary test data and results are presented.
Forging T-Lymphocyte Identity: Intersecting Networks of Transcriptional Control

PubMed Central

Rothenberg, Ellen V.; Ungerbäck, Jonas; Champhekar, Ameya

2016-01-01

T lymphocyte development branches off from other lymphoid developmental programs through its requirement for sustained environmental signals through the Notch pathway. In the thymus, Notch signaling induces a succession of T-lineage regulatory factors that collectively create the T-cell identity through distinct steps. This process involves both the staged activation of T-cell identity genes and the staged repression of progenitor-cell-inherited regulatory genes once their roles in self-renewal and population expansion are no longer needed. With the recent characterization of Innate Lymphoid Cells (ILCs) that share transcriptional regulation programs extensively with T cell subsets, T-cell identity can increasingly be seen as defined in modular terms, as the processes selecting and actuating effector function are potentially detachable from the processes generating and selecting clonally unique T-cell receptor structures. The developmental pathways of different classes of T cells and ILCs are distinguished by the numbers of prerequisites of gene rearrangement, selection, and antigen contact before the cells gain access to nearly-common regulatory mechanisms for choosing effector function. Here, the major classes of transcription factors that interact with Notch signals during T-lineage specification are discussed in terms of their roles in these programs, the evidence for their spectra of target genes at different stages, and their cross-regulatory and cooperative actions with each other. Specific topics include Notch modulation of PU.1 and GATA-3, PU.1-Notch competition, the relationship between PU.1 and GATA-3, and the roles of E proteins, Bcl11b, and GATA-3 in guiding acquisition of T-cell identity while avoiding redirection to an ILC fate. PMID:26791859
Tracing Activity Across the Whole Brain Neural Network with Optogenetic Functional Magnetic Resonance Imaging

PubMed Central

Lee, Jin Hyung

2011-01-01

Despite the overwhelming need, there has been a relatively large gap in our ability to trace network level activity across the brain. The complex dense wiring of the brain makes it extremely challenging to understand cell-type specific activity and their communication beyond a few synapses. Recent development of the optogenetic functional magnetic resonance imaging (ofMRI) provides a new impetus for the study of brain circuits by enabling causal tracing of activities arising from defined cell types and firing patterns across the whole brain. Brain circuit elements can be selectively triggered based on their genetic identity, cell body location, and/or their axonal projection target with temporal precision while the resulting network response is monitored non-invasively with unprecedented spatial and temporal accuracy. With further studies including technological innovations to bring ofMRI to its full potential, ofMRI is expected to play an important role in our system-level understanding of the brain circuit mechanism. PMID:22046160
Ubiquitin Ligases and Deubiquitinating Enzymes in CD4+ T Cell Effector Fate Choice and Function.

PubMed

Layman, Awo A K; Oliver, Paula M

2016-05-15

The human body is exposed to potentially pathogenic microorganisms at barrier sites such as the skin, lungs, and gastrointestinal tract. To mount an effective response against these pathogens, the immune system must recruit the right cells with effector responses that are appropriate for the task at hand. Several types of CD4(+) T cells can be recruited, including Th cells (Th1, Th2, and Th17), T follicular helper cells, and regulatory T cells. These cells help to maintain normal immune homeostasis in the face of constantly changing microbes in the environment. Because these cells differentiate from a common progenitor, the composition of their intracellular milieu of proteins changes to appropriately guide their effector function. One underappreciated process that impacts the levels and functions of effector fate-determining factors is ubiquitylation. This review details our current understanding of how ubiquitylation regulates CD4(+) T cell effector identity and function. Copyright © 2016 by The American Association of Immunologists, Inc.
Detection of AIDS Virus in Macrophages in Brain Tissue from AIDS Patients with Encephalopathy

NASA Astrophysics Data System (ADS)

Koenig, Scott; Gendelman, Howard E.; Orenstein, Jan M.; Canto, Mauro C.; Pezeshkpour, Gholam H.; Yungbluth, Margaret; Janotta, Frank; Aksamit, Allen; Martin, Malcolm A.; Fauci, Anthony S.

1986-09-01

One of the common neurological complications in patients with the acquired immune deficiency syndrome (AIDS) is a subacute encephalopathy with progressive dementia. By using the techniques of cocultivation for virus isolation, in situ hybridization, immunocytochemistry, and transmission electron microscopy, the identity of an important cell type that supports replication of the AIDS retrovirus in brain tissue was determined in two affected individuals. These cells were mononucleated and multinucleated macrophages that actively synthesized viral RNA and produced progeny virions in the brains of the patients. Infected brain macrophages may serve as a reservoir for virus and as a vehicle for viral dissemination in the infected host.
Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting.

PubMed

Chen, Fuqiang; Ding, Xiao; Feng, Yongmei; Seebeck, Timothy; Jiang, Yanfang; Davis, Gregory D

2017-04-07

Bacterial CRISPR-Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR-Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR-Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification.
Targeted activation of diverse CRISPR-Cas systems for mammalian genome editing via proximal CRISPR targeting

PubMed Central

Chen, Fuqiang; Ding, Xiao; Feng, Yongmei; Seebeck, Timothy; Jiang, Yanfang; Davis, Gregory D.

2017-01-01

Bacterial CRISPR–Cas systems comprise diverse effector endonucleases with different targeting ranges, specificities and enzymatic properties, but many of them are inactive in mammalian cells and are thus precluded from genome-editing applications. Here we show that the type II-B FnCas9 from Francisella novicida possesses novel properties, but its nuclease function is frequently inhibited at many genomic loci in living human cells. Moreover, we develop a proximal CRISPR (termed proxy-CRISPR) targeting method that restores FnCas9 nuclease activity in a target-specific manner. We further demonstrate that this proxy-CRISPR strategy is applicable to diverse CRISPR–Cas systems, including type II-C Cas9 and type V Cpf1 systems, and can facilitate precise gene editing even between identical genomic sites within the same genome. Our findings provide a novel strategy to enable use of diverse otherwise inactive CRISPR–Cas systems for genome-editing applications and a potential path to modulate the impact of chromatin microenvironments on genome modification. PMID:28387220
Cytochrome components of nitrate- and sulfate-respiring Desulfovibrio desulfuricans ATCC 27774.

PubMed Central

Liu, M C; Costa, C; Coutinho, I B; Moura, J J; Moura, I; Xavier, A V; LeGall, J

1988-01-01

Three multiheme c-type cytochromes--the tetraheme cytochrome c3 (molecular weight [MW] 13,500), a dodecaheme cytochrome c (MW 40,800), and a "split-Soret" cytochrome c (MW 51,540), which is a dimer with 2 hemes per subunit (MW 26,300)--were isolated from the soluble fraction of Desulfovibrio desulfuricans (ATCC 27774) grown under nitrate- or sulfate-respiring conditions. Two of them, the dodecaheme and the split-Soret cytochromes, showed no similarities to any of the c-type cytochromes isolated from other sulfate-reducing bacteria, while the tetraheme cytochrome c3 appeared to be analogous to the cytochrome c3 found in other sulfate-reducing bacteria. For all three multiheme c-type cytochromes isolated, the homologous proteins from nitrate- and sulfate-grown cells were indistinguishable in amino acid composition, physical properties, and spectroscopic characteristics. It therefore appears that the same c-type cytochrome components are present when D. desulfuricans ATCC 27774 cells are grown under either condition. This is in contrast to the considerable difference found in Pseudomonas perfectomarina (Liu et al., J. Bacteriol. 154:278-286, 1983), a marine denitrifier, when the cells are grown on nitrate or oxygen as the terminal electron acceptor. In addition, two spectroscopy methods capable of revealing minute structural variations in proteins provided identical information about the tetraheme cytochrome c3 from nitrate-grown and sulfate-grown cells. PMID:2848008
The roadmap for estimation of cell-type-specific neuronal activity from non-invasive measurements

PubMed Central

Uhlirova, Hana; Kılıç, Kıvılcım; Tian, Peifang; Sakadžić, Sava; Thunemann, Martin; Desjardins, Michèle; Saisan, Payam A.; Nizar, Krystal; Yaseen, Mohammad A.; Hagler, Donald J.; Vandenberghe, Matthieu; Djurovic, Srdjan; Andreassen, Ole A.; Silva, Gabriel A.; Masliah, Eliezer; Vinogradov, Sergei; Buxton, Richard B.; Einevoll, Gaute T.; Boas, David A.; Dale, Anders M.; Devor, Anna

2016-01-01

The computational properties of the human brain arise from an intricate interplay between billions of neurons connected in complex networks. However, our ability to study these networks in healthy human brain is limited by the necessity to use non-invasive technologies. This is in contrast to animal models where a rich, detailed view of cellular-level brain function with cell-type-specific molecular identity has become available due to recent advances in microscopic optical imaging and genetics. Thus, a central challenge facing neuroscience today is leveraging these mechanistic insights from animal studies to accurately draw physiological inferences from non-invasive signals in humans. On the essential path towards this goal is the development of a detailed ‘bottom-up’ forward model bridging neuronal activity at the level of cell-type-specific populations to non-invasive imaging signals. The general idea is that specific neuronal cell types have identifiable signatures in the way they drive changes in cerebral blood flow, cerebral metabolic rate of O2 (measurable with quantitative functional Magnetic Resonance Imaging), and electrical currents/potentials (measurable with magneto/electroencephalography). This forward model would then provide the ‘ground truth’ for the development of new tools for tackling the inverse problem—estimation of neuronal activity from multimodal non-invasive imaging data. This article is part of the themed issue ‘Interpreting BOLD: a dialogue between cognitive and cellular neuroscience’. PMID:27574309

An Acvr1 R206H knock-in mouse has fibrodysplasia ossificans progressiva

PubMed Central

Chakkalakal, Salin A.; Zhang, Deyu; Culbert, Andria L.; Convente, Michael R.; Caron, Robert J.; Wright, Alexander C.; Maidment, Andrew D.A.; Kaplan, Frederick S.; Shore, Eileen M.

2013-01-01

Fibrodysplasia ossificans progressiva (FOP; MIM #135100) is a debilitating genetic disorder of dysregulated cellular differentiation characterized by malformation of the great toes during embryonic skeletal development and by progressive heterotopic endochondral ossification post-natally. Patients with these classic clinical features of FOP have the identical heterozygous single nucleotide substitution (c.617G>A; R206H) in the gene encoding ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor. Gene targeting was used to develop a knock-in mouse model for FOP (Acvr1R206H/+). Radiographic analysis of Acvr1R206H/+ chimeric mice revealed that this mutation induced malformed first digits in the hind limbs and post-natal extra-skeletal bone formation, recapitulating the human disease. Histological analysis of murine lesions showed inflammatory infiltration and apoptosis of skeletal muscle followed by robust formation of heterotopic bone through an endochondral pathway, identical to that seen in patients. Progenitor cells of a Tie2+ lineage participated in each stage of endochondral osteogenesis. We further determined that both wild-type and mutant cells are present within the ectopic bone tissue, an unexpected finding that indicates that although the mutation is necessary to induce the bone formation process, the mutation is not required for progenitor cell contribution to bone and cartilage. This unique knock-in mouse model provides novel insight into the genetic regulation of heterotopic ossification and establishes the first direct in vivo evidence that the R206H mutation in ACVR1 causes FOP. PMID:22508565
Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA.

PubMed Central

de la Cruz, J; Vioque, A

2001-01-01

tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans-translation reaction that contributes to the recycling of stalled ribosomes at the 3' end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperature-sensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Synechocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells (deltassrA) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of deltassrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the deltassrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics. PMID:11780628
Single-Cell Tracking Reveals a Role for Pre-Existing CCR5+ Memory Th1 Cells in the Control of Rhinovirus-A39 After Experimental Challenge in Humans.

PubMed

Muehling, Lyndsey M; Turner, Ronald B; Brown, Kenneth B; Wright, Paul W; Patrie, James T; Lahtinen, Sampo J; Lehtinen, Markus J; Kwok, William W; Woodfolk, Judith A

2018-01-17

Little is known about T cells that respond to human rhinovirus in vivo, due to timing of infection, viral diversity, and complex T-cell specificities. We tracked circulating CD4+ T cells with identical epitope specificities that responded to intranasal challenge with rhinovirus (RV)-A39, and we assessed T-cell signatures in the nose. Cells were monitored using a mixture of 2 capsid-specific major histocompatibility complex II tetramers over a 7-week period, before and after RV-A39 challenge, in 16 human leukocyte antigen-DR4+ subjects who participated in a trial of Bifidobacterium lactis (Bl-04) supplementation. Pre-existing tetramer+ T cells were linked to delayed viral shedding, enriched for activated CCR5+ Th1 effectors, and included a minor interleukin-21+ T follicular helper cell subset. After RV challenge, expansion and activation of virus-specific CCR5+ Th1 effectors was restricted to subjects who had a rise in neutralizing antibodies, and tetramer-negative CCR5+ effector memory types were comodulated. In the nose, CXCR3-CCR5+ T cells present during acute infection were activated effector memory type, whereas CXCR3+ cells were central memory type, and cognate chemokine ligands were elevated over baseline. Probiotic had no T-cell effects. We conclude that virus-specific CCR5+ effector memory CD4+ T cells primed by previous exposure to related viruses contribute to the control of rhinovirus. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.
Cellular programming and reprogramming: sculpting cell fate for the production of dopamine neurons for cell therapy.

PubMed

Aguila, Julio C; Hedlund, Eva; Sanchez-Pernaute, Rosario

2012-01-01

Pluripotent stem cells are regarded as a promising cell source to obtain human dopamine neurons in sufficient amounts and purity for cell replacement therapy. Importantly, the success of clinical applications depends on our ability to steer pluripotent stem cells towards the right neuronal identity. In Parkinson disease, the loss of dopamine neurons is more pronounced in the ventrolateral population that projects to the sensorimotor striatum. Because synapses are highly specific, only neurons with this precise identity will contribute, upon transplantation, to the synaptic reconstruction of the dorsal striatum. Thus, understanding the developmental cell program of the mesostriatal dopamine neurons is critical for the identification of the extrinsic signals and cell-intrinsic factors that instruct and, ultimately, determine cell identity. Here, we review how extrinsic signals and transcription factors act together during development to shape midbrain cell fates. Further, we discuss how these same factors can be applied in vitro to induce, select, and reprogram cells to the mesostriatal dopamine fate.
Maitotoxin Is a Potential Selective Activator of the Endogenous Transient Receptor Potential Canonical Type 1 Channel in Xenopus laevis Oocytes

PubMed Central

Flores, Pedro L.; Rodríguez, Emma; Zapata, Estrella; Carbó, Roxana; Farías, José María; Martínez, Martín

2017-01-01

Maitotoxin (MTX) is the most potent marine toxin known to date. It is responsible for a particular human intoxication syndrome called ciguatera fish poisoning (CFP). Several reports indicate that MTX is an activator of non-selective cation channels (NSCC) in different cell types. The molecular identity of these channels is still an unresolved topic, and it has been proposed that the transient receptor potential (TRP) channels are involved in this effect. In Xenopus laevis oocytes, MTX at picomolar (pM) concentrations induces the activation of NSCC with functional and pharmacological properties that resemble the activity of TRP channels. The purpose of this study was to characterize the molecular identity of the TRP channel involved in the MTX response, using the small interference RNA (siRNA) approach and the two-electrode voltage-clamp technique (TEVC). The injection of a specifically designed siRNA to silence the transient receptor potential canonical type 1 (TRPC1) protein expression abolished the MTX response. MTX had no effect on oocytes, even at doses 20-fold higher compared to cells without injection. Total mRNA and protein levels of TRPC1 were notably diminished. The TRPC4 siRNA did not change the MTX effect, even though it was important to note that the protein level was reduced by the silencing of TRPC4. Our results suggest that MTX could be a selective activator of TRPC1 channels in X. laevis oocytes and a useful pharmacological tool for further studies on these TRP channels. PMID:28672825
Maitotoxin Is a Potential Selective Activator of the Endogenous Transient Receptor Potential Canonical Type 1 Channel in Xenopus laevis Oocytes.

PubMed

Flores, Pedro L; Rodríguez, Emma; Zapata, Estrella; Carbó, Roxana; Farías, José María; Martínez, Martín

2017-06-25

Maitotoxin (MTX) is the most potent marine toxin known to date. It is responsible for a particular human intoxication syndrome called ciguatera fish poisoning (CFP). Several reports indicate that MTX is an activator of non-selective cation channels (NSCC) in different cell types. The molecular identity of these channels is still an unresolved topic, and it has been proposed that the transient receptor potential (TRP) channels are involved in this effect. In Xenopus laevis oocytes, MTX at picomolar (pM) concentrations induces the activation of NSCC with functional and pharmacological properties that resemble the activity of TRP channels. The purpose of this study was to characterize the molecular identity of the TRP channel involved in the MTX response, using the small interference RNA (siRNA) approach and the two-electrode voltage-clamp technique (TEVC). The injection of a specifically designed siRNA to silence the transient receptor potential canonical type 1 (TRPC1) protein expression abolished the MTX response. MTX had no effect on oocytes, even at doses 20-fold higher compared to cells without injection. Total mRNA and protein levels of TRPC1 were notably diminished. The TRPC4 siRNA did not change the MTX effect, even though it was important to note that the protein level was reduced by the silencing of TRPC4. Our results suggest that MTX could be a selective activator of TRPC1 channels in X. laevis oocytes and a useful pharmacological tool for further studies on these TRP channels.
Human YKL39 (chitinase 3-like protein 2), an osteoarthritis-associated gene, enhances proliferation and type II collagen expression in ATDC5 cells

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

Miyatake, Kazumasa; Tsuji, Kunikazu, E-mail: ktsuji.gcoe@tmd.ac.jp; Yamaga, Mika

Highlights: ► hYKL-39 expression is increased in osteoarthritic articular chondrocytes. ► To examine the molecular functions of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in chondrocytic ATDC5 cells. ► hYKL-39 enhanced proliferation and colony formation in ATDC5 cells. ► hYKL-39 increased type II collagen expression in ATDC5 cells treated with chondrogenic medium. -- Abstract: Human YKL39 (chitinase 3-like protein 2/CHI3L2) is a secreted 39 kDa protein produced by articular chondrocytes and synoviocytes. Recent studies showed that hYKL-39 expression is increased in osteoarthritic articular chondrocytes suggesting the involvement of hYKL-39 in the progression of osteoarthritis (OA). However little is known regarding themore » molecular function of hYKL-39 in joint homeostasis. Sequence analyses indicated that hYKL-39 has significant identity with the human chitotorisidase family molecules, although it is considered that hYKL-39 has no enzymatic activity since it lacks putative chitinase catalytic motif. In this study, to examine the molecular function of hYKL-39 in chondrocytes, we overexpressed hYKL-39 in ATDC5 cells. Here we report that hYKL-39 enhances colony forming activity, cell proliferation, and type II collagen expression in these cells. These data suggest that hYKL-39 is a novel growth and differentiation factor involved in cartilage homeostasis.« less
The structure of people's hair.

PubMed

Yang, Fei-Chi; Zhang, Yuchen; Rheinstädter, Maikel C

2014-01-01

Hair is a filamentous biomaterial consisting mainly of proteins in particular keratin. The structure of human hair is well known: the medulla is a loosely packed, disordered region near the centre of the hair surrounded by the cortex, which contains the major part of the fibre mass, mainly consisting of keratin proteins and structural lipids. The cortex is surrounded by the cuticle, a layer of dead, overlapping cells forming a protective layer around the hair. The corresponding structures have been studied extensively using a variety of different techniques, such as light, electron and atomic force microscopes, and also X-ray diffraction. We were interested in the question how much the molecular hair structure differs from person to person, between male and female hair, hair of different appearances such as colour and waviness. We included hair from parent and child, identical and fraternal twins in the study to see if genetically similar hair would show similar structural features. The molecular structure of the hair samples was studied using high-resolution X-ray diffraction, which covers length scales from molecules up to the organization of secondary structures. Signals due to the coiled-coil phase of α-helical keratin proteins, intermediate keratin filaments in the cortex and from the lipid layers in the cell membrane complex were observed in the specimen of all individuals, with very small deviations. Despite the relatively small number of individuals (12) included in this study, some conclusions can be drawn. While the general features were observed in all individuals and the corresponding molecular structures were almost identical, additional signals were observed in some specimen and assigned to different types of lipids in the cell membrane complex. Genetics seem to play a role in this composition as identical patterns were observed in hair from father and daughter and identical twins, however, not for fraternal twins. Identification and characterization of these features is an important step towards the detection of abnormalities in the molecular structure of hair as a potential diagnostic tool for certain diseases.
The cytological manifestation of gene amplification in multidrug-resistant mouse leukemia P388 sublines is correlated with amplicon content

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

Il`inskaya, G.V.; Kopnin, B.P.; Demidova, N.S.

1995-10-01

Previously, we showed that development of multidrug resistance (MDR) in mouse P388 leukemia cells is often associated with the appearance of newly-formed chromosomelike structures that contain amplified copies of the mdr1 gene. In the present study, we compared amplicon content in P388 sublines showing different types of these structures. A strong correlation between the formation of specific acentric markers consisting of two identical arms and the absence of the sorcin gene coamplification was found. In all the sublines containing other types of chromosomelike structures, the sorcin gene is coamplified. 9 refs., 2 figs., 1 tab.
Zebrafish pronephros tubulogenesis and epithelial identity maintenance are reliant on the polarity proteins Prkc iota and zeta

PubMed Central

Gerlach, Gary F.; Wingert, Rebecca A.

2014-01-01

The zebrafish pronephros provides an excellent in vivo system to study the mechanisms of vertebrate nephron development. When and how renal progenitors in the zebrafish embryo undergo tubulogenesis to form nephrons is poorly understood, but is known to involve a mesenchymal to epithelial transition (MET) and the acquisition of polarity. Here, we determined the precise timing of these events in pronephros tubulogenesis. As the ternary polarity complex is an essential regulator of epithelial cell polarity across tissues, we performed gene knockdown studies to assess the roles of the related factors atypical protein kinase C iota and zeta (prkcι, prkcζ). We found that prkcι and prkcζ serve partially redundant functions to establish pronephros tubule epithelium polarity. Further, the loss of prkcι or the combined knockdown of prkcι/ζ disrupted proximal tubule morphogenesis and podocyte migration due to cardiac defects that prevented normal fluid flow to the kidney. Surprisingly, tubule cells in prkcι/ζ morphants displayed ectopic expression of the transcription factor pax2a and the podocyte-associated genes wt1a, wt1b, and podxl, suggesting that prkcι/ζ are needed to maintain renal epithelial identity. Knockdown of genes essential for cardiac contractility and vascular flow to the kidney, such as tnnt2a, or elimination of pronephros fluid output through knockdown of the intraflagellar transport gene ift88, was not associated with ectopic pronephros gene expression, thus suggesting a unique role for prkcι/ζ in maintaining tubule epithelial identity separate from the consequence of disruptions to renal fluid flow. Interestingly, knockdown of pax2a, but not wt1a, was sufficient to rescue ectopic tubule gene expression in prkcι/ζ morphants. These data suggest a model in which the redundant activities of prkcι and prkcζ are essential to establish tubule epithelial polarity and also serve to maintain proper epithelial cell type identity in the tubule by inhibiting pax2a expression. These studies provide a valuable foundation for further analysis of MET during nephrogenesis, and have implications for understanding the pathways that affect nephron epithelial cells during kidney disease and regeneration. PMID:25446529
Ribbon Synaptic Plasticity in Gravity Sensors of Rats Flown on Neurolab

NASA Technical Reports Server (NTRS)

Ross, Muriel D.; Varelas, Joseph

2003-01-01

Previous spaceflight experiments (Space Life Sciences-1 and -2 (SLS-1 and SLS-2)) first demonstrated the extraordinary ability of gravity sensor hair cells to change the number, kind, and distribution of connections (synapses) they make to other cells while in weightlessness. The number of synapses in hair cells in one part of the inner ear (the utricle) was markedly elevated on flight day 13 (FD13) of SLS-2. Unanswered questions, however, were whether these increases in synapses occur rapidly and whether they remain stable in weightlessness. The answers have implications for long-duration human space travel. If gravity sensors can adapt quickly, crews may be able to move easily between different gravity levels, since the sensors will adapt rapidly to weightlessness on the spacecraft and then back to Earth's gravity when the mission ends. This ability to adapt is also important for recovery from balance disorders. To further our understanding of this adaptive potential (a property called neuronal synaptic plasticity), the present Neurolab research was undertaken. Our experiment examined whether: (a) increases in synapses would remain stable throughout the flight, (b) changes in the number of synapses were uniform across different portions of the gravity sensors (the utricle and saccule), and (c) synaptic changes were similar for the different types of hair cells (Type I and Type II). Utricular and saccular maculae (the gravity-sensing portions of the inner ear) were collected in flight from rats on FD2 and FD14. Samples were also collected from control rats on the ground. Tissues were prepared for ultrastructural study. Hair cells and their ribbon synapses were examined in a transmission electron microscope. Synapses were counted in all hair cells in 50 consecutive sections that crossed the striolar zone. Results indicate that utricular hair cell synapses initially increased significantly in number in both types of hair cells by FD2. Counts declined by FD14, but the mean number of synapses in utricular Type II cells remained significantly higher than in the ground control rats. For saccular samples, synaptic number in Type I and Type II cells declined on FD2, but returned to near-baseline values by FD14. These findings indicate that: (a) synaptic plasticity occurs rapidly in weightlessness, and (b) synaptic changes are not identical for the two types of hair cells or for the two maculae.
Impact of donor and recipient sex and parity on outcomes of HLA-identical sibling allogeneic hematopoietic stem cell transplantation.

PubMed

Loren, Alison W; Bunin, Greta R; Boudreau, Christian; Champlin, Richard E; Cnaan, Avital; Horowitz, Mary M; Loberiza, Fausto R; Porter, David L

2006-07-01

Allogeneic hematopoietic stem cell transplantation (SCT) may cure patients with hematologic malignancies, but it carries significant risks. Careful donor selection is an important component of the clinical transplantation decision-making process and includes evaluation of HLA typing and other criteria, the most controversial of which is parity. We examined the effect of donor sex and parity on outcomes of HLA-identical sibling SCT. Because the effect of recipient sex/parity has never been explicitly evaluated, we also analyzed the effect of recipient sex/parity on outcomes of transplantation. We found that (1) parous female donors result in an increased risk of chronic graft-versus-host disease (GVHD) in all recipients, (2) the magnitude of this increased risk is similar in male and female recipients, and (3) nulliparous female donors increase the risk of chronic GVHD in male recipients to a degree comparable to that from parous donors. A decrease in the risk of relapse was not observed, and there was no effect on overall survival, acute GVHD, or transplant-related mortality. Recipient parity had no independent effect on any endpoint. Until the effects of pregnancy on the maternal immune system are better understood, it is appropriate whenever possible to avoid parous female donors and to choose male donors for male recipients in HLA-identical related donor SCT.
View-Tolerant Face Recognition and Hebbian Learning Imply Mirror-Symmetric Neural Tuning to Head Orientation.

PubMed

Leibo, Joel Z; Liao, Qianli; Anselmi, Fabio; Freiwald, Winrich A; Poggio, Tomaso

2017-01-09

The primate brain contains a hierarchy of visual areas, dubbed the ventral stream, which rapidly computes object representations that are both specific for object identity and robust against identity-preserving transformations, like depth rotations [1, 2]. Current computational models of object recognition, including recent deep-learning networks, generate these properties through a hierarchy of alternating selectivity-increasing filtering and tolerance-increasing pooling operations, similar to simple-complex cells operations [3-6]. Here, we prove that a class of hierarchical architectures and a broad set of biologically plausible learning rules generate approximate invariance to identity-preserving transformations at the top level of the processing hierarchy. However, all past models tested failed to reproduce the most salient property of an intermediate representation of a three-level face-processing hierarchy in the brain: mirror-symmetric tuning to head orientation [7]. Here, we demonstrate that one specific biologically plausible Hebb-type learning rule generates mirror-symmetric tuning to bilaterally symmetric stimuli, like faces, at intermediate levels of the architecture and show why it does so. Thus, the tuning properties of individual cells inside the visual stream appear to result from group properties of the stimuli they encode and to reflect the learning rules that sculpted the information-processing system within which they reside. Copyright © 2017 Elsevier Ltd. All rights reserved.
Gender differences in the self-defining activities and identity experiences of adolescents and emerging adults.

PubMed

Sharp, Erin Hiley; Coatsworth, J Douglas; Darling, Nancy; Cumsille, Patricio; Ranieri, Sonia

2007-04-01

Activity participation provides a unique context for adolescents and emerging adults to explore interests, talents, and skills and for identity work to occur. Research has found consistent gender differences in the types of activities in which males and females participate. The current study drew on Eudaimonistic identity theory to examine the subjective identity-related experiences of personal expressiveness, flow experiences, and goal-directed behaviour [Waterman, 1984; Waterman, 2004. Finding someone to be: Studies on the role of intrinsic motivation in identity formation. Identity, 4, 209-228] within a special type of activity, self-defining activities, or those activities that participants identify as being important to who they are as a person. This study also tested for gender and country differences in a sample of 572 adolescents and emerging adults from the United States, Italy, and Chile. Findings indicate gender and country differences in the types of self-defining activities for males and females, but no gender differences in the reported identity-related experiences within those activities. This finding held across the three countries. Results from Multivariate Analyses of Variance also indicate that identity-related experiences differ significantly across seven broad activity classes. Findings are discussed in the context of the growing literature on adolescent activity involvement and time use, gender, and their relations to identity exploration.
In Vitro Assays for Mouse Müller Cell Phenotyping Through microRNA Profiling in the Damaged Retina.

PubMed

Reyes-Aguirre, Luis I; Quintero, Heberto; Estrada-Leyva, Brenda; Lamas, Mónica

2018-01-01

microRNA profiling has identified cell-specific expression patterns that could represent molecular signatures triggering the acquisition of a specific phenotype; in other words, of cellular identity and its associated function. Several groups have hypothesized that retinal cell phenotyping could be achieved through the determination of the global pattern of miRNA expression across specific cell types in the adult retina. This is especially relevant for Müller glia in the context of retinal damage, as these cells undergo dramatic changes of gene expression in response to injury, that render them susceptible to acquire a progenitor-like phenotype and be a source of new neurons.We describe a method that combines an experimental protocol for excitotoxic-induced retinal damage through N-methyl-D-aspartate subretinal injection with magnetic-activated cell sorting (MACS) of Müller cells and RNA isolation for microRNA profiling. Comparison of microRNA patterns of expression should allow Müller cell phenotyping under different experimental conditions.
Distributed coupling and multi-frequency microwave accelerators

DOEpatents

Tantawi, Sami G.; Li, Zenghai; Borchard, Philipp

2016-07-05

A microwave circuit for a linear accelerator has multiple metallic cell sections, a pair of distribution waveguide manifolds, and a sequence of feed arms connecting the manifolds to the cell sections. The distribution waveguide manifolds are connected to the cell sections so that alternating pairs of cell sections are connected to opposite distribution waveguide manifolds. The distribution waveguide manifolds have concave modifications of their walls opposite the feed arms, and the feed arms have portions of two distinct widths. In some embodiments, the distribution waveguide manifolds are connected to the cell sections by two different types of junctions adapted to allow two frequency operation. The microwave circuit may be manufactured by making two quasi-identical parts, and joining the two parts to form the microwave circuit, thereby allowing for many manufacturing techniques including electron beam welding, and thereby allowing the use of un-annealled copper alloys, and hence greater tolerance to high gradient operation.
Identification of 6H1 as a P2Y purinoceptor: P2Y5.

PubMed

Webb, T E; Kaplan, M G; Barnard, E A

1996-02-06

We have determined the identity of the orphan G-protein coupled receptor cDNA, 6H1, present in activated chicken T cells, as a subtype of P2Y purinoceptor. This identification is based on first on the degree of sequence identity shared with recently cloned members of the P2Y receptor family and second on the pharmacological profile. Upon transient expression in COS-7 cells the 6H1 receptor bound the radiolabel [35S]dATP alpha S specifically and with high affinity (Kd, 10 nM). This specific binding could be competitively displaced by a range of ligands active at P2 purinoceptors, with ATP being the most active (K (i)), 116 nM). Such competition studies have established the following rank order of activity: ATP ADP 2-methylthioATP alpha, beta-methylene ATP, UTP, thus confirming 6H1 as a member of the growing family of P2Y purinoceptors. As the fifth receptor of this type to be identified we suggest that it be named P2Y5.
Forging T-Lymphocyte Identity: Intersecting Networks of Transcriptional Control.

PubMed

Rothenberg, Ellen V; Ungerbäck, Jonas; Champhekar, Ameya

2016-01-01

T-lymphocyte development branches off from other lymphoid developmental programs through its requirement for sustained environmental signals through the Notch pathway. In the thymus, Notch signaling induces a succession of T-lineage regulatory factors that collectively create the T-cell identity through distinct steps. This process involves both the staged activation of T-cell identity genes and the staged repression of progenitor-cell-inherited regulatory genes once their roles in self-renewal and population expansion are no longer needed. With the recent characterization of innate lymphoid cells (ILCs) that share transcriptional regulation programs extensively with T-cell subsets, T-cell identity can increasingly be seen as defined in modular terms, as the processes selecting and actuating effector function are potentially detachable from the processes generating and selecting clonally unique T-cell receptor structures. The developmental pathways of different classes of T cells and ILCs are distinguished by the numbers of prerequisites of gene rearrangement, selection, and antigen contact before the cells gain access to nearly common regulatory mechanisms for choosing effector function. Here, the major classes of transcription factors that interact with Notch signals during T-lineage specification are discussed in terms of their roles in these programs, the evidence for their spectra of target genes at different stages, and their cross-regulatory and cooperative actions with each other. Specific topics include Notch modulation of PU.1 and GATA-3, PU.1-Notch competition, the relationship between PU.1 and GATA-3, and the roles of E proteins, Bcl11b, and GATA-3 in guiding acquisition of T-cell identity while avoiding redirection to an ILC fate. © 2016 Elsevier Inc. All rights reserved.
Transformation of Mouse Macrophages by Simian Virus 40

PubMed Central

Stone, Lawrence B.; Takemoto, Kenneth K.

1970-01-01

Studies were undertaken to prove that simian virus 40 (SV40) can transform the mouse macrophage, a cell type naturally restricted from deoxyribonucleic acid (DNA) replication. Balb/C macrophages infected with SV40 demonstrated T-antigen production and induced DNA synthesis simultaneously. In the absence of apparent division, these cells remained T antigen-positive for at least 45 days. SV40 could be rescued from nondividing, unaltered macrophages during the T antigen-producing period. Proliferating transformants appeared at an average of 66 days post-SV40 infection. Established cell lines were T antigen-positive and were negative for infectious virus, but yielded SV40 after fusion with African green monkey kidney cells. Their identity as transformed macrophages was substantiated by evaluation of cellular morphology, phagocytosis, acid phosphatase, β1c synthesis, and aminoacridine incorporation. Images PMID:4320698
Exploiting genomics and natural genetic variation to decode macrophage enhancers

PubMed Central

Romanoski, Casey E.; Link, Verena M.; Heinz, Sven; Glass, Christopher K.

2015-01-01

The mammalian genome contains on the order of a million enhancer-like regions that are required to establish the identities and functions of specific cell types. Here, we review recent studies in immune cells that have provided insight into the mechanisms that selectively activate certain enhancers in response to cell lineage and environmental signals. We describe a working model wherein distinct classes of transcription factors define the repertoire of active enhancers in macrophages through collaborative and hierarchical interactions, and discuss important challenges to this model, specifically providing examples from T cells. We conclude by discussing the use of natural genetic variation as a powerful approach for decoding transcription factor combinations that play dominant roles in establishing the enhancer landscapes, and the potential that these insights have for advancing our understanding of the molecular causes of human disease. PMID:26298065

Molecular cloning of a murine homologue of membrane cofactor protein (CD46): preferential expression in testicular germ cells.

PubMed Central

Tsujimura, A; Shida, K; Kitamura, M; Nomura, M; Takeda, J; Tanaka, H; Matsumoto, M; Matsumiya, K; Okuyama, A; Nishimune, Y; Okabe, M; Seya, T

1998-01-01

Human membrane cofactor protein (MCP, CD46) has been suggested, although no convincing evidence has been proposed, to be a fertilization-associated protein, in addition to its primary functions as a complement regulator and a measles virus receptor. We have cloned a cDNA encoding the murine homologue of MCP. This cDNA showed 45% identity in deduced protein sequence and 62% identity in nucleotide sequence with human MCP. Its ectodomains were four short consensus repeats and a serine/threonine-rich domain, and it appeared to be a type 1 membrane protein with a 23-amino acid transmembrane domain and a short cytoplasmic tail. The protein expressed on Chinese hamster ovary cell transfectants was 47 kDa on SDS/PAGE immunoblotting, approximately 6 kDa larger than the murine testis MCP. It served as a cofactor for factor I-mediated inactivation of the complement protein C3b in a homologous system and, to a lesser extent, in a human system. Strikingly, the major message of murine MCP was 1.5 kb and was expressed predominantly in the testis. It was not detected in mice defective in spermatogenesis or with immature germ cells (until 23 days old). Thus, murine MCP may be a sperm-dominant protein the message of which is expressed selectively in spermatids during germ-cell differentiation. PMID:9461505
Matched unrelated donor allogeneic transplantation provides comparable long-term outcome to HLA-identical sibling transplantation in relapsed diffuse large B-cell lymphoma.

PubMed

Avivi, I; Canals, C; Vernant, J-P; Wulf, G; Nagler, A; Hermine, O; Petersen, E; Yakoub-Agha, I; Craddock, C; Schattenberg, A; Niederwieser, D; Thomson, K; Blaise, D; Attal, M; Pfreundschuh, M; Passweg, J; Russell, N; Dreger, P; Sureda, A

2014-05-01

The objective of this retrospective analysis was to compare outcomes of patients with diffuse large B-cell lymphoma (DLBCL) who received either a matched sibling (sib) or an unrelated donor (URD) allogeneic hematopoietic cell transplantation (allo-HCT). Long-term outcome of 172 DLBCL patients receiving URD-HCT between 2000 and 2007 and reported to the European Group for Blood and Marrow Transplantation, was compared with that of 301 subjects, allografted from sib-HCT. With a median follow-up of 45 months, 3-year PFS approached 35% for both groups; overall survival (OS) was 42% for sib-HCT versus 37% for URD (NS). Multivariate analyses confirmed that donor type was not associated with differences in non-relapse mortality (NRM), relapse rate (RR), PFS or OS. Poor performance status (PS) and refractory disease adversely affected PFS and OS. Prior auto-SCT and multiple previous therapies predicted for shorter PFS. NRM was adversely affected by older age (⩾50 years), poor PS and refractory disease, and RR by time from diagnosis to allo-HCT of <36 months, prior auto-SCT, refractory disease, poor PS and in vivo T-cell depletion with alemtuzumab. This large study shows for the first time that URD-HCT is not inferior to sib-HCT, providing a reasonable therapeutic approach for DLBCL patients, having no HLA-identical sibling available.
Insights into Host Cell Modulation and Induction of New Cells by the Corn Smut Ustilago maydis.

PubMed

Redkar, Amey; Matei, Alexandra; Doehlemann, Gunther

2017-01-01

Many filamentous fungal pathogens induce drastic modulation of host cells causing abnormal infectious structures such as galls, or tumors that arise as a result of re-programming in the original developmental cell fate of a colonized host cell. Developmental consequences occur predominantly with biotrophic phytopathogens. This suggests that these host structures result as an outcome of efficient defense suppression and intimate fungal-host interaction to suit the pathogen's needs for completion of its infection cycle. This mini-review mainly summarizes host cell re-programming that occurs in the Ustilago maydis - maize interaction, in which the pathogen deploys cell-type specific effector proteins with varying activities. The fungus senses the physiological status and identity of colonized host cells and re-directs the endogenous developmental program of its host. The disturbance of host cell physiology and cell fate leads to novel cell shapes, increased cell size, and/or the number of host cells. We particularly highlight the strategies of U. maydis to induce physiologically varied host organs to form the characteristic tumors in both vegetative and floral parts of maize.
The Genotype of the Donor for the (GT)n Polymorphism in the Promoter/Enhancer of FOXP3 Is Associated with the Development of Severe Acute GVHD but Does Not Affect the GVL Effect after Myeloablative HLA-Identical Allogeneic Stem Cell Transplantation

PubMed Central

Buces, Elena; Pion, Marjorie; Sánchez-Hernández, Noemí; Martín-Antonio, Beatriz; Guillem, Vicent; Bosch-Vizcaya, Anna; Bento, Leyre; González-Rivera, Milagros; Balsalobre, Pascual; Kwon, Mi; Serrano, David; Gayoso, Jorge; de la Cámara, Rafael; Brunet, Salut; Rojas-Contreras, Rafael; Nieto, José B.; Martínez, Carmen; Gónzalez, Marcos; Espigado, Ildefonso; Vallejo, Juan C.; Sampol, Antonia; Jiménez-Velasco, Antonio; Urbano-Ispizua, Alvaro; Solano, Carlos; Gallardo, David; Díez-Martín, José L.; Buño, Ismael

2015-01-01

The FOXP3 gene encodes for a protein (Foxp3) involved in the development and functional activity of regulatory T cells (CD4+/CD25+/Foxp3+), which exert regulatory and suppressive roles over the immune system. After allogeneic stem cell transplantation, regulatory T cells are known to mitigate graft versus host disease while probably maintaining a graft versus leukemia effect. Short alleles (≤(GT)15) for the (GT)n polymorphism in the promoter/enhancer of FOXP3 are associated with a higher expression of FOXP3, and hypothetically with an increase of regulatory T cell activity. This polymorphism has been related to the development of auto- or alloimmune conditions including type 1 diabetes or graft rejection in renal transplant recipients. However, its impact in the allo-transplant setting has not been analyzed. In the present study, which includes 252 myeloablative HLA-identical allo-transplants, multivariate analysis revealed a lower incidence of grade III-IV acute graft versus host disease (GVHD) in patients transplanted from donors harboring short alleles (OR = 0.26, CI 0.08–0.82, p = 0.021); without affecting chronic GVHD or graft versus leukemia effect, since cumulative incidence of relapse, event free survival and overall survival rates are similar in both groups of patients. PMID:26473355
The Genotype of the Donor for the (GT)n Polymorphism in the Promoter/Enhancer of FOXP3 Is Associated with the Development of Severe Acute GVHD but Does Not Affect the GVL Effect after Myeloablative HLA-Identical Allogeneic Stem Cell Transplantation.

PubMed

Noriega, Víctor; Martínez-Laperche, Carolina; Buces, Elena; Pion, Marjorie; Sánchez-Hernández, Noemí; Martín-Antonio, Beatriz; Guillem, Vicent; Bosch-Vizcaya, Anna; Bento, Leyre; González-Rivera, Milagros; Balsalobre, Pascual; Kwon, Mi; Serrano, David; Gayoso, Jorge; de la Cámara, Rafael; Brunet, Salut; Rojas-Contreras, Rafael; Nieto, José B; Martínez, Carmen; Gónzalez, Marcos; Espigado, Ildefonso; Vallejo, Juan C; Sampol, Antonia; Jiménez-Velasco, Antonio; Urbano-Ispizua, Alvaro; Solano, Carlos; Gallardo, David; Díez-Martín, José L; Buño, Ismael

2015-01-01

The FOXP3 gene encodes for a protein (Foxp3) involved in the development and functional activity of regulatory T cells (CD4+/CD25+/Foxp3+), which exert regulatory and suppressive roles over the immune system. After allogeneic stem cell transplantation, regulatory T cells are known to mitigate graft versus host disease while probably maintaining a graft versus leukemia effect. Short alleles (≤(GT)15) for the (GT)n polymorphism in the promoter/enhancer of FOXP3 are associated with a higher expression of FOXP3, and hypothetically with an increase of regulatory T cell activity. This polymorphism has been related to the development of auto- or alloimmune conditions including type 1 diabetes or graft rejection in renal transplant recipients. However, its impact in the allo-transplant setting has not been analyzed. In the present study, which includes 252 myeloablative HLA-identical allo-transplants, multivariate analysis revealed a lower incidence of grade III-IV acute graft versus host disease (GVHD) in patients transplanted from donors harboring short alleles (OR = 0.26, CI 0.08-0.82, p = 0.021); without affecting chronic GVHD or graft versus leukemia effect, since cumulative incidence of relapse, event free survival and overall survival rates are similar in both groups of patients.
Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium.

PubMed

Wagner, Diane R; Lindsey, Derek P; Li, Kelvin W; Tummala, Padmaja; Chandran, Sheena E; Smith, R Lane; Longaker, Michael T; Carter, Dennis R; Beaupre, Gary S

2008-05-01

This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.
Stem cell sources for clinical islet transplantation in type 1 diabetes: embryonic and adult stem cells.

PubMed

Miszta-Lane, Helena; Mirbolooki, Mohammadreza; James Shapiro, A M; Lakey, Jonathan R T

2006-01-01

Lifelong immunosuppressive therapy and inadequate sources of transplantable islets have led the islet transplantation benefits to less than 0.5% of type 1 diabetics. Whereas the potential risk of infection by animal endogenous viruses limits the uses of islet xeno-transplantation, deriving islets from stem cells seems to be able to overcome the current problems of islet shortages and immune compatibility. Both embryonic (derived from the inner cell mass of blastocysts) and adult stem cells (derived from adult tissues) have shown controversial results in secreting insulin in vitro and normalizing hyperglycemia in vivo. ESCs research is thought to have much greater developmental potential than adult stem cells; however it is still in the basic research phase. Existing ESC lines are not believed to be identical or ideal for generating islets or beta-cells and additional ESC lines have to be established. Research with ESCs derived from humans is controversial because it requires the destruction of a human embryo and/or therapeutic cloning, which some believe is a slippery slope to reproductive cloning. On the other hand, adult stem cells are already in some degree specialized, recipients may receive their own stem cells. They are flexible but they have shown mixed degree of availability. Adult stem cells are not pluripotent. They may not exist for all organs. They are difficult to purify and they cannot be maintained well outside the body. In order to draw the future avenues in this field, existent discrepancies between the results need to be clarified. In this study, we will review the different aspects and challenges of using embryonic or adult stem cells in clinical islet transplantation for the treatment of type 1 diabetes.
Demethylation of induced pluripotent stem cells from type 1 diabetic patients enhances differentiation into functional pancreatic β cells.

PubMed

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

2017-08-25

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

PubMed Central

He, Yisheng; Ding, Peng; Kao, Jui-Chun; Lee, Tzumin

2010-01-01

Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain. PMID:20808769
Argonne National Laboratory Li-alloy/FeS cell testing and R and D programs

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

Gay, E.C.

1982-01-01

Groups of 12 or more identical Li-alloy/FeS cells fabricated by Eagle-Picher Industries, Inc. and Gould Inc. were operated at Argonne National Laboratory (ANL) in the status cell test program to obtain data for statistical analysis of cell cycle life and failure modes. The cells were full-size electric vehicle battery cells (150 to 350 Ah capacity) and they were cycled at the 4-h discharge rate and 8-h charge rate. The end of life was defined as a 20% loss of capacity or a decrease in the coulombic efficiency to less than 95%. Seventy-four cells (six groups of identical cells) were cycle-lifemore » tested and the results were analyzed statistically. The ultimate goal of this analysis was to predict cell and battery reliability. Testing of groups of identical cells also provided a means of identifying common failure modes which were eliminated by cell design changes. Mean time to failure (MTTF) for the cells based on the Weibull distribution is presented.« less
Induced pluripotent stem cells for regenerative cardiovascular therapies and biomedical discovery.

PubMed

Nsair, Ali; MacLellan, W Robb

2011-04-30

The discovery of induced pluripotent stem cells (iPSC) has, in the short time since their discovery, revolutionized the field of stem cell biology. This technology allows the generation of a virtually unlimited supply of cells with pluripotent potential similar to that of embryonic stem cells (ESC). However, in contrast to ESC, iPSC are not subject to the same ethical concerns and can be easily generated from living individuals. For the first time, patient-specific iPSC can be generated and offer a supply of genetically identical cells that can be differentiated into all somatic cell types for potential use in regenerative therapies or drug screening and testing. As the techniques for generation of iPSC lines are constantly evolving, new uses for human iPSC are emerging from in-vitro disease modeling to high throughput drug discovery and screening. This technology promises to revolutionize the field of medicine and offers new hope for understanding and treatment of numerous diseases. Copyright © 2011 Elsevier B.V. All rights reserved.
A GRAPH PARTITIONING APPROACH TO PREDICTING PATTERNS IN LATERAL INHIBITION SYSTEMS

PubMed Central

RUFINO FERREIRA, ANA S.; ARCAK, MURAT

2017-01-01

We analyze spatial patterns on networks of cells where adjacent cells inhibit each other through contact signaling. We represent the network as a graph where each vertex represents the dynamics of identical individual cells and where graph edges represent cell-to-cell signaling. To predict steady-state patterns we find equitable partitions of the graph vertices and assign them into disjoint classes. We then use results from monotone systems theory to prove the existence of patterns that are structured in such a way that all the cells in the same class have the same final fate. To study the stability properties of these patterns, we rely on the graph partition to perform a block decomposition of the system. Then, to guarantee stability, we provide a small-gain type criterion that depends on the input-output properties of each cell in the reduced system. Finally, we discuss pattern formation in stochastic models. With the help of a modal decomposition we show that noise can enhance the parameter region where patterning occurs. PMID:29225552
Tissue engineering, stem cells and cloning: current concepts and changing trends.

PubMed

Atala, Anthony

2005-07-01

Organ damage or loss can occur from congenital disorders, cancer, trauma, infection, inflammation, iatrogenic injuries or other conditions and often necessitates reconstruction or replacement. Replacement may take the form of organ transplant. At present, there is a severe shortage of donor organs that is worsening with the aging of the population. Tissue engineering follows the principles of cell transplantation, materials science and engineering towards the development of biological substitutes that can restore and maintain normal tissue function. Therapeutic cloning involves the introduction of a nucleus from a donor cell into an enucleated oocyte to generate embryonic stem cell lines whose genetic material is identical to that of its source. These autologous stem cells have the potential to become almost any type of cell in the adult body, and thus would be useful in tissue and organ replacement applications. This paper reviews recent advances in stem cell research and regenerative medicine, and describes the clinical applications of these technologies as novel therapies for tissue or organ loss.
Crystal structure of tandem type III fibronectin domains from Drosophila neuroglian at 2.0 A.

PubMed

Huber, A H; Wang, Y M; Bieber, A J; Bjorkman, P J

1994-04-01

We report the crystal structure of two adjacent fibronectin type III repeats from the Drosophila neural cell adhesion molecule neuroglian. Each domain consists of two antiparallel beta sheets and is folded topologically identically to single fibronectin type III domains from the extracellular matrix proteins tenascin and fibronectin. beta bulges and left-handed polyproline II helices disrupt the regular beta sheet structure of both neuroglian domains. The hydrophobic interdomain interface includes a metal-binding site, presumably involved in stabilizing the relative orientation between domains and predicted by sequence comparision to be present in the vertebrate homolog molecule L1. The neuroglian domains are related by a near perfect 2-fold screw axis along the longest molecular dimension. Using this relationship, a model for arrays of tandem fibronectin type III repeats in neuroglian and other molecules is proposed.
Identification of a type-D feruloyl esterase from Neurospora crassa.

PubMed

Crepin, V F; Faulds, C B; Connerton, I F

2004-02-01

Feruloyl esterases constitute an interesting group of enzymes that have the potential for use over a broad range of applications in the agri-food industries. In order to expand the range of available enzymes, we have examined the presence of feruoyl esterase genes present in the genome sequence of the filamentous fungus Neurospora crassa. We have identified an orphan gene (contig 3.544), the translation of which shows sequence identity with known feruloyl esterases. This gene was cloned and the corresponding recombinant protein expressed in Pichia pastoris to confirm that the enzyme (NcFaeD-3.544) exhibits feruloyl esterase activity. Unusually the enzyme was capable of p-coumaric acid release from untreated crude plant cell wall materials. The substrate utilisation preferences of the recombinant enzyme place it in the recently recognised type-D sub-class of feruloyl esterase.
Permissive Sense and Antisense Transcription from the 5′ and 3′ Long Terminal Repeats of Human T-Cell Leukemia Virus Type 1

PubMed Central

Polakowski, Nicholas; Hoang, Kimson

2016-01-01

ABSTRACT Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus, and, as such, its genome becomes chromosomally integrated following infection. The resulting provirus contains identical 5′ and 3′ peripheral long terminal repeats (LTRs) containing bidirectional promoters. Antisense transcription from the 3′ LTR regulates expression of a single gene, hbz, while sense transcription from the 5′ LTR controls expression of all other viral genes, including tax. Both the HBZ and Tax proteins are implicated in the development of adult T-cell leukemia (ATL), a T-cell malignancy caused by HTLV-1 infection. However, these proteins appear to harbor opposing molecular functions, indicating that they may act independently and at different time points prior to leukemogenesis. Here, we used bidirectional reporter constructs to test whether transcriptional interference serves as a mechanism that inhibits simultaneous expression of Tax and HBZ. We found that sense transcription did not interfere with antisense transcription from the 3′ LTR and vice versa, even with strong transcription emanating from the opposing direction. Therefore, bidirectional transcription across the provirus might not restrict hbz or tax expression. Single-cell analyses revealed that antisense transcription predominates in the absence of Tax, which transactivates viral sense transcription. Interestingly, a population of Tax-expressing cells exhibited antisense but not activated sense transcription. Consistent with the ability of Tax to induce cell cycle arrest, this population was arrested in G0/G1 phase. These results imply that cell cycle arrest inhibits Tax-mediated activation of sense transcription without affecting antisense transcription, which may be important for long-term viral latency. IMPORTANCE The chromosomally integrated form of the retrovirus human T-cell leukemia virus type 1 (HTLV-1) contains identical DNA sequences, known as long terminal repeats (LTRs), at its 5′ and 3′ ends. The LTRs modulate transcription in both forward (sense) and reverse (antisense) directions. We found that sense transcription from the 5′ LTR does not interfere with antisense transcription from the 3′ LTR, allowing viral genes encoded on opposite DNA strands to be simultaneously transcribed. Two such genes are tax and hbz, and while they are thought to function at different times during the course of infection to promote leukemogenesis of infected T cells, our results indicate that they can be simultaneously transcribed. We also found that the ability of Tax to induce cell cycle arrest inhibits its fundamental function of activating viral sense transcription but does not affect antisense transcription. This regulatory mechanism may be important for long-term HTLV-1 infection. PMID:26792732
Reduced Abd-B Hox function during kidney development results in lineage infidelity.

PubMed

Magella, Bliss; Mahoney, Robert; Adam, Mike; Potter, S Steven

2018-06-15

Hox genes can function as key drivers of segment identity, with Hox mutations in Drosophila often resulting in dramatic homeotic transformations. In addition, however, they can serve other essential functions. In mammals, the study of Hox gene roles in development is complicated by the presence of four Hox clusters with a total of 39 genes showing extensive functional overlap. In this study, in order to better understand shared core Hox functions, we examined kidney development in mice with frameshift mutations of multiple Abd-B type Hox genes. The resulting phenotypes included dramatically reduced branching morphogenesis of the ureteric bud, premature depletion of nephron progenitors and abnormal development of the stromal compartment. Most unexpected, however, we also observed a cellular level lineage infidelity in nephron segments. Scattered cells within the proximal tubules, for example, expressed genes normally expressed only in collecting ducts. Multiple combinations of inappropriate nephron segment specific marker expression were found. In some cases, cells within a tubule showed incorrect identity, while in other cases cells showed ambiguous character, with simultaneous expression of genes associated with more than one nephron segment. These results give evidence that Hox genes have an overlapping core function at the cellular level in driving and/or maintaining correct differentiation decisions. Copyright © 2018 Elsevier Inc. All rights reserved.
Predicting Bone Metastatic Potential of Prostate Cancer via Computational Modeling of TGF-Beta Signaling

DTIC Science & Technology

2008-05-01

receptor trafficking. Smad phosphorylation Although the receptors for TGF-β signal through both Smad2 and Smad3 proteins in epithelial cells, we...select Smad2 to represent the R-Smads, because the two are virtually identical kinetically; furthermore, Smad2 is ~12-fold more abundant than Smad3 (11...the type of R-Smads. For example, regarding nuclear import, it has been proposed that Smad2, Smad3 , and Smad4 enter the nucleus by direct
Administration of Interleukin-6 Stimulates Multilineage Hematopoiesis and Accelerates Recovery from Radiation-Induced Hematopoietic Depression

DTIC Science & Technology

1991-02-01

Htirano T, Kishimoto T, Nakahata T. Asano S: In vitro hernatopoietic growth factors. J1 Natl Cancer Inst 81: t370. 1989 expansion of the murine...a.i phas S. %’ink A. Billiau A. VanSnick 1: Identification of the nutohlbyrcmiatntrekn.Cllmuolt12. human Zh-kd protein, interferon beta ,, as a B... beta . B-cell %timulatory factor type 2 shares identity T. Takaku F. Akivama Y: In vivo effects of recombinant human with rnsanc~tc-derived
Human T-cell leukemia virus type 1 infects multiple lineage hematopoietic cells in vivo

PubMed Central

Sugata, Kenji; Ueno, Takaharu; Koh, Ki-Ryang; Higuchi, Yusuke; Matsuda, Fumihiko; Melamed, Anat; Bangham, Charles R.

2017-01-01

Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4+CCR4+ effector/memory T cells in vivo. However, it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells. Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells. Therefore, we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 (STLV-1), whose virological attributes closely resemble those of HTLV-1. Although the tax transcript was detected only in certain tissues, Tax expression was much higher in the bone marrow, indicating the possibility of de novo infection. Furthermore, Tax expression of non-T cells was suspected in bone marrow. These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow. To explore the possibility that HTLV-1 infects hematopoietic stem cells (HSCs), we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a HTLV-1 carrier using the high-throughput sequencing method. Identical integration sites were detected in neutrophils, monocytes, B cells, CD8+ T cells and CD4+ T cells, indicating that HTLV-1 infects HSCs in vivo. We also detected Tax protein in myeloperoxidase positive neutrophils. Furthermore, dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells, indicating that infected monocytes are implicated in viral spreading in vivo. Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points, indicating that infected HSCs persist and differentiate in vivo. This study demonstrates that HTLV-1 infects HSCs, and infected stem cells differentiate into diverse cell lineages. These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo. PMID:29186194

Vulnerability- and Diversity-Aware Anonymization of Personally Identifiable Information for Improving User Privacy and Utility of Publishing Data.

PubMed

Majeed, Abdul; Ullah, Farman; Lee, Sungchang

2017-05-08

Personally identifiable information (PII) affects individual privacy because PII combinations may yield unique identifications in published data. User PII such as age, race, gender, and zip code contain private information that may assist an adversary in determining the user to whom such information relates. Each item of user PII reveals identity differently, and some types of PII are highly identity vulnerable. More vulnerable types of PII enable unique identification more easily, and their presence in published data increases privacy risks. Existing privacy models treat all types of PII equally from an identity revelation point of view, and they mainly focus on hiding user PII in a crowd of other users. Ignoring the identity vulnerability of each type of PII during anonymization is not an effective method of protecting user privacy in a fine-grained manner. This paper proposes a new anonymization scheme that considers the identity vulnerability of PII to effectively protect user privacy. Data generalization is performed adaptively based on the identity vulnerability of PII as well as diversity to anonymize data. This adaptive generalization effectively enables anonymous data, which protects user identity and private information disclosures while maximizing the utility of data for performing analyses and building classification models. Additionally, the proposed scheme has low computational overheads. The simulation results show the effectiveness of the scheme and verify the aforementioned claims.
Vulnerability- and Diversity-Aware Anonymization of Personally Identifiable Information for Improving User Privacy and Utility of Publishing Data

PubMed Central

Majeed, Abdul; Ullah, Farman; Lee, Sungchang

2017-01-01

Personally identifiable information (PII) affects individual privacy because PII combinations may yield unique identifications in published data. User PII such as age, race, gender, and zip code contain private information that may assist an adversary in determining the user to whom such information relates. Each item of user PII reveals identity differently, and some types of PII are highly identity vulnerable. More vulnerable types of PII enable unique identification more easily, and their presence in published data increases privacy risks. Existing privacy models treat all types of PII equally from an identity revelation point of view, and they mainly focus on hiding user PII in a crowd of other users. Ignoring the identity vulnerability of each type of PII during anonymization is not an effective method of protecting user privacy in a fine-grained manner. This paper proposes a new anonymization scheme that considers the identity vulnerability of PII to effectively protect user privacy. Data generalization is performed adaptively based on the identity vulnerability of PII as well as diversity to anonymize data. This adaptive generalization effectively enables anonymous data, which protects user identity and private information disclosures while maximizing the utility of data for performing analyses and building classification models. Additionally, the proposed scheme has low computational overheads. The simulation results show the effectiveness of the scheme and verify the aforementioned claims. PMID:28481298
Linking Prenatal Androgens to Gender-Related Attitudes, Identity, and Activities: Evidence From Girls With Congenital Adrenal Hyperplasia.

PubMed

Endendijk, Joyce J; Beltz, Adriene M; McHale, Susan M; Bryk, Kristina; Berenbaum, Sheri A

2016-10-01

Key questions for developmentalists concern the origins of gender attitudes and their implications for behavior. We examined whether prenatal androgen exposure was related to gender attitudes, and whether and how the links between attitudes and gendered activity interest and participation were mediated by gender identity and moderated by hormones. Gender attitudes (i.e., gender-role attitudes and attitudes about being a girl), gender identity, and gender-typed activities were reported by 54 girls aged 10-13 years varying in degree of prenatal androgen exposure, including 40 girls with classical congenital adrenal hyperplasia (C-CAH) exposed to high prenatal androgens and 14 girls with non-classical (NC) CAH exposed to low, female-typical, prenatal androgens. Both girls with C-CAH and NC-CAH reported positive attitudes about being a girl and egalitarian gender attitudes, consistent with their female-typical gender identity. In contrast, girls with C-CAH had more male-typed activity interest and participation than girls with NC-CAH. Gender attitudes were linked to activities in both groups, with gender identity mediating the links. Specifically, gender-role attitudes and positive attitudes about being a girl were associated with feminine gender identity, which in turn was associated with decreased male-typed activity interests and participation, and increased female-typed activity interests. Our results are consistent with schema theories, with attitudes more closely associated with gender identity than with prenatal androgens.
Identity Formation of American Indian Adolescents: Local, National, and Global Considerations

ERIC Educational Resources Information Center

Markstrom, Carol A.

2011-01-01

A conceptual model is presented that approaches identity formation of American Indian adolescents according to 3 levels of social contextual influence--local, national, and global--relative to types of identity, dynamics of identity, and sources of influence. Ethnic identity of American Indians is embedded within the local cultural milieu and…
HIV-1 dynamics revisited: biphasic decay by cytotoxic T lymphocyte killing?

PubMed Central

Arnaout, R A; Nowak, M A; Wodarz, D

2000-01-01

The biphasic decay of blood viraemia in patients being treated for human immunodeficiency virus type 1 (HIV-1) infection has been explained as the decay of two distinct populations of cells: the rapid death of productively infected cells followed by the much slower elimination of a second population the identity of which remains unknown. Here we advance an alternative explanation based on the immune response against a single population of infected cells. We show that the biphasic decay can be explained simply, without invoking multiple compartments: viral load falls quickly while cytotoxic T lymphocytes (CTL) are still abundant, and more slowly as CTL disappear. We propose a method to test this idea, and develop a framework that is readily applicable to treatment of other infections. PMID:10972131
Broad H3K4me3 is associated with increased transcription elongation and enhancer activity at tumor-suppressor genes.

PubMed

Chen, Kaifu; Chen, Zhong; Wu, Dayong; Zhang, Lili; Lin, Xueqiu; Su, Jianzhong; Rodriguez, Benjamin; Xi, Yuanxin; Xia, Zheng; Chen, Xi; Shi, Xiaobing; Wang, Qianben; Li, Wei

2015-10-01

Tumor suppressors are mostly defined by inactivating mutations in tumors, yet little is known about their epigenetic features in normal cells. Through integrative analysis of 1,134 genome-wide epigenetic profiles, mutations from >8,200 tumor-normal pairs and our experimental data from clinical samples, we discovered broad peaks for trimethylation of histone H3 at lysine 4 (H3K4me3; wider than 4 kb) as the first epigenetic signature for tumor suppressors in normal cells. Broad H3K4me3 is associated with increased transcription elongation and enhancer activity, which together lead to exceptionally high gene expression, and is distinct from other broad epigenetic features, such as super-enhancers. Genes with broad H3K4me3 peaks conserved across normal cells may represent pan-cancer tumor suppressors, such as TP53 and PTEN, whereas genes with cell type-specific broad H3K4me3 peaks may represent cell identity genes and cell type-specific tumor suppressors. Furthermore, widespread shortening of broad H3K4me3 peaks in cancers is associated with repression of tumor suppressors. Thus, the broad H3K4me3 epigenetic signature provides mutation-independent information for the discovery and characterization of new tumor suppressors.
The association of personal semantic memory to identity representations: insight into higher-order networks of autobiographical contents.

PubMed

Grilli, Matthew D

2017-11-01

Identity representations are higher-order knowledge structures that organise autobiographical memories on the basis of personality and role-based themes of one's self-concept. In two experiments, the extent to which different types of personal semantic content are reflected in these higher-order networks of memories was investigated. Healthy, young adult participants generated identity representations that varied in remoteness of formation and verbally reflected on these themes in an open-ended narrative task. The narrative responses were scored for retrieval of episodic, experience-near personal semantic and experience-far (i.e., abstract) personal semantic contents. Results revealed that to reflect on remotely formed identity representations, experience-far personal semantic contents were retrieved more than experience-near personal semantic contents. In contrast, to reflect on recently formed identity representations, experience-near personal semantic contents were retrieved more than experience-far personal semantic contents. Although episodic memory contents were retrieved less than both personal semantic content types to reflect on remotely formed identity representations, this content type was retrieved at a similar frequency as experience-far personal semantic content to reflect on recently formed identity representations. These findings indicate that the association of personal semantic content to identity representations is robust and related to time since acquisition of these knowledge structures.
Super-enhancers: Asset management in immune cell genomes.

PubMed

Witte, Steven; O'Shea, John J; Vahedi, Golnaz

2015-09-01

Super-enhancers (SEs) are regions of the genome consisting of clusters of regulatory elements bound with very high amounts of transcription factors, and this architecture appears to be the hallmark of genes and noncoding RNAs linked with cell identity. Recent studies have identified SEs in CD4(+) T cells and have further linked these regions to single nucleotide polymorphisms (SNPs) associated with immune-mediated disorders, pointing to an important role for these structures in the T cell differentiation and function. Here we review the features that define SEs, and discuss their function within the broader understanding of the mechanisms that define immune cell identity and function. We propose that SEs present crucial regulatory hubs, coordinating intrinsic and extrinsic differentiation signals, and argue that delineating these regions will provide important insight into the factors and mechanisms that define immune cell identity. Copyright © 2015 Elsevier Ltd. All rights reserved.
CXCR6 plays a critical role in angiotensin II-induced renal injury and fibrosis.

PubMed

Xia, Yunfeng; Jin, Xiaogao; Yan, Jingyin; Entman, Mark L; Wang, Yanlin

2014-07-01

Recent studies have shown that angiotensin II (Ang II) plays a critical role in the pathogenesis and progression of hypertensive kidney disease. However, the signaling mechanisms are poorly understood. In this study, we investigated the role of CXCR6 in Ang II-induced renal injury and fibrosis. Wild-type and CXCR6-green fluorescent protein (GFP) knockin mice were treated with Ang II via subcutaneous osmotic minipumps at 1500 ng/kg per minute after unilateral nephrectomy for ≤ 4 weeks. Wild-type and CXCR6-GFP knockin mice had virtually identical blood pressure at baseline. Ang II treatment led to an increase in blood pressure that was similar between wild-type and CXCR6-GFP knockin mice. CXCR6-GFP knockin mice were protected from Ang II-induced renal dysfunction, proteinuria, and fibrosis. CXCR6-GFP knockin mice accumulated fewer bone marrow-derived fibroblasts and myofibroblasts and produced less extracellular matrix protein in the kidneys after Ang II treatment. Furthermore, CXCR6-GFP knockin mice exhibited fewer F4/80(+) macrophages and CD3(+) T cells and expressed less proinflammatory cytokines in the kidneys after Ang II treatment. Finally, wild-type mice engrafted with CXCR6(-/-) bone marrow cells displayed fewer bone marrow-derived fibroblasts, macrophages, and T cells in the kidney after Ang II treatment when compared with wild-type mice engrafted with CXCR6(+/+) bone marrow cells. Our results indicate that CXCR6 plays a pivotal role in the development of Ang II-induced renal injury and fibrosis through regulation of macrophage and T-cell infiltration and bone marrow-derived fibroblast accumulation. © 2014 American Heart Association, Inc.
10.2% power conversion efficiency polymer tandem solar cells consisting of two identical sub-cells.

PubMed

You, Jingbi; Chen, Chun-Chao; Hong, Ziruo; Yoshimura, Ken; Ohya, Kenichiro; Xu, Run; Ye, Shenglin; Gao, Jing; Li, Gang; Yang, Yang

2013-08-07

Polymer tandem solar cells with 10.2% power conversion efficiency are demonstrated via stacking two PDTP-DFBT:PC₇₁ BM bulk heterojunctions, connected by MoO₃/PEDOT:PSS/ZnO as an interconnecting layer. The tandem solar cells increase the power conversion efficiency of the PDTP-DFBT:PC₇₁ BM system from 8.1% to 10.2%, successfully demonstrating polymer tandem solar cells with identical sub-cells of double-digit efficiency. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Identification of a basic protein of Mr 75,000 as an accessory desmosomal plaque protein in stratified and complex epithelia.

PubMed

Kapprell, H P; Owaribe, K; Franke, W W

1988-05-01

Desmosomes are intercellular adhering junctions characterized by a special structure and certain obligatory constituent proteins such as the cytoplasmic protein, desmoglein. Desmosomal fractions from bovine muzzle epidermis contain, in addition, a major polypeptide of Mr approximately 75,000 ("band 6 protein") which differs from all other desmosomal proteins so far identified by its positive charge (isoelectric at pH approximately 8.5 in the denatured state) and its avidity to bind certain type I cytokeratins under stringent conditions. We purified this protein from bovine muzzle epidermis and raised antibodies to it. Using affinity-purified antibodies, we identified a protein of identical SDS-PAGE mobility and isoelectric pH in all epithelia of higher complexity, including representatives of stratified, complex (pseudostratified) and transitional epithelia as well as benign and malignant human tumors derived from such epithelia. Immunolocalization studies revealed the location of this protein along cell boundaries in stratified and complex epithelia, often resolved into punctate arrays. In some epithelia it seemed to be restricted to certain cell types and layers; in rat cornea, for example, it was only detected in upper strata. Electron microscopic immunolocalization showed that this protein is a component of the desmosomal plaque. However, it was not found in the desmosomes of all simple epithelia examined, in the tumors and cultured cells derived thereof, in myocardiac and Purkinje fiber cells, in arachnoideal cells and meningiomas, and in dendritic reticulum cells of lymphoid tissue, i.e., all cells containing typical desmosomes. The protein was also absent in all nondesmosomal adhering junctions. From these results we conclude that this basic protein is not an obligatory desmosomal plaque constituent but an accessory component specific to the desmosomes of certain kinds of epithelial cells with stratified tissue architecture. This suggests that the Mr 75,000 basic protein does not serve general desmosomal functions but rather cell type-specific ones and that the composition of the desmosomal plaque can be different in different cell types. The possible diagnostic value of this protein as a marker in cell typing is discussed.
Effects of Lubrol detergents on adenylate cyclases.

PubMed

Bär, H P; Kulshrestha, S

1975-04-01

The nonionic detergent Lubrol WX showed diverse, concentration-dependent effects onbasal and stimulated adenylate cyclases. Above concentrations of 0.001-0.01% Lubrol WX, the basal activity of cyclase from Ehrlich ascites cells was inhibed about 50%, and that from rat fat cells was doubled. In both cases, hormonal sensitivity was lost at 0.01%. These effects were reversed upon dilution of the detergent. It is suggested that solubilization of adenylate cyclases at such low concentrations of Lubrol should be attempted since it is conceivable that loss of hormone sensitivity may then be reversible. Different Lubrol-type detergents may also offer centain advantages, since Lubrol PX effects were not identical with those of Lubrol WX.
Identification of a mouse B-type cyclin which exhibits developmentally regulated expression in the germ line

NASA Technical Reports Server (NTRS)

Chapman, D. L.; Wolgemuth, D. J.

1992-01-01

To begin to examine the function of cyclins in mammalian germ cells, we have screened an adult mouse testis cDNA library for the presence of B-type cyclins. We have isolated cDNAs that encode a murine B-type cyclin, which has been designated cycB1. cycB1 was shown to be expressed in several adult tissues and in the midgestation mouse embryo. In the adult tissues, the highest levels of cycB1 transcripts were seen in the testis and ovary, which contain germ cells at various stages of differentiation. The major transcripts corresponding to cycB1 are 1.7 and 2.5 kb, with the 1.7 kb species being the predominant testicular transcript and the 2.5 kb species more abundant in the ovary. Examination of cDNAs corresponding to the 2.5 kb and 1.7 kb mRNAs revealed that these transcripts encode identical proteins, differing only in the polyadenylation signal used and therefore in the length of their 3' untranslated regions. Northern blot and in situ hybridization analyses revealed that the predominant sites of cycB1 expression in the testis and ovary were in the germinal compartment, particularly in early round spermatids in the testis and growing oocytes in the ovary. Thus cycB1 is expressed in both meiotic and postmeiotic cells. This pattern of cycB1 expression further suggests that cycB1 may have different functions in the two cell types, only one of which correlates with progression of the cell cycle.
Gender Identity and Adjustment: Understanding the Impact of Individual and Normative Differences in Sex Typing

ERIC Educational Resources Information Center

Lurye, Leah E.; Zosuls, Kristina M.; Ruble, Diane N.

2008-01-01

The relationship among gender identity, sex typing, and adjustment has attracted the attention of social and developmental psychologists for many years. However, they have explored this issue with different assumptions and different approaches. Generally the approaches differ regarding whether sex typing is considered adaptive versus maladaptive,…
A Narrative Inquiry of Identity Formation of EFL University Teachers

ERIC Educational Resources Information Center

Cheng, Xiangli

2016-01-01

Drawing on narrative inquiry, the present study aims to investigate the trajectory of identity formation of EFL university teachers. Two types of data are collected. One type comes from life histories of Hyland (2014), Nunan (2011) and Widdowson (2009), and the other type comes from semi-structured interviews with three excellent university…
Pericytes of Multiple Organs Do Not Behave as Mesenchymal Stem Cells In Vivo.

PubMed

Guimarães-Camboa, Nuno; Cattaneo, Paola; Sun, Yunfu; Moore-Morris, Thomas; Gu, Yusu; Dalton, Nancy D; Rockenstein, Edward; Masliah, Eliezer; Peterson, Kirk L; Stallcup, William B; Chen, Ju; Evans, Sylvia M

2017-03-02

Pericytes are widely believed to function as mesenchymal stem cells (MSCs), multipotent tissue-resident progenitors with great potential for regenerative medicine. Cultured pericytes isolated from distinct tissues can differentiate into multiple cell types in vitro or following transplantation in vivo. However, the cell fate plasticity of endogenous pericytes in vivo remains unclear. Here, we show that the transcription factor Tbx18 selectively marks pericytes and vascular smooth muscle cells in multiple organs of adult mouse. Fluorescence-activated cell sorting (FACS)-purified Tbx18-expressing cells behaved as MSCs in vitro. However, lineage-tracing experiments using an inducible Tbx18-CreERT2 line revealed that pericytes and vascular smooth muscle cells maintained their identity in aging and diverse pathological settings and did not significantly contribute to other cell lineages. These results challenge the current view of endogenous pericytes as multipotent tissue-resident progenitors and suggest that the plasticity observed in vitro or following transplantation in vivo arises from artificial cell manipulations ex vivo. Copyright © 2016 Elsevier Inc. All rights reserved.
The pore of the leaf cavity of Azolla species: teat cell differentiation and cell wall projections.

PubMed

Veys, P; Lejeune, A; Van Hove, C

2002-02-01

The differentiation of the specialized secretory teat cells of the leaf cavity pore of Azolla species was investigated at the ultrastructural level with emphasis on their peculiar cell wall projections. The results indicated that the projections are formed as soon as the teat cells complete their differentiation and that their production is principally associated with changes in endoplasmic reticulum profiles. The number of projections increases with the teat cell age and is stimulated under salt and P deficiency stresses. Salt stress also promotes their emergence on Azolla species that under normal conditions do not produce projections. Cytochemical tests on different Azolla species showed that the projection composition is almost identical: proteins, acidic polysaccharides, and pectin are always detected. This study revealed that Azolla teat cell projections differ fundamentally from other types of hitherto described cell wall projections that are considered as remnant structures from cell separation. In contrast, in Azolla teat cells projections are actively produced and compounds are excreted by an exocytotic mechanism. The possible role of the projections in the symbiosis of Azolla spp. with Anabaena azollae is discussed.
Nutrient shielding in clusters of cells

NASA Astrophysics Data System (ADS)

Lavrentovich, Maxim O.; Koschwanez, John H.; Nelson, David R.

2013-06-01

Cellular nutrient consumption is influenced by both the nutrient uptake kinetics of an individual cell and the cells' spatial arrangement. Large cell clusters or colonies have inhibited growth at the cluster's center due to the shielding of nutrients by the cells closer to the surface. We develop an effective medium theory that predicts a thickness ℓ of the outer shell of cells in the cluster that receives enough nutrient to grow. The cells are treated as partially absorbing identical spherical nutrient sinks, and we identify a dimensionless parameter ν that characterizes the absorption strength of each cell. The parameter ν can vary over many orders of magnitude among different cell types, ranging from bacteria and yeast to human tissue. The thickness ℓ decreases with increasing ν, increasing cell volume fraction ϕ, and decreasing ambient nutrient concentration ψ∞. The theoretical results are compared with numerical simulations and experiments. In the latter studies, colonies of budding yeast, Saccharomyces cerevisiae, are grown on glucose media and imaged under a confocal microscope. We measure the growth inside the colonies via a fluorescent protein reporter and compare the experimental and theoretical results for the thickness ℓ.
Six commercially available angiotensin II AT1 receptor antibodies are non-specific.

PubMed

Benicky, Julius; Hafko, Roman; Sanchez-Lemus, Enrique; Aguilera, Greti; Saavedra, Juan M

2012-11-01

Commercially available Angiotensin II AT1 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, six commercially available AT1 receptor antibodies were characterized by established criteria: sc-1173 and sc-579 from Santa Cruz Biotechnology, Inc., AAR-011 from Alomone Labs, Ltd., AB15552 from Millipore, and ab18801 and ab9391 from Abcam. The immunostaining patterns observed were different for every antibody tested, and were unrelated to the presence or absence of AT1 receptors. The antibodies detected a 43 kDa band in western blots, corresponding to the predicted size of the native AT1 receptor. However, identical bands were observed in wild-type mice and in AT1A knock-out mice not expressing the target protein. Moreover, immunoreactivity detected in rat hypothalamic 4B cells not expressing AT1 receptors or transfected with AT1A receptor construct was identical, as revealed by western blotting and immunocytochemistry in cultured 4B cells. Additional prominent immunoreactive bands above and below 43 kDa were observed by western blotting in extracts from tissues of AT1A knock-out and wild-type mice and in 4B cells with or without AT1 receptor expression. In all cases, the patterns of immunoreactivity were independent of the AT1 receptor expression and different for each antibody studied. We conclude that, in our experimental setup, none of the commercially available AT1 receptor antibodies tested met the criteria for specificity and that competitive radioligand binding remains the only reliable approach to study AT1 receptor physiology in the absence of full antibody characterization.
Six Commercially Available Angiotensin II AT1 Receptor Antibodies are Non-specific

PubMed Central

Benicky, Julius; Hafko, Roman; Sanchez-Lemus, Enrique; Aguilera, Greti

2012-01-01

Commercially available Angiotensin II AT1 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, six commercially available AT1 receptor antibodies were characterized by established criteria: sc-1173 and sc-579 from Santa Cruz Biotechnology, Inc., AAR-011 from Alomone Labs, Ltd., AB15552 from Millipore, and ab18801 and ab9391 from Abcam. The immunostaining patterns observed were different for every antibody tested, and were unrelated to the presence or absence of AT1 receptors. The antibodies detected a 43 kDa band in western blots, corresponding to the predicted size of the native AT1 receptor. However, identical bands were observed in wild-type mice and in AT1A knock-out mice not expressing the target protein. Moreover, immunoreactivity detected in rat hypothalamic 4B cells not expressing AT1 receptors or transfected with AT1A receptor construct was identical, as revealed by western blotting and immunocytochemistry in cultured 4B cells. Additional prominent immunoreactive bands above and below 43 kDa were observed by western blotting in extracts from tissues of AT1A knock-out and wild-type mice and in 4B cells with or without AT1 receptor expression. In all cases, the patterns of immunoreactivity were independent of the AT1 receptor expression and different for each antibody studied. We conclude that, in our experimental setup, none of the commercially available AT1 receptor antibodies tested met the criteria for specificity and that competitive radioligand binding remains the only reliable approach to study AT1 receptor physiology in the absence of full antibody characterization. PMID:22843099

Complete genome sequences of avian paramyxovirus type 8 strains goose/Delaware/1053/76 and pintail/Wakuya/20/78

PubMed Central

Paldurai, Anandan; Subbiah, Madhuri; Kumar, Sachin; Collins, Peter L.; Samal, Siba K.

2009-01-01

Complete consensus genome sequences were determined for avian paramyxovirus type 8 (APMV-8) strains goose/Delaware/1053/76 (prototype strain) and pintail/Wakuya/20/78. The genome of each strain is 15,342 nucleotides (nt) long, which follows the “rule of six”. The genome consists of six genes in the order of 3′-N-P/V/W-M-F-HN-L-5′. The genes are flanked on either side by conserved transcription start and stop signals, and have intergenic regions ranging from 1 to 30 nt. The genome contains a 55 nt leader region at the 3′-end and a 171 nt trailer region at the 5′-end. Comparison of sequences of strains Delaware and Wakuya showed nucleotide identity of 96.8% at the genome level and amino acid identities of 99.3%, 96.5%, 98.6%, 99.4%, 98.6% and 99.1% for the predicted N, P, M, F, HN and L proteins, respectively. Both strains grew in embryonated chicken eggs and in primary chicken embryo kidney cells, and 293T cells. Both strains contained only a single basic residue at the cleavage activation site of the F protein and their efficiency of replication in vitro depended on and was augmented by, the presence of exogenous protease in most cell lines. Sequence alignment and phylogenic analysis of the predicted amino acid sequence of APMV-8 strain Delaware proteins with the cognate proteins of other available APMV serotypes showed that APMV-8 is more closely related to APMV-2 and -6 than to APMV-1, -3 and -4. PMID:19341613
Biciliated ependymal cell proliferation contributes to spinal cord growth

PubMed Central

Alfaro-Cervello, Clara; Soriano-Navarro, Mario; Mirzadeh, Zaman; Alvarez-Buylla, Arturo; Garcia-Verdugo, Jose Manuel

2013-01-01

Two neurogenic regions have been described in the adult brain, the lateral ventricle subventricular zone and the dentate gyrus subgranular zone. It has been suggested that neural stem cells also line the central canal of the adult spinal cord. Using transmission and scanning electron microscopy and immunostaining, we describe here the organization and cell types of the central canal epithelium in adult mice. The identity of dividing cells was determined by three-dimensional ultrastructural reconstructions of [3H]thymidine-labeled cells and confocal analysis of bromodeoxyuridine labeling. The most common cell type lining the central canal had two long motile (9+2) cilia and was vimentin+, CD24+, FoxJ1+, Sox2+ and CD133+, but nestin- and glial fibrillary acidic protein (GFAP)-. These biciliated ependymal cells of the central canal (Ecc) resembled E2 cells of the lateral ventricles, but their basal bodies were different from that of E2 or E1 cells. Interestingly, we frequently found Ecc cells with two nuclei and four cilia, suggesting they are formed by incomplete cytokinesis or cell fusion. GFAP+ astrocytes with a single cilium and an orthogonally oriented centriole were also observed. The majority of dividing cells corresponded to biciliated Ecc cells. Central canal proliferation was most common during the active period of spinal cord growth. Pairs of labeled Ecc cells were observed within the central canal in adult mice 2.5 weeks post-labeling. Our work suggests that the vast majority of postnatal dividing cells in the central canal are Ecc cells and their proliferation is associated with the growth of the spinal cord. PMID:22434575
Quantum dot agglomerates in biological media and their characterization by asymmetrical flow field-flow fractionation.

PubMed

Moquin, Alexandre; Neibert, Kevin D; Maysinger, Dusica; Winnik, Françoise M

2015-01-01

The molecular composition of the biological environment of nanoparticles influences their physical properties and changes their pristine physicochemical identity. In order to understand, or predict, the interactions of cells with specific nanoparticles, it is critical to know their size, shape, and agglomeration state not only in their nascent state but also in biological media. Here, we use asymmetrical flow field-flow fractionation (AF4) with on-line multiangle light scattering (MALS), dynamic light scattering (DLS) and UV-Visible absorption detections to determine the relative concentration of isolated nanoparticles and agglomerates in the case of three types of semi-conductor quantum dots (QDs) dispersed in Dulbecco's Modified Eagle Media (DMEM) containing 10% of fetal bovine serum (DMEM-FBS). AF4 analysis also yielded the size and size distribution of the agglomerates as a function of the time of QDs incubation in DMEM-FBS. The preferred modes of internalization of the QDs are assessed for three cell-types, N9 microglia, human hepatocellular carcinoma cells (HepG2) and human embryonic kidney cells (Hek293), by confocal fluorescence imaging of live cells, quantitative determination of the intracellular QD concentration, and flow cytometry. There is an excellent correlation between the agglomeration status of the three types of QDs in DMEM-FBS determined by AF4 analysis and their preferred mode of uptake by the three cell lines, which suggests that AF4 yields an accurate description of the nanoparticles as they encounter cells and advocates its use as a means to characterize particles under evaluation. Copyright © 2014 Elsevier B.V. All rights reserved.
A homologue of the defender against the apoptotic death gene (dad1 )in UV-exposed Chlamydomonas cells is downregulated with the onset of programmed cell death.

PubMed

Moharikar, Swati; D'Souza, Jacinta S; Rao, Basuthkar J

2007-03-01

We report here the isolation of a homologue of the potential anti-apoptotic gene, defender against apoptotic death (dad1 )from Chlamydomonas reinhardtii cells.Using polymerase chain reaction (PCR),we investigated its expression in the execution process of programmed cell death (PCD)in UV-C exposed dying C.reinhardtii cells.Reverse- transcriptase (RT)-PCR showed that C.reinhardtii dad1 amplification was drastically reduced in UV-C exposed dying C.reinhardtii cells.We connect the downregulation of dad1 with the upregulation of apoptosis protease activating factor-1 (APAF-1)and the physiological changes that occur in C.reinhardtii cells upon exposure to 12 J/m 2 UV-C in order to show a reciprocal relationship between proapoptotic and inhibitor of apoptosis factors.The temporal changes indicate a correlation between the onset of cell death and dad1 downregulation.The sequence of the PCR product of the cDNA encoding the dad1 homologue was aligned with the annotated dad1 (C_20215)from the Chlamydomonas database (http://genome.jgi-psf.org:8080/annotator/servlet/jgi.annotation.Annotation?pDb=chlre2); Annotation?pDb=chlre2 );this sequence was found to show 100% identity,both at the nucleotide and amino acid level. The 327 bp transcript showed an open reading frame of 87 amino acid residues.The deduced amino acid sequence of the putative C.reinhardtii DAD1 homologue showed 54% identity with Oryza sativa, 56 identity with Drosophila melanogaster, 66% identity with Xenopus laevis, and 64% identity with Homo sapiens,Sus scrofa,Gallus gallus,Rattus norvegicus and Mus musculus.
Development of an efficient Procedure for Resist Wall Space Experiment

NASA Astrophysics Data System (ADS)

Matsumoto, Shouhei; Kumasaki, Saori; Higuchi, Sayoko; Kirihata, Kuniaki; Inoue, Yasue; Fujie, Miho; Soga, Kouichi; Wakabayashi, Kazuyuki; Hoson, Takayuki

The Resist Wall space experiment aims to examine the role of the cortical microtubule-plasma membrane-cell wall continuum in plant resistance to the gravitational force, thereby clarifying the mechanism of gravity resistance. For this purpose, we will cultivate Arabidopsis mutants defective in organization of cortical microtubules (tua6 ) or synthesis of membrane sterols (hmg1 ) as well as the wild type under microgravity and 1 g conditions in the European Modular Cultivation System on the International Space Station up to reproductive stage, and compare phenotypes on growth and development. We will also analyze cell wall properties and gene expression levels using collected materials. However, the amounts of materials collected will be severely limited, and we should develop an efficient procedure for this space experiment. In the present study, we examined the possibility of analyzing various parameters successively using the identical material. On orbit, plant materials will be fixed with RNAlater solution, kept at 4° C for several days and then frozen in a freezer at -20° C. We first examined whether the cell wall extensibility of inflorescence stems can be measured after RNAlater fixation. The gradient of the cell wall extensibility along inflorescence stems was detected in RNAlater-fixed materials as in methanol-killed ones. The sufficient amounts of RNA to analyze the gene expression were also obtained from the materials after measurement of the cell wall extensibility. Furthermore, the levels and composition of cell wall polysaccharides could be measured using the materials after extraction of RNA. These results show that we can analyze the physical and chemical properties of the cell wall as well as gene expression using the identical material obtained in the space experiments.
ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing.

PubMed

Chen, Xingqi; Shen, Ying; Draper, Will; Buenrostro, Jason D; Litzenburger, Ulrike; Cho, Seung Woo; Satpathy, Ansuman T; Carter, Ava C; Ghosh, Rajarshi P; East-Seletsky, Alexandra; Doudna, Jennifer A; Greenleaf, William J; Liphardt, Jan T; Chang, Howard Y

2016-12-01

Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.
Quantitative Analysis of Human Pluripotency and Neural Specification by In-Depth (Phospho)Proteomic Profiling.

PubMed

Singec, Ilyas; Crain, Andrew M; Hou, Junjie; Tobe, Brian T D; Talantova, Maria; Winquist, Alicia A; Doctor, Kutbuddin S; Choy, Jennifer; Huang, Xiayu; La Monaca, Esther; Horn, David M; Wolf, Dieter A; Lipton, Stuart A; Gutierrez, Gustavo J; Brill, Laurence M; Snyder, Evan Y

2016-09-13

Controlled differentiation of human embryonic stem cells (hESCs) can be utilized for precise analysis of cell type identities during early development. We established a highly efficient neural induction strategy and an improved analytical platform, and determined proteomic and phosphoproteomic profiles of hESCs and their specified multipotent neural stem cell derivatives (hNSCs). This quantitative dataset (nearly 13,000 proteins and 60,000 phosphorylation sites) provides unique molecular insights into pluripotency and neural lineage entry. Systems-level comparative analysis of proteins (e.g., transcription factors, epigenetic regulators, kinase families), phosphorylation sites, and numerous biological pathways allowed the identification of distinct signatures in pluripotent and multipotent cells. Furthermore, as predicted by the dataset, we functionally validated an autocrine/paracrine mechanism by demonstrating that the secreted protein midkine is a regulator of neural specification. This resource is freely available to the scientific community, including a searchable website, PluriProt. Published by Elsevier Inc.
Chromosomal abnormalities in HPV-16-immortalized oral epithelial cells.

PubMed

Oda, D; Bigler, L; Mao, E J; Disteche, C M

1996-09-01

Human papilloma virus (HPV) type 16 has an established association with anogenital carcinoma, and to some extent with human oral squamous cell carcinoma. We hypothesize that HPV type 16 is capable of inducing chromosomal and cell cycle changes in cultured oral epithelial cells. Normal human oral epithelia] cells were immortalized with recombinant retrovirus containing the E6/E7 open reading frames of HPV type 16. These cells have been in culture for more than 350 passages and over 4 years. Flow cytometry demonstrated an average of 42% nuclear aneuploidy in HPV 16-immortalized cells; 16% in normal controls (probably tetrasomy). Cytogenetic analysis demonstrated significant progression of chromosomal abnormalities. Cells at early passage (p10) showed trisomy 20, with no other major changes. At passage 18, trisomy 1q and monosomy 13 were seen in addition to trisomy 20. At passage 61 there were two distinct cell populations ('a' and 'b'), with multiple chromosomal changes including trisomy 5q,14,20 in one line and 7p,9q,llq in the other. Both populations had monosomy 3p, with monosomy 8p in one population and monosomy 13 in the other. At passage 136, the cells were essentially identical to population 'b' of passage 61. At this passage, mutation of the p53 gene was detected at codon 273 of exon 8, with G to T conversion (Arg to Leu). This was absent in the normal cells from which this line was developed. Passage 262 contained the two major cell populations, each with a sub-group with additional chromosomal changes such as 10p monosomy. Cells from passages 217 and 305 were injected into nude mice a year apart. Both failed to produce tumors, as did normal cells. In conclusion, we present an HPV type 16-immortalized oral epithelial cell line (IHGK) with extensive and progressive chromosomal abnormalities, invasive growth in culture and yet no tumor formation in nude mice. We suggest that the question as to whether HPV alone can induce transformation is still open.
The effects of cyclic tensile strain on the organisation and expression of cytoskeletal elements in bovine intervertebral disc cells: an in vitro study.

PubMed

Li, S; Jia, X; Duance, V C; Blain, E J

2011-06-20

It is still relatively unclear how intervertebral disc (IVD) cells sense a mechanical stimulus and convert this signal into a biochemical response. Previous studies demonstrated that the cytoskeletal elements are mechano-responsive in many cell types and may contribute to mechano-signalling pathways. The objective of this study was to determine the response of cells from the outer annulus fibrosus (OAF) to physiological levels of cyclic tensile strain; further, cells from the nucleus pulposus (NP) were also subjected to an identical loading regime to compare biological responses across the IVD populations. We determined whether the organisation and expression of the major cytoskeletal elements and their associated accessory proteins are responsive to mechanical stimulation in these cells, and whether these changes correlated with either a catabolic or anabolic phenotype. OAF and NP cells from immature bovine IVD were seeded onto Flexcell® type I collagen coated plates. Cells were subjected to cyclic tensile strain (10 %, 1 Hz) for 60 minutes. Post-loading, cells were processed for immunofluorescence microscopy, RNA extracted for quantitative PCR and protein extracted for Western blotting analysis. F-actin reorganisation was evident in OAF and NP cells subjected to tensile strain; strain induced β-actin at the transcriptional and translational level in OAF cells. β-tubulin mRNA and protein synthesis increased in strained OAF cells, but vimentin expression was significantly inhibited. Cytoskeletal element organisation and expression were less responsive to strain in NP cells. Tensile strain increased type I collagen and differentially regulated extracellular matrix (ECM)-degrading enzymes' mRNA levels in OAF cells. Strain induced type II collagen transcription in NP cells, but had no effect on the transcription of any other genes analysed. Tensile strain induces different mechano-responses in the organisation and/or expression of cytoskeletal elements and on markers of IVD metabolism. Differential mechano-regulation of anabolic and catabolic ECM components in the OAF and NP populations reflects their respective mechanical environments in situ.
30 CFR 41.11 - Notification by operator.

Code of Federal Regulations, 2010 CFR

2010-07-01

... ADMINISTRATIVE REQUIREMENTS NOTIFICATION OF LEGAL IDENTITY Notification of Legal Identity § 41.11 Notification by... of the legal identity of the operator in accordance with the applicable provisions of paragraph (b... proprietorship, partnership, or corporation, the operator shall state: (1) The nature and type, or legal identity...
30 CFR 41.11 - Notification by operator.

Code of Federal Regulations, 2011 CFR

2011-07-01

... ADMINISTRATIVE REQUIREMENTS NOTIFICATION OF LEGAL IDENTITY Notification of Legal Identity § 41.11 Notification by... of the legal identity of the operator in accordance with the applicable provisions of paragraph (b... proprietorship, partnership, or corporation, the operator shall state: (1) The nature and type, or legal identity...
Proteome Analysis of Thyroid Cancer Cells After Long-Term Exposure to a Random Positioning Machine

NASA Astrophysics Data System (ADS)

Pietsch, Jessica; Bauer, Johann; Weber, Gerhard; Nissum, Mikkel; Westphal, Kriss; Egli, Marcel; Grosse, Jirka; Schönberger, Johann; Eilles, Christoph; Infanger, Manfred; Grimm, Daniela

2011-11-01

Annulling gravity during cell culturing triggers various types of cells to change their protein expression in a time dependent manner. We therefore decided to determine gravity sensitive proteins and their period of sensitivity to the effects of gravity. In this study, thyroid cancer cells of the ML-1 cell line were cultured under normal gravity (1 g) or in a random positioning machine (RPM), which simulated near weightlessness for 7 and 11 days. Cells were then sonicated and proteins released into the supernatant were separated from those that remained attached to the cell fragments. Subsequently, both types of proteins were fractionated by free-flow isoelectric focussing (FF-IEF). The fractions obtained were further separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to which comparable FF-IEF fractions derived from cells cultured either under 1 g or on the RPM had been applied side by side. The separation resulted in pairs of lanes, on which a number of identical bands were observed. Selected gel pieces were excised and their proteins determined by mass spectrometry. Equal proteins from cells cultured under normal gravity and the RPM, respectively, were detected in comparable gel pieces. However, many of these proteins had received different Mascot scores. Quantifying heat shock cognate 71 kDa protein, glutathione S-transferase P, nucleoside diphosphate kinase A and annexin-2 by Western blotting using whole cell lysates indicated usefulness of Mascot scores for selecting the most efficient antibodies.
Size, Shape, and Arrangement of Cellulose Microfibril in Higher Plant Cell Walls

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

Ding, S. Y.

2013-01-01

Plant cell walls from maize (Zea mays L.) are imaged using atomic force microscopy (AFM) at the sub-nanometer resolution. We found that the size and shape of fundamental cellulose elementary fibril (CEF) is essentially identical in different cell wall types, i.e., primary wall (PW), parenchyma secondary wall (pSW), and sclerenchyma secondary wall (sSW), which is consistent with previously proposed 36-chain model (Ding et al., 2006, J. Agric. Food Chem.). The arrangement of individual CEFs in these wall types exhibits two orientations. In PW, CEFs are horizontally associated through their hydrophilic faces, and the planar faces are exposed, forming ribbon-like macrofibrils.more » In pSW and sSW, CEFs are vertically oriented, forming layers, in which hemicelluloses are interacted with the hydrophobic faces of the CEF and serve as spacers between CEFs. Lignification occurs between CEF-hemicelluloses layers in secondary walls. Furthermore, we demonstrated quantitative analysis of plant cell wall accessibility to and digestibility by different cellulase systems at real-time using chemical imaging (e.g., stimulated Raman scattering) and fluorescence microscopy of labeled cellulases (Ding et al., 2012, Science, in press).« less
Mitochondrial depolarization in yeast zygotes inhibits clonal expansion of selfish mtDNA.

PubMed

Karavaeva, Iuliia E; Golyshev, Sergey A; Smirnova, Ekaterina A; Sokolov, Svyatoslav S; Severin, Fedor F; Knorre, Dmitry A

2017-04-01

Non-identical copies of mitochondrial DNA (mtDNA) compete with each other within a cell and the ultimate variant of mtDNA present depends on their relative replication rates. Using yeast Saccharomyces cerevisiae cells as a model, we studied the effects of mitochondrial inhibitors on the competition between wild-type mtDNA and mutant selfish mtDNA in heteroplasmic zygotes. We found that decreasing mitochondrial transmembrane potential by adding uncouplers or valinomycin changes the competition outcomes in favor of the wild-type mtDNA. This effect was significantly lower in cells with disrupted mitochondria fission or repression of the autophagy-related genes ATG8 , ATG32 or ATG33 , implying that heteroplasmic zygotes activate mitochondrial degradation in response to the depolarization. Moreover, the rate of mitochondrially targeted GFP turnover was higher in zygotes treated with uncoupler than in haploid cells or untreated zygotes. Finally, we showed that vacuoles of zygotes with uncoupler-activated autophagy contained DNA. Taken together, our data demonstrate that mitochondrial depolarization inhibits clonal expansion of selfish mtDNA and this effect depends on mitochondrial fission and autophagy. These observations suggest an activation of mitochondria quality control mechanisms in heteroplasmic yeast zygotes. © 2017. Published by The Company of Biologists Ltd.
Resolving stem and progenitor cells in the adult mouse incisor through gene co-expression analysis

PubMed Central

Seidel, Kerstin; Marangoni, Pauline; Tang, Cynthia; Houshmand, Bahar; Du, Wen; Maas, Richard L; Murray, Steven; Oldham, Michael C; Klein, Ophir D

2017-01-01

Investigations into stem cell-fueled renewal of an organ benefit from an inventory of cell type-specific markers and a deep understanding of the cellular diversity within stem cell niches. Using the adult mouse incisor as a model for a continuously renewing organ, we performed an unbiased analysis of gene co-expression relationships to identify modules of co-expressed genes that represent differentiated cells, transit-amplifying cells, and residents of stem cell niches. Through in vivo lineage tracing, we demonstrated the power of this approach by showing that co-expression module members Lrig1 and Igfbp5 define populations of incisor epithelial and mesenchymal stem cells. We further discovered that two adjacent mesenchymal tissues, the periodontium and dental pulp, are maintained by distinct pools of stem cells. These findings reveal novel mechanisms of incisor renewal and illustrate how gene co-expression analysis of intact biological systems can provide insights into the transcriptional basis of cellular identity. DOI: http://dx.doi.org/10.7554/eLife.24712.001 PMID:28475038
Laser Capture and Single Cell Genotyping from Frozen Tissue Sections.

PubMed

Kroneis, Thomas; Ye, Jody; Gillespie, Kathleen

2016-01-01

There is an increasing requirement for genetic analysis of individual cells from tissue sections. This is particularly the case for analysis of tumor cells but is also a requirement for analysis of cells in pancreas from individuals with type 1 diabetes where there is evidence of viral infection or in the analysis of chimerism in pancreas; either post-transplant or as a result of feto-maternal cell transfer.This protocol describes a strategy to isolate cells using laser microdissection and to run a 17plex PCR to discriminate between cells of haplo-identical origin (i.e., fetal and maternal cells) in pancreas tissue but other robust DNA tests could be used. In short, snap-frozen tissues are cryo-sectioned and mounted onto membrane-coated slides. Target cells are harvested from the tissue sections by laser microdissection and pressure catapulting (LMPC) prior to DNA profiling. This is based on amplification of highly repetitive yet stably inherited loci (short tandem repeats, STR) as well as the amelogenin locus for sex determination and separation of PCR products by capillary electrophoresis.
A Factor Linking Floral Organ Identity and Growth Revealed by Characterization of the Tomato Mutant unfinished flower development (ufd).

PubMed

Poyatos-Pertíñez, Sandra; Quinet, Muriel; Ortíz-Atienza, Ana; Yuste-Lisbona, Fernando J; Pons, Clara; Giménez, Estela; Angosto, Trinidad; Granell, Antonio; Capel, Juan; Lozano, Rafael

2016-01-01

Floral organogenesis requires coordinated interactions between genes specifying floral organ identity and those regulating growth and size of developing floral organs. With the aim to isolate regulatory genes linking both developmental processes (i.e., floral organ identity and growth) in the tomato model species, a novel mutant altered in the formation of floral organs was further characterized. Under normal growth conditions, floral organ primordia of mutant plants were correctly initiated, however, they were unable to complete their development impeding the formation of mature and fertile flowers. Thus, the growth of floral buds was blocked at an early stage of development; therefore, we named this mutant as unfinished flower development ( ufd ). Genetic analysis performed in a segregating population of 543 plants showed that the abnormal phenotype was controlled by a single recessive mutation. Global gene expression analysis confirmed that several MADS-box genes regulating floral identity as well as other genes participating in cell division and different hormonal pathways were affected in their expression patterns in ufd mutant plants. Moreover, ufd mutant inflorescences showed higher hormone contents, particularly ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and strigol compared to wild type. Such results indicate that UFD may have a key function as positive regulator of the development of floral primordia once they have been initiated in the four floral whorls. This function should be performed by affecting the expression of floral organ identity and growth genes, together with hormonal signaling pathways.
DISTAG/TBCCd1 Is Required for Basal Cell Fate Determination in Ectocarpus[OPEN

PubMed Central

Godfroy, Olivier; Uji, Toshiki; Nagasato, Chikako; Colin, Sebastien; Mignerot, Laure; Motomura, Taizo

2017-01-01

Brown algae are one of the most developmentally complex groups within the eukaryotes. As in many land plants and animals, their main body axis is established early in development, when the initial cell gives rise to two daughter cells that have apical and basal identities, equivalent to shoot and root identities in land plants, respectively. We show here that mutations in the Ectocarpus DISTAG (DIS) gene lead to loss of basal structures during both the gametophyte and the sporophyte generations. Several abnormalities were observed in the germinating initial cell in dis mutants, including increased cell size, disorganization of the Golgi apparatus, disruption of the microtubule network, and aberrant positioning of the nucleus. DIS encodes a TBCCd1 protein, which has a role in internal cell organization in animals, Chlamydomonas reinhardtii, and trypanosomes. Our study highlights the key role of subcellular events within the germinating initial cell in the determination of apical/basal cell identities in a brown alga and emphasizes the remarkable functional conservation of TBCCd1 in regulating internal cell organization across extremely distant eukaryotic groups. PMID:29208703
Characterization and classification of one new cytoplasmic male sterility (CMS) line based on morphological, cytological and molecular markers in non-heading Chinese cabbage (Brassica rapa L.).

PubMed

Heng, Shuangping; Shi, Dianyi; Hu, Zhenhua; Huang, Tao; Li, Jinping; Liu, Liyan; Xia, Chunxiu; Yuan, Zhenzhen; Xu, Yuejin; Fu, Tingdong; Wan, Zhengjie

2015-09-01

A new non-heading Chinese cabbage CMS line M119A was characterized and specific molecular markers were developed to classify different CMS types. One new non-heading Chinese cabbage (Brassica rapa L.) cytoplasmic male sterile (CMS) line M119A was obtained by interspecific crosses between the recently discovered hau CMS line of Brassica juncea and B. rapa. Furthermore, the line was characterized and compared with other five isonuclear-alloplasmic CMS lines. The M119A line produced six stamens without pollen and only two stamen fused together in fewer flowers. Tissue section indicated that anther abortion in M119A may have occurred during differentiation of the archesporial cells without pollen sac. All the six CMS lines were grouped into three types based on the presence of three PCR fragments of 825, 465 and 772 bp amplified with different mitochondrial genes specific primers. The 825-bp fragment was amplified both in 09-10A and H201A using the specific primer pair P-orf224-atp6, and showed 100 % identity with the mitochondrial gene of pol CMS. The 465-bp fragment was amplified in 30A and 105A using the primer pair P-orf138 and shared 100 % identity with the mitochondrial gene of ogu CMS. The 772-bp fragment was amplified in M119A and H203A using the primer pair P-orf288 and showed 100 % identity with the mitochondrial gene of hau CMS. Therefore, these markers could efficiently distinguish different types of isonuclear-alloplasmic CMS lines of non-heading Chinese cabbage, which were useful for improving the efficiency of cross-breeding and heterosis utilization in cruciferous vegetables.
Comparative transcriptome analysis in induced neural stem cells reveals defined neural cell identities in vitro and after transplantation into the adult rodent brain.

PubMed

Hallmann, Anna-Lena; Araúzo-Bravo, Marcos J; Zerfass, Christina; Senner, Volker; Ehrlich, Marc; Psathaki, Olympia E; Han, Dong Wook; Tapia, Natalia; Zaehres, Holm; Schöler, Hans R; Kuhlmann, Tanja; Hargus, Gunnar

2016-05-01

Reprogramming technology enables the production of neural progenitor cells (NPCs) from somatic cells by direct transdifferentiation. However, little is known on how neural programs in these induced neural stem cells (iNSCs) differ from those of alternative stem cell populations in vitro and in vivo. Here, we performed transcriptome analyses on murine iNSCs in comparison to brain-derived neural stem cells (NSCs) and pluripotent stem cell-derived NPCs, which revealed distinct global, neural, metabolic and cell cycle-associated marks in these populations. iNSCs carried a hindbrain/posterior cell identity, which could be shifted towards caudal, partially to rostral but not towards ventral fates in vitro. iNSCs survived after transplantation into the rodent brain and exhibited in vivo-characteristics, neural and metabolic programs similar to transplanted NSCs. However, iNSCs vastly retained caudal identities demonstrating cell-autonomy of regional programs in vivo. These data could have significant implications for a variety of in vitro- and in vivo-applications using iNSCs. Copyright © 2016 Roslin Cells Ltd. Published by Elsevier B.V. All rights reserved.

Centromere Transcription: Means and Motive.

PubMed

Duda, Zachary; Trusiak, Sarah; O'Neill, Rachel

2017-01-01

The chromosome biology field at large has benefited from studies of the cell cycle components, protein cascades and genomic landscape that are required for centromere identity, assembly and stable transgenerational inheritance. Research over the past 20 years has challenged the classical descriptions of a centromere as a stable, unmutable, and transcriptionally silent chromosome component. Instead, based on studies from a broad range of eukaryotic species, including yeast, fungi, plants, and animals, the centromere has been redefined as one of the more dynamic areas of the eukaryotic genome, requiring coordination of protein complex assembly, chromatin assembly, and transcriptional activity in a cell cycle specific manner. What has emerged from more recent studies is the realization that the transcription of specific types of nucleic acids is a key process in defining centromere integrity and function. To illustrate the transcriptional landscape of centromeres across eukaryotes, we focus this review on how transcripts interact with centromere proteins, when in the cell cycle centromeric transcription occurs, and what types of sequences are being transcribed. Utilizing data from broadly different organisms, a picture emerges that places centromeric transcription as an integral component of centromere function.
Synthetic virus seeds for improved vaccine safety: Genetic reconstruction of poliovirus seeds for a PER.C6 cell based inactivated poliovirus vaccine.

PubMed

Sanders, Barbara P; Edo-Matas, Diana; Papic, Natasa; Schuitemaker, Hanneke; Custers, Jerome H H V

2015-10-13

Safety of vaccines can be compromised by contamination with adventitious agents. One potential source of adventitious agents is a vaccine seed, typically derived from historic clinical isolates with poorly defined origins. Here we generated synthetic poliovirus seeds derived from chemically synthesized DNA plasmids encoding the sequence of wild-type poliovirus strains used in marketed inactivated poliovirus vaccines. The synthetic strains were phenotypically identical to wild-type polioviruses as shown by equivalent infectious titers in culture supernatant and antigenic content, even when infection cultures are scaled up to 10-25L bioreactors. Moreover, the synthetic seeds were genetically stable upon extended passaging on the PER.C6 cell culture platform. Use of synthetic seeds produced on the serum-free PER.C6 cell platform ensures a perfectly documented seed history and maximum control over starting materials. It provides an opportunity to maximize vaccine safety which increases the prospect of a vaccine end product that is free from adventitious agents. Copyright © 2015 Elsevier Ltd. All rights reserved.
Characterization of herpes simplex virus type 2 latency-associated transcription in human sacral ganglia and in cell culture.

PubMed

Croen, K D; Ostrove, J M; Dragovic, L; Straus, S E

1991-01-01

The ability of herpes simplex virus type 2 (HSV-2) to establish latency in and reactivate from sacral dorsal root sensory ganglia is the basis for recurrent genital herpes. The expression of HSV-2 genes in latently infected human sacral ganglia was investigated by in situ hybridization. Hybridizations with a probe from the long repeat region of HSV-2 revealed strong nuclear signals overlying neurons in sacral ganglia from five of nine individuals. The RNA detected overlaps with the transcript for infected cell protein O but in the opposite, or "anti-sense," orientation. These observations mimic those made previously with HSV-1 in human trigeminal ganglia and confirm the recent findings during latency in HSV-2-infected mice and guinea pigs. Northern hybridization of RNA from infected Vero cells showed that an HSV-2 latency-associated transcript was similar in size to the larger (1.85 kb) latency transcript of HSV-1. Thus, HSV-1 and HSV-2 latency in human sensory ganglia are similar, if not identical, in terms of their cellular localization and pattern of transcription.
Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model.

PubMed

Bednar, Kyle J; Shanina, Elena; Ballet, Romain; Connors, Edward P; Duan, Shiteng; Juan, Joana; Arlian, Britni M; Kulis, Michael D; Butcher, Eugene C; Fung-Leung, Wai-Ping; Rao, Tadimeti S; Paulson, James C; Macauley, Matthew S

2017-11-01

CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca 2+ ) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22 -/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22 -/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22 -/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22. Copyright © 2017 by The American Association of Immunologists, Inc.
Role of Gender, Sex Role Identity, and Type A Behavior in Anger Expression and Mental Health Functioning.

ERIC Educational Resources Information Center

Kopper, Beverly A.

1993-01-01

Investigated relationship of gender, sex role identity, Type A behavior to multiple dimensions of anger expression and mental health functioning among 407 female and 222 male college students. Found significant multivariate effects for sex role and behavior pattern type for anger expression. Significant gender differences were not observed.…
Identities, Social Representations and Critical Thinking

ERIC Educational Resources Information Center

Lopez-Facal, Ramon; Jimenez-Aleixandre, Maria Pilar

2009-01-01

This comment on L. Simonneaux and J. Simonneaux paper focuses on the role of "identities" in dealing with socio-scientific issues. We argue that there are two types of identities (social representations) influencing the students' positions: On the one hand their social representations of the bears' and wolves' identities as belonging to…
A mechanistic link between gene regulation and genome architecture in mammalian development.

PubMed

Bonora, Giancarlo; Plath, Kathrin; Denholtz, Matthew

2014-08-01

The organization of chromatin within the nucleus and the regulation of transcription are tightly linked. Recently, mechanisms underlying this relationship have been uncovered. By defining the organizational hierarchy of the genome, determining changes in chromatin organization associated with changes in cell identity, and describing chromatin organization within the context of linear genomic features (such as chromatin modifications and transcription factor binding) and architectural proteins (including Cohesin, CTCF, and Mediator), a new paradigm in genome biology was established wherein genomes are organized around gene regulatory factors that govern cell identity. As such, chromatin organization plays a central role in establishing and maintaining cell state during development, with gene regulation and genome organization being mutually dependent effectors of cell identity. Copyright © 2014 Elsevier Ltd. All rights reserved.
Implementing the LIM code: the structural basis for cell type-specific assembly of LIM-homeodomain complexes

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

Bhati, Mugdha; Lee, Christopher; Nancarrow, Amy L.

2008-09-03

LIM-homeodomain (LIM-HD) transcription factors form a combinatorial 'LIM code' that contributes to the specification of cell types. In the ventral spinal cord, the binary LIM homeobox protein 3 (Lhx3)/LIM domain-binding protein 1 (Ldb1) complex specifies the formation of V2 interneurons. The additional expression of islet-1 (Isl1) in adjacent cells instead specifies the formation of motor neurons through assembly of a ternary complex in which Isl1 contacts both Lhx3 and Ldb1, displacing Lhx3 as the binding partner of Ldb1. However, little is known about how this molecular switch occurs. Here, we have identified the 30-residue Lhx3-binding domain on Isl1 (Isl1{sub LBD}).more » Although the LIM interaction domain of Ldb1 (Ldb1{sub LID}) and Isl1{sub LBD} share low levels of sequence homology, X-ray and NMR structures reveal that they bind Lhx3 in an identical manner, that is, Isl1{sub LBD} mimics Ldb1{sub LID}. These data provide a structural basis for the formation of cell type-specific protein-protein interactions in which unstructured linear motifs with diverse sequences compete to bind protein partners. The resulting alternate protein complexes can target different genes to regulate key biological events.« less
Two subpopulations of Crocosphaera watsonii have distinct distributions in the North and South Pacific.

PubMed

Bench, Shellie R; Frank, Ildiko; Robidart, Julie; Zehr, Jonathan P

2016-02-01

Crocosphaera watsonii is a unicellular nitrogen (N2)-fixing cyanobacterium with ecological importance in oligotrophic oceans. In cultivated strains there are two phenotypes of C. watsonii (large and small cells) with differences that could differentially impact biogeochemical processes. Recent work has shown the phenotypes diverged through loss or addition of type-specific genes in a fraction of their genomes, whereas the rest of the genomes were maintained at 99-100% DNA identity. Previous molecular assays for C. watsonii abundances targeted the conserved regions and therefore could not differentiate between phenotypes, so their relative distributions in natural communities were unknown. To determine phenotype distributions, this study developed and applied type-specific quantitative polymerase chain reaction assays to samples from the North and South Pacific. Abundances of both Crocosphaera types declined sharply with depth between 45 and 75 m in both sites. In surface water small cells were 10-100 times more abundant than large cells in the N. Pacific, whereas in the S. Pacific the two phenotypes were nearly equal. Evidence for large cell aggregation was only found in N. Pacific samples. The differences in C. watsonii sub-populations in the North and South Pacific have direct implications for biogeochemistry and carbon export in oligotrophic gyres. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.
Uptake and bio-reactivity of polystyrene nanoparticles is affected by surface modifications, ageing and LPS adsorption: in vitro studies on neural tissue cells

NASA Astrophysics Data System (ADS)

Murali, Kumarasamy; Kenesei, Kata; Li, Yang; Demeter, Kornél; Környei, Zsuzsanna; Madarász, Emilia

2015-02-01

Because of their capacity of crossing an intact blood-brain barrier and reaching the brain through an injured barrier or via the nasal epithelium, nanoparticles have been considered as vehicles to deliver drugs and as contrast materials for brain imaging. The potential neurotoxicity of nanoparticles, however, is not fully explored. Using particles with a biologically inert polystyrene core material, we investigated the role of the chemical composition of particle surfaces in the in vitro interaction with different neural cell types. PS NPs within a size-range of 45-70 nm influenced the metabolic activity of cells depending on the cell-type, but caused toxicity only at extremely high particle concentrations. Neurons did not internalize particles, while microglial cells ingested a large amount of carboxylated but almost no PEGylated NPs. PEGylation reduced the protein adsorption, toxicity and cellular uptake of NPs. After storage (shelf-life >6 months), the toxicity and cellular uptake of NPs increased. The altered biological activity of ``aged'' NPs was due to particle aggregation and due to the adsorption of bioactive compounds on NP surfaces. Aggregation by increasing the size and sedimentation velocity of NPs results in increased cell-targeted NP doses. The ready endotoxin adsorption which cannot be prevented by PEG coating, can render the particles toxic. The age-dependent changes in otherwise harmless NPs could be the important sources for variability in the effects of NPs, and could explain the contradictory data obtained with ``identical'' NPs.Because of their capacity of crossing an intact blood-brain barrier and reaching the brain through an injured barrier or via the nasal epithelium, nanoparticles have been considered as vehicles to deliver drugs and as contrast materials for brain imaging. The potential neurotoxicity of nanoparticles, however, is not fully explored. Using particles with a biologically inert polystyrene core material, we investigated the role of the chemical composition of particle surfaces in the in vitro interaction with different neural cell types. PS NPs within a size-range of 45-70 nm influenced the metabolic activity of cells depending on the cell-type, but caused toxicity only at extremely high particle concentrations. Neurons did not internalize particles, while microglial cells ingested a large amount of carboxylated but almost no PEGylated NPs. PEGylation reduced the protein adsorption, toxicity and cellular uptake of NPs. After storage (shelf-life >6 months), the toxicity and cellular uptake of NPs increased. The altered biological activity of ``aged'' NPs was due to particle aggregation and due to the adsorption of bioactive compounds on NP surfaces. Aggregation by increasing the size and sedimentation velocity of NPs results in increased cell-targeted NP doses. The ready endotoxin adsorption which cannot be prevented by PEG coating, can render the particles toxic. The age-dependent changes in otherwise harmless NPs could be the important sources for variability in the effects of NPs, and could explain the contradictory data obtained with ``identical'' NPs. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr06849a
Specification of spatial identities of cerebellar neuron progenitors by ptf1a and atoh1 for proper production of GABAergic and glutamatergic neurons.

PubMed

Yamada, Mayumi; Seto, Yusuke; Taya, Shinichiro; Owa, Tomoo; Inoue, Yukiko U; Inoue, Takayoshi; Kawaguchi, Yoshiya; Nabeshima, Yo-Ichi; Hoshino, Mikio

2014-04-02

In the cerebellum, the bHLH transcription factors Ptf1a and Atoh1 are expressed in distinct neuroepithelial regions, the ventricular zone (VZ) and the rhombic lip (RL), and are required for producing GABAergic and glutamatergic neurons, respectively. However, it is unclear whether Ptf1a or Atoh1 is sufficient for specifying GABAergic or glutamatergic neuronal fates. To test this, we generated two novel knock-in mouse lines, Ptf1a(Atoh1) and Atoh1(Ptf1a), that are designed to express Atoh1 and Ptf1a ectopically in the VZ and RL, respectively. In Ptf1a(Atoh1) embryos, ectopically Atoh1-expressing VZ cells produced glutamatergic neurons, including granule cells and deep cerebellar nuclei neurons. Correspondingly, in Atoh1(Ptf1a) animals, ectopically Ptf1a-expressing RL cells produced GABAergic populations, such as Purkinje cells and GABAergic interneurons. Consistent results were also obtained from in utero electroporation of Ptf1a or Atoh1 into embryonic cerebella, suggesting that Ptf1a and Atoh1 are essential and sufficient for GABAergic versus glutamatergic specification in the neuroepithelium. Furthermore, birthdating analyses with BrdU in the knock-in mice or with electroporation studies showed that ectopically produced fate-changed neuronal types were generated at temporal schedules closely simulating those of the wild-type RL and VZ, suggesting that the VZ and RL share common temporal information. Observations of knock-in brains as well as electroporated brains revealed that Ptf1a and Atoh1 mutually negatively regulate their expression, probably contributing to formation of non-overlapping neuroepithelial domains. These findings suggest that Ptf1a and Atoh1 specify spatial identities of cerebellar neuron progenitors in the neuroepithelium, leading to appropriate production of GABAergic and glutamatergic neurons, respectively.
Identification and characterization of the p35 gene of Bombyx mori nuclear polyhedrosis virus that prevents virus-induced apoptosis.

PubMed Central

Kamita, S G; Majima, K; Maeda, S

1993-01-01

Nucleotide sequence analysis of the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome revealed the existence of a gene homologous to the p35 gene of Autographa californica NPV (AcNPV), which has been shown to prevent virus-induced apoptosis. The BmNPV p35 gene showed 96.1% nucleotide and 89.6% predicted amino acid sequence identity to the AcNPV p35 gene. A mutant BmNPV (BmP35Z) lacking a functional p35 gene induced apoptosis-like cell degradation in infected BmN cells. However, unlike the p35-deleted AcNPV mutant (vAcAnh), BmP35Z replicated normally and produced polyhedral inclusion bodies. The patterns of protein synthesis and the percentages of viable BmN cells remaining following infection with either wild-type BmNPV or BmP35Z were nearly identical. BmP35Z also replicated in silkworm larvae without showing any apparent apoptotic response in infected hemocytes, fat body, or other tissues. Time to death of larvae infected with BmP35Z was similar to that for wild-type-infected larvae, and significant numbers of polyhedral inclusion bodies were produced. These results indicate that viral factors (or genes) other than p35 or host cell factors play a role in inducing, accelerating, or interfering with apoptotic processes. The evolution of baculovirus genomes is also discussed with reference to comparative analysis of the p35 and p94 gene sequences. The p94 gene is found immediately upstream of p35 in AcNPV; in BmNPV, however, the p94 gene was nearly completely missing, presumably because of large deletions in a BmNPV ancestor virus having a gene similar to the AcNPV p94 gene. Images PMID:8416377
Differential transcriptional regulation of orthologous dps genes from two closely related heterocyst-forming cyanobacteria.

PubMed

Li, Xin; Sandh, Gustaf; Nenninger, Anja; Muro-Pastor, Alicia M; Stensjö, Karin

2015-03-01

In cyanobacteria, DNA-binding proteins from starved cells (Dps) play an important role in the cellular response to oxidative and nutritional stresses. In this study, we have characterized the cell-type specificity and the promoter regions of two orthologous dps genes, Npun_R5799 in Nostoc punctiforme and alr3808 in Anabaena sp. PCC 7120. A transcriptional start site (TSS), identical in location to the previously identified proximal TSS of alr3808, was identified for Npun_R5799 under both combined nitrogen supplemented and N2-fixing growth conditions. However, only alr3808 was also transcribed from a second distal TSS. Sequence homologies suggest that the promoter region containing the distal TSS is not conserved upstream of orthologous genes among heterocyst-forming cyanobacteria. The analysis of promoter GFP-reporter strains showed a different role in governing cell-type specificity between the proximal and distal promoter of alr3808. We here confirmed the heterocyst specificity of the distal promoter of alr3808 and described a very early induction of its expression during proheterocyst differentiation. In contrast, the complete promoters of both genes were active in all cells. Even though Npun_R5799 and alr3808 are orthologs, the regulation of their respective expression differs, indicating distinctions in the function of these cyanobacterial Dps proteins depending on the strain and cell type. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Establishment of a canine model of human type 2 diabetes mellitus by overexpressing phosphoenolypyruvate carboxykinase.

PubMed

Jeong, Yeon Woo; Lee, Geun-Shik; Kim, Joung Joo; Park, Sun Woo; Ko, Kyeong Hee; Kang, Mina; Kim, Yu Kyung; Jung, Eui-Man; Hyun, Sang Hwan; Shin, Taeyoung; Jeung, Eui-Bae; Hwang, Woo Suk

2012-08-01

Dogs are useful models for studying human metabolic diseases such as type 2 diabetes mellitus due to similarities in physiology, anatomy and life styles with humans. Somatic cell nuclear transfer (SCNT) facilitates the production of transgenic dogs. In this study, we generated transgenic dogs expressing the phosphoenolpyruvate carboxykinase (PEPCK) gene, which is closely involved in the pathogenesis of type 2 diabetes mellitus. In addition, we assessed the cloning efficiency associated with adult or fetal (cloned or natural mating) fibroblasts as a nuclear source. Cloning efficiency was determined by the fusion, pregnancy and cloning rates. The fusion rates were significantly high for fibroblasts from cloned fetuses, but the pregnancy and cloning rates were relatively high for cells from normal fetuses. Based on these data, fetal fibroblasts were selected as the nuclear donor for SCNT and genetically engineered to overexpress the PEPCK gene and dual selection marker genes controlled by the PEPCK promoter. The transgenic cells were introduced into oocytes and transferred into five recipient dogs, resulting in two pregnancies. Finally, three puppies were born and confirmed by microsatellite analysis to be genetically identical to the donor. One puppy successfully overexpressed PEPCK mRNA and protein in the liver. This canine disease model may be useful for studying the pathogenesis and/or therapeutic targets of type 2 diabetes mellitus.
Developmental and Subcellular Organization of Single-Cell C₄ Photosynthesis in Bienertia sinuspersici Determined by Large-Scale Proteomics and cDNA Assembly from 454 DNA Sequencing.

PubMed

Offermann, Sascha; Friso, Giulia; Doroshenk, Kelly A; Sun, Qi; Sharpe, Richard M; Okita, Thomas W; Wimmer, Diana; Edwards, Gerald E; van Wijk, Klaas J

2015-05-01

Kranz C4 species strictly depend on separation of primary and secondary carbon fixation reactions in different cell types. In contrast, the single-cell C4 (SCC4) species Bienertia sinuspersici utilizes intracellular compartmentation including two physiologically and biochemically different chloroplast types; however, information on identity, localization, and induction of proteins required for this SCC4 system is currently very limited. In this study, we determined the distribution of photosynthesis-related proteins and the induction of the C4 system during development by label-free proteomics of subcellular fractions and leaves of different developmental stages. This was enabled by inferring a protein sequence database from 454 sequencing of Bienertia cDNAs. Large-scale proteome rearrangements were observed as C4 photosynthesis developed during leaf maturation. The proteomes of the two chloroplasts are different with differential accumulation of linear and cyclic electron transport components, primary and secondary carbon fixation reactions, and a triose-phosphate shuttle that is shared between the two chloroplast types. This differential protein distribution pattern suggests the presence of a mRNA or protein-sorting mechanism for nuclear-encoded, chloroplast-targeted proteins in SCC4 species. The combined information was used to provide a comprehensive model for NAD-ME type carbon fixation in SCC4 species.
Diversity in spatial scope of contrast adaptation among mouse retinal ganglion cells.

PubMed

Khani, Mohammad Hossein; Gollisch, Tim

2017-12-01

Retinal ganglion cells adapt to changes in visual contrast by adjusting their response kinetics and sensitivity. While much work has focused on the time scales of these adaptation processes, less is known about the spatial scale of contrast adaptation. For example, do small, localized contrast changes affect a cell's signal processing across its entire receptive field? Previous investigations have provided conflicting evidence, suggesting that contrast adaptation occurs either locally within subregions of a ganglion cell's receptive field or globally over the receptive field in its entirety. Here, we investigated the spatial extent of contrast adaptation in ganglion cells of the isolated mouse retina through multielectrode-array recordings. We applied visual stimuli so that ganglion cell receptive fields contained regions where the average contrast level changed periodically as well as regions with constant average contrast level. This allowed us to analyze temporal stimulus integration and sensitivity separately for stimulus regions with and without contrast changes. We found that the spatial scope of contrast adaptation depends strongly on cell identity, with some ganglion cells displaying clear local adaptation, whereas others, in particular large transient ganglion cells, adapted globally to contrast changes. Thus, the spatial scope of contrast adaptation in mouse retinal ganglion cells appears to be cell-type specific. This could reflect differences in mechanisms of contrast adaptation and may contribute to the functional diversity of different ganglion cell types. NEW & NOTEWORTHY Understanding whether adaptation of a neuron in a sensory system can occur locally inside the receptive field or whether it always globally affects the entire receptive field is important for understanding how the neuron processes complex sensory stimuli. For mouse retinal ganglion cells, we here show that both local and global contrast adaptation exist and that this diversity in spatial scope can contribute to the functional diversity of retinal ganglion cell types. Copyright © 2017 the American Physiological Society.
The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle.

PubMed Central

Minshull, J; Golsteyn, R; Hill, C S; Hunt, T

1990-01-01

Cyclins play a key role in the induction of mitosis. In this paper we report the isolation of a cyclin A cDNA clone from Xenopus eggs. Its cognate mRNA encodes a protein that shows characteristic accumulation and destruction during mitotic cell cycles. The cyclin A polypeptide is associated with a protein that cross-reacts with an antibody against the conserved 'PSTAIR' epitope of p34cdc2, and the cyclin A-cdc2 complex exhibits protein kinase activity that oscillates with the cell cycle. This kinase activity rises more smoothly than that of the cyclin B-cdc2 complexes and reaches a peak earlier in the cell cycle; indeed, cyclin A is destroyed before nuclear envelope breakdown. None of the cyclin-cdc2 complexes show simple relationships between the concentration of the cyclin moiety and the kinase activity. All three cyclin associated kinases (A, B1 and B2) phosphorylate identical sites on histones with the consensus XSPXK/R, although they show significant differences in their substrate preferences. We discuss possible models for the different roles of the A- and B-type cyclins in the control of cell division. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:2143983
L-Dopa decarboxylase expression profile in human cancer cells.

PubMed

Chalatsa, Ioanna; Nikolouzou, Eleftheria; Fragoulis, Emmanuel G; Vassilacopoulou, Dido

2011-02-01

L-Dopa decarboxylase (DDC) catalyses the decarboxylation of L-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5'-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5'UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.
Nucleotide sequence of the beta-lactamase gene from Enterococcus faecalis HH22 and its similarity to staphylococcal beta-lactamase genes.

PubMed Central

Zscheck, K K; Murray, B E

1991-01-01

The nucleotide sequence of the constitutively produced beta-lactamase (Bla) gene from Enterococcus faecalis HH22 was shown to be identical to the published sequences of three of four staphylococcal type A beta-lactamase genes; more differences were seen with the genes for staphylococcal type C and D enzymes. One hundred forty nucleotides upstream of the beta-lactamase start codon were determined for an inducible staphylococcal beta-lactamase and were identical to those of the constitutively expressed enterococcal gene, indicating that the changes resulting in constitutive expression are not due to changes in the promoter or operator region. Moreover, complementation studies indicated that production of the enterococcal enzyme could be repressed. The genes for the enterococcal Bla and an inducible staphylococcal Bla were each cloned into a shuttle vector and transformed into enterococcal and staphylococcal recipients. The major difference between the backgrounds of the two hosts was that more enzyme was produced by the staphylococcal host, regardless of the source of the gene. The location of the enzyme was found to be host dependent, since each cloned gene generated extracellular (free) enzyme in the staphylococcus and cell-bound enzyme in the enterococcus. On the basis of the identities of the enterococcal Bla and several staphylococcal Bla sequences, these data suggest the recent spread of beta-lactamase to enterococci and also suggest the loss of a functional repressor. PMID:1952840
In Vitro Expanded Stem Cells from the Developing Retina Fail to Generate Photoreceptors but Differentiate into Myelinating Oligodendrocytes

PubMed Central

Czekaj, Magdalena; Haas, Jochen; Gebhardt, Marlen; Müller-Reichert, Thomas; Humphries, Peter; Farrar, Jane; Bartsch, Udo; Ader, Marius

2012-01-01

Cell transplantation to treat retinal degenerative diseases represents an option for the replacement of lost photoreceptor cells. In vitro expandable cells isolated from the developing mammalian retina have been suggested as a potential source for the generation of high numbers of donor photoreceptors. In this study we used standardized culture conditions based on the presence of the mitogens FGF-2 and EGF to generate high numbers of cells in vitro from the developing mouse retina. These presumptive ‘retinal stem cells’ (‘RSCs’) can be propagated as monolayer cultures over multiple passages, express markers of undifferentiated neural cells, and generate neuronal and glial cell types upon withdrawal of mitogens in vitro or following transplantation into the adult mouse retina. The proportion of neuronal differentiation can be significantly increased by stepwise removal of mitogens and inhibition of the notch signaling pathway. However, ‘RSCs’, by contrast to their primary counterparts in vivo, i.e. retinal progenitor cells, loose the expression of retina-specific progenitor markers like Rax and Chx10 after passaging and fail to differentiate into photoreceptors both in vitro or after intraretinal transplantation. Notably, ‘RSCs’ can be induced to differentiate into myelinating oligodendrocytes, a cell type not generated by primary retinal progenitor cells. Based on these findings we conclude that ‘RSCs’ expanded in high concentrations of FGF-2 and EGF loose their retinal identity and acquire features of in vitro expandable neural stem-like cells making them an inappropriate cell source for strategies aimed at replacing photoreceptor cells in the degenerated retina. PMID:22848612

Homologs of CD83 from elasmobranch and teleost fish.

PubMed

Ohta, Yuko; Landis, Eric; Boulay, Thomas; Phillips, Ruth B; Collet, Bertrand; Secombes, Chris J; Flajnik, Martin F; Hansen, John D

2004-10-01

Dendritic cells are one of the most important cell types connecting innate and adaptive immunity, but very little is known about their evolutionary origins. To begin to study dendritic cells from lower vertebrates, we isolated and characterized CD83 from the nurse shark (Ginglymostoma cirratum (Gici)) and rainbow trout (Oncorhynchus mykiss (Onmy)). The open reading frames for Gici-CD83 (194 aa) and Onmy-CD83 (218 aa) display approximately 28-32% identity to mammalian CD83 with the presence of two conserved N-linked glycosylation sites. Identical with mammalian CD83 genes, Gici-CD83 is composed of five exons including conservation of phase for the splice sites. Mammalian CD83 genes contain a split Ig superfamily V domain that represents a unique sequence feature for CD83 genes, a feature conserved in both Gici- and Onmy-CD83. Gici-CD83 and Onmy-CD83 are not linked to the MHC, an attribute shared with mouse but not human CD83. Gici-CD83 is expressed rather ubiquitously with highest levels in the epigonal tissue, a primary site for lymphopoiesis in the nurse shark, whereas Onmy-CD83 mRNA expression largely paralleled that of MHC class II but at lower levels. Finally, Onmy-CD83 gene expression is up-regulated in virus-infected trout, and the promoter is responsive to trout IFN regulatory factor-1. These results suggest that the role of CD83, an adhesion molecule for cell-mediated immunity, has been conserved over 450 million years of vertebrate evolution.
Variation in Sexual Identification Among Behaviorally Bisexual Women in the Midwestern United States: Challenging the Established Methods for Collecting Data on Sexual Identity and Orientation.

PubMed

Baldwin, Aleta; Schick, Vanessa R; Dodge, Brian; van Der Pol, Barbara; Herbenick, Debby; Sanders, Stephanie A; Fortenberry, J Dennis

2017-07-01

Collecting information on sexual identity is critical to ensuring the visibility of minority populations who face stigmatization and discrimination related to sexual identities. However, it is challenging to capture the nuances of sexual identity with traditional survey research methods. Using a mixed-methods approach, we gathered data on the sexual identities of 80 behaviorally bisexual women in the Midwestern United States through an online survey. When provided different types of measures (e.g., open ended and fixed response) and different contexts in which to identify (e.g., private and public), participants varied in how they reported their sexual identities. Qualitative analysis of participant narratives around identity change finds partitioning and ranking of attraction is a key component in understanding behaviorally bisexual women's identities. We further identify a division regarding the desired outcomes of identity development processes. Given the multiple ways in which participants identified depending upon the type of measure and the context specified, and the variation in identification over time, results support reconsidering the capability of typical measures and methods used in survey research to capture sexual identity information. Additionally, findings highlight the utility of including multiple, context-specific measures of sexual identities in future research.
Verifying Parentage and Confirming Identity in Blackberry with a Fingerprinting Set

USDA-ARS?s Scientific Manuscript database

Parentage and identity confirmation is an important aspect of clonally propagated crops outcrossing. Potential errors resulting misidentification include off-type pollination events, labeling errors, or sports of clones. DNA fingerprinting sets are an excellent solution to quickly identify off-type ...
Nuclei of Tsuga canadensis: Role of Flavanols in Chromatin Organization

PubMed Central

Feucht, Walter; Schmid, Markus; Treutter, Dieter

2011-01-01

Needle primordia of Tsuga canadensis (hemlock) arising from flank meristems of a shoot apex, form cell lineages consisting of four or eight cells. Within a recently established lineage there is striking uniformity in the pattern of nuclear flavanols. This fact points to an identical transcriptional expression of these flavanols during cell cycling. However two lineages, even if located close together within the same meristem, can be very different in the expression of both cell shape and nuclear flavanol pattern, indicating that epigenetic positional signals are operating in a collective specification of cell lineage development. There is a wide range of nuclear flavanol patterning from a mosaic-like distribution in an activated cell type to a homogenous appearance in silenced cell types. Single cells deriving from lineages are desynchronized because they underlie a signaling network at a higher tissue level which results in stronger epigenetic modifications of their nuclear flavanols. As an extreme case of epigenetic modulation, transient drought conditions caused a drastic reduction of nuclear flavanols. Upon treatment with sucrose or cytokinin, these nuclear flavanols could be fully restored. Analytical determination of the flavanols revealed 3.4 mg/g DW for newly sprouting needles and 19.6 mg/g DW for anthers during meiosis. The roughly 6-fold difference in flavanols is apparently a reflection of the highly diverging organogenetic processes. Collectively, the studies provide strong evidence for combinatorial interplay between cell fate and nuclear flavanols. PMID:22072922
Regulation of the mouse Treacher Collins syndrome homolog (Tcof1) promoter through differential repression of constitutive expression.

PubMed

Shows, Kathryn H; Shiang, Rita

2008-11-01

Treacher Collins syndrome is an autosomal-dominant mandibulofacial dysostosis caused by haploinsufficiency of the TCOF1 gene product treacle. Mouse Tcof1 protein is approximately 61% identical and 71% similar to treacle, and heterozygous knockout of Tcof1 causes craniofacial malformation. Tcof1 expression is high in developing neural crest, but much lower in other tissues. To investigate this dual regulation, highly conserved regions upstream of TCOF1 homologs were tested through deletion and mutation reporter assays, and conserved predicted transcription factor binding sites were assessed through chromatin binding studies. Assays were performed in mouse P19 embryonic carcinoma cells and in HEK293 cells to determine differential activation in cell types at different stages of differentiation. Binding of Cebpb, Zfp161, and Sp1 transcription factors was specific to the Tcof1 regulatory region in P19 cells. The Zfp161 binding site demonstrated P19 cell-specific repression, while the Sp1/Sp3 candidate site demonstrated HEK293 cell-specific activation. Moreover, presence of c-myb and Zfp161 transcripts was specific to P19 cells. A minimal promoter fragment from -253 to +43 bp directs constitutive expression in both cell types, and dual regulation of Tcof1 appears to be through differential repression of this minimal promoter. The CpG island at the transcription start site remains unmethylated in P19 cells, 11.5 dpc mouse embryonic tissue, and adult mouse ear, which supports constitutive activation of the Tcof1 promoter.
Cell-bound lipases from Burkholderia sp. ZYB002: gene sequence analysis, expression, enzymatic characterization, and 3D structural model.

PubMed

Shu, Zhengyu; Lin, Hong; Shi, Shaolei; Mu, Xiangduo; Liu, Yanru; Huang, Jianzhong

2016-05-03

The whole-cell lipase from Burkholderia cepacia has been used as a biocatalyst in organic synthesis. However, there is no report in the literature on the component or the gene sequence of the cell-bound lipase from this species. Qualitative analysis of the cell-bound lipase would help to illuminate the regulation mechanism of gene expression and further improve the yield of the cell-bound lipase by gene engineering. Three predictive cell-bound lipases, lipA, lipC21 and lipC24, from Burkholderia sp. ZYB002 were cloned and expressed in E. coli. Both LipA and LipC24 displayed the lipase activity. LipC24 was a novel mesophilic enzyme and displayed preference for medium-chain-length acyl groups (C10-C14). The 3D structural model of LipC24 revealed the open Y-type active site. LipA displayed 96 % amino acid sequence identity with the known extracellular lipase. lipA-inactivation and lipC24-inactivation decreased the total cell-bound lipase activity of Burkholderia sp. ZYB002 by 42 % and 14 %, respectively. The cell-bound lipase activity from Burkholderia sp. ZYB002 originated from a multi-enzyme mixture with LipA as the main component. LipC24 was a novel lipase and displayed different enzymatic characteristics and structural model with LipA. Besides LipA and LipC24, other type of the cell-bound lipases (or esterases) should exist.
H3K4me1 marks DNA regions hypomethylated during aging in human stem and differentiated cells.

PubMed

Fernández, Agustín F; Bayón, Gustavo F; Urdinguio, Rocío G; Toraño, Estela G; García, María G; Carella, Antonella; Petrus-Reurer, Sandra; Ferrero, Cecilia; Martinez-Camblor, Pablo; Cubillo, Isabel; García-Castro, Javier; Delgado-Calle, Jesús; Pérez-Campo, Flor M; Riancho, José A; Bueno, Clara; Menéndez, Pablo; Mentink, Anouk; Mareschi, Katia; Claire, Fabian; Fagnani, Corrado; Medda, Emanuela; Toccaceli, Virgilia; Brescianini, Sonia; Moran, Sebastián; Esteller, Manel; Stolzing, Alexandra; de Boer, Jan; Nisticò, Lorenza; Stazi, Maria A; Fraga, Mario F

2015-01-01

In differentiated cells, aging is associated with hypermethylation of DNA regions enriched in repressive histone post-translational modifications. However, the chromatin marks associated with changes in DNA methylation in adult stem cells during lifetime are still largely unknown. Here, DNA methylation profiling of mesenchymal stem cells (MSCs) obtained from individuals aged 2 to 92 yr identified 18,735 hypermethylated and 45,407 hypomethylated CpG sites associated with aging. As in differentiated cells, hypermethylated sequences were enriched in chromatin repressive marks. Most importantly, hypomethylated CpG sites were strongly enriched in the active chromatin mark H3K4me1 in stem and differentiated cells, suggesting this is a cell type-independent chromatin signature of DNA hypomethylation during aging. Analysis of scedasticity showed that interindividual variability of DNA methylation increased during aging in MSCs and differentiated cells, providing a new avenue for the identification of DNA methylation changes over time. DNA methylation profiling of genetically identical individuals showed that both the tendency of DNA methylation changes and scedasticity depended on nongenetic as well as genetic factors. Our results indicate that the dynamics of DNA methylation during aging depend on a complex mixture of factors that include the DNA sequence, cell type, and chromatin context involved and that, depending on the locus, the changes can be modulated by genetic and/or external factors. © 2015 Fernández et al.; Published by Cold Spring Harbor Laboratory Press.
Improved process conditions for increasing expression of MHC class II protein from a stable Drosophila S2 cell line.

PubMed

Shen, Xiao; Dojcinovic, Danijel; Baldi, Lucia; Hacker, David L; Luescher, Immanuel F; Wurm, Florian M

2018-01-01

To investigate the effects of operational process conditions on expression of MHC class II protein from a stable Drosophila S2 cell line. When the Drosophila S2 cells were grown in vented orbitally shaken TubeSpin bioreactor 600 containers, cell growth was improved three-fold and the yield of recombinant major histocompatibility (MHC) class II protein (HLA-DR1 2xHis ) increased four-fold over the levels observed for the same cells cultivated in roller bottles (RB) without vented caps. Culturing in RB with vented caps while increasing the rotation speed from 6 rpm to 18 rpm also improved cell growth five-fold and protein productivity three-fold which is comparable to the levels observed in the orbitally shaken containers. Protein activity was found to be almost identical between the two vessel systems tested. Optimized cell culture conditions and a more efficient vessel type can enhance gas transfer and mixing and lead to substantial improvement of recombinant product yields from S2 cells.
Mutually exclusive signaling signatures define the hepatic and pancreatic progenitor cell lineage divergence

PubMed Central

Rodríguez-Seguel, Elisa; Mah, Nancy; Naumann, Heike; Pongrac, Igor M.; Cerdá-Esteban, Nuria; Fontaine, Jean-Fred; Wang, Yongbo; Chen, Wei; Andrade-Navarro, Miguel A.; Spagnoli, Francesca M.

2013-01-01

Understanding how distinct cell types arise from multipotent progenitor cells is a major quest in stem cell biology. The liver and pancreas share many aspects of their early development and possibly originate from a common progenitor. However, how liver and pancreas cells diverge from a common endoderm progenitor population and adopt specific fates remains elusive. Using RNA sequencing (RNA-seq), we defined the molecular identity of liver and pancreas progenitors that were isolated from the mouse embryo at two time points, spanning the period when the lineage decision is made. The integration of temporal and spatial gene expression profiles unveiled mutually exclusive signaling signatures in hepatic and pancreatic progenitors. Importantly, we identified the noncanonical Wnt pathway as a potential developmental regulator of this fate decision and capable of inducing the pancreas program in endoderm and liver cells. Our study offers an unprecedented view of gene expression programs in liver and pancreas progenitors and forms the basis for formulating lineage-reprogramming strategies to convert adult hepatic cells into pancreatic cells. PMID:24013505
Dicer maintains the identity and function of proprioceptive sensory neurons

PubMed Central

O’Toole, Sean M.; Ferrer, Monica M.; Mekonnen, Jennifer; Zhang, Haihan; Shima, Yasuyuki; Ladle, David R.

2017-01-01

Neuronal cell identity is established during development and must be maintained throughout an animal’s life (Fishell G, Heintz N. Neuron 80: 602–612, 2013). Transcription factors critical for establishing neuronal identity can be required for maintaining it (Deneris ES, Hobert O. Nat Neurosci 17: 899–907, 2014). Posttranscriptional regulation also plays an important role in neuronal differentiation (Bian S, Sun T. Mol Neurobiol 44: 359–373, 2011), but its role in maintaining cell identity is less established. To better understand how posttranscriptional regulation might contribute to cell identity, we examined the proprioceptive neurons in the dorsal root ganglion (DRG), a highly specialized sensory neuron class, with well-established properties that distinguish them from other neurons in the ganglion. By conditionally ablating Dicer in mice, using parvalbumin (Pvalb)-driven Cre recombinase, we impaired posttranscriptional regulation in the proprioceptive sensory neuron population. Knockout (KO) animals display a progressive form of ataxia at the beginning of the fourth postnatal week that is accompanied by a cell death within the DRG. Before cell loss, expression profiling shows a reduction of proprioceptor specific genes and an increased expression of nonproprioceptive genes normally enriched in other ganglion neurons. Furthermore, although central connections of these neurons are intact, the peripheral connections to the muscle are functionally impaired. Posttranscriptional regulation is therefore necessary to retain the transcriptional identity and support functional specialization of the proprioceptive sensory neurons. NEW & NOTEWORTHY We have demonstrated that selectively impairing Dicer in parvalbumin-positive neurons, which include the proprioceptors, triggers behavioral changes, a lack of muscle connectivity, and a loss of transcriptional identity as observed through RNA sequencing. These results suggest that Dicer and, most likely by extension, microRNAs are crucially important for maintaining proprioception. Additionally, this study hints at the larger question of how neurons maintain their functional and molecular specificity. PMID:28003412
Dicer maintains the identity and function of proprioceptive sensory neurons.

PubMed

O'Toole, Sean M; Ferrer, Monica M; Mekonnen, Jennifer; Zhang, Haihan; Shima, Yasuyuki; Ladle, David R; Nelson, Sacha B

2017-03-01

Neuronal cell identity is established during development and must be maintained throughout an animal's life (Fishell G, Heintz N. Neuron 80: 602-612, 2013). Transcription factors critical for establishing neuronal identity can be required for maintaining it (Deneris ES, Hobert O. Nat Neurosci 17: 899-907, 2014). Posttranscriptional regulation also plays an important role in neuronal differentiation (Bian S, Sun T. Mol Neurobiol 44: 359-373, 2011), but its role in maintaining cell identity is less established. To better understand how posttranscriptional regulation might contribute to cell identity, we examined the proprioceptive neurons in the dorsal root ganglion (DRG), a highly specialized sensory neuron class, with well-established properties that distinguish them from other neurons in the ganglion. By conditionally ablating Dicer in mice, using parvalbumin (Pvalb)-driven Cre recombinase, we impaired posttranscriptional regulation in the proprioceptive sensory neuron population. Knockout (KO) animals display a progressive form of ataxia at the beginning of the fourth postnatal week that is accompanied by a cell death within the DRG. Before cell loss, expression profiling shows a reduction of proprioceptor specific genes and an increased expression of nonproprioceptive genes normally enriched in other ganglion neurons. Furthermore, although central connections of these neurons are intact, the peripheral connections to the muscle are functionally impaired. Posttranscriptional regulation is therefore necessary to retain the transcriptional identity and support functional specialization of the proprioceptive sensory neurons. NEW & NOTEWORTHY We have demonstrated that selectively impairing Dicer in parvalbumin-positive neurons, which include the proprioceptors, triggers behavioral changes, a lack of muscle connectivity, and a loss of transcriptional identity as observed through RNA sequencing. These results suggest that Dicer and, most likely by extension, microRNAs are crucially important for maintaining proprioception. Additionally, this study hints at the larger question of how neurons maintain their functional and molecular specificity. Copyright © 2017 the American Physiological Society.
Cancer-associated fibroblasts in a human HEp-2 established laryngeal xenografted tumor are not derived from cancer cells through epithelial-mesenchymal transition, phenotypically activated but karyotypically normal.

PubMed

Wang, Mei; Wu, Chun-Ping; Pan, Jun-Yan; Zheng, Wen-Wei; Cao, Xiao-Juan; Fan, Guo-Kang

2015-01-01

Cancer-associated fibroblasts (CAFs) play a crucial role in cancer progression and even initiation. However, the origins of CAFs in various cancer types remain controversial, and one of the important hypothesized origins is through epithelial-mesenchymal transition (EMT) from cancer cells. In this study, we investigated whether the HEp-2 laryngeal cancer cells are able to generate CAFs via EMT during tumor formation, which is now still unknown. The laryngeal xenografted tumor model was established by inoculating the HEp-2 laryngeal cancer cell line in nude mice. Primary cultured CAFs from the tumor nodules and matched normal fibroblasts (NFs) from the adjacent connective tissues were subcultured, purified, and verified by immunofluorescence. Migration, invasion, and proliferation potentials were compared between the CAFs and NFs. A co-culture of CAFs with HEp-2 cells and a co-injection of CAFs with HEp-2 cells in nude mice were performed to examine the cancer-promoting potential of CAFs to further verify their identity. Karyotypic analyses of the CAFs, NFs, and HEp-2 cells were conducted. A co-culture of NFs with HEp-2 cells was also performed to examine the expression of activated markers of CAFs. A pathological examination confirmed that the laryngeal xenografted tumor model was successfully established, containing abundant CAFs. Immunocytochemical staining verified the purities and identities of the CAFs and NFs. Although the CAFs manifested higher migration, invasion, proliferation, and cancer-promoting capacities compared with the NFs, an analysis of chromosomes revealed that both the CAFs and NFs showed typical normal mouse karyotypes. In addition, the NFs co-cultured with HEp-2 cells did not show induced expressions of activated markers of CAFs. Our findings reveal that the CAFs in the HEp-2 established laryngeal xenografted tumor are not of laryngeal cancer origin but of mouse origin, indicating that the HEp-2 laryngeal cancer cells cannot generate their own CAFs via EMT in this model.
Functional impact of splice isoform diversity in individual cells

PubMed Central

Yap, Karen; Makeyev, Eugene V.

2016-01-01

Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a ‘splicing noise’, co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities. PMID:27528755
Functional impact of splice isoform diversity in individual cells.

PubMed

Yap, Karen; Makeyev, Eugene V

2016-08-15

Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a 'splicing noise', co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities. © 2016 The Author(s).
Effects of freezing-induced cell-fluid-matrix interactions on the cells and extracellular matrix of engineered tissues.

PubMed

Teo, Ka Yaw; DeHoyos, Tenok O; Dutton, J Craig; Grinnell, Frederick; Han, Bumsoo

2011-08-01

The two most significant challenges for successful cryopreservation of engineered tissues (ETs) are preserving tissue functionality and controlling highly tissue-type dependent preservation outcomes. In order to address these challenges, freezing-induced cell-fluid-matrix interactions should be understood, which determine the post-thaw cell viability and extracellular matrix (ECM) microstructure. However, the current understanding of this tissue-level biophysical interaction is still limited. In this study, freezing-induced cell-fluid-matrix interactions and their impact on the cells and ECM microstructure of ETs were investigated using dermal equivalents as a model ET. The dermal equivalents were constructed by seeding human dermal fibroblasts in type I collagen matrices with varying cell seeding density and collagen concentration. While these dermal equivalents underwent an identical freeze/thaw condition, their spatiotemporal deformation during freezing, post-thaw ECM microstructure, and cellular level cryoresponse were characterized. The results showed that the extent and characteristics of freezing-induced deformation were significantly different among the experimental groups, and the ETs with denser ECM microstructure experienced a larger deformation. The magnitude of the deformation was well correlated to the post-thaw ECM structure, suggesting that the freezing-induced deformation is a good indicator of post-thaw ECM structure. A significant difference in the extent of cellular injury was also noted among the experimental groups, and it depended on the extent of freezing-induced deformation of the ETs and the initial cytoskeleton organization. These results suggest that the cells have been subjected to mechanical insult due to the freezing-induced deformation as well as thermal insult. These findings provide insight on tissue-type dependent cryopreservation outcomes, and can help to design and modify cryopreservation protocols for new types of tissues from a pre-developed cryopreservation protocol. Copyright © 2011 Elsevier Ltd. All rights reserved.
Combinatorial regulation of a Blimp1 (Prdm1) enhancer in the mouse retina

PubMed Central

Mills, Taylor S.; Eliseeva, Tatiana; Bersie, Stephanie M.; Randazzo, Grace; Nahreini, Jhenya; Park, Ko Uoon

2017-01-01

The mouse retina comprises seven major cell types that exist in differing proportions. They are generated from multipotent progenitors in a stochastic manner, such that the relative frequency of any given type generated changes over time. The mechanisms determining the proportions of each cell type are only partially understood. Photoreceptors and bipolar interneurons are derived from cells that express Otx2. Within this population, Blimp1 (Prdm1) helps set the balance between photoreceptors and bipolar cells by suppressing bipolar identity in most of the cells. How only a subset of these Otx2+ cells decides to upregulate Blimp1 and adopt photoreceptor fate is unknown. To understand this, we investigated how Blimp1 transcription is regulated. We identified several potential Blimp1 retinal enhancer elements using DNase hypersensitivity sequencing. Only one of the elements recapitulated Blimp1 spatial and temporal expression in cultured explant assays and within the retinas of transgenic mice. Mutagenesis of this retinal Blimp1 enhancer element revealed four discrete sequences that were each required for its activity. These included highly conserved Otx2 and ROR (retinoic acid receptor related orphan receptor) binding sites. The other required sequences do not appear to be controlled by Otx2 or ROR factors, increasing the complexity of the Blimp1 gene regulatory network. Our results show that the intersection of three or more transcription factors is required to correctly regulate the spatial and temporal features of Blimp1 enhancer expression. This explains how Blimp1 expression can diverge from Otx2 and set the balance between photoreceptor and bipolar fates. PMID:28829770
Generation of Mast Cells from Mouse Fetus: Analysis of Differentiation and Functionality, and Transcriptome Profiling Using Next Generation Sequencer

PubMed Central

Fukuishi, Nobuyuki; Igawa, Yuusuke; Kunimi, Tomoyo; Hamano, Hirofumi; Toyota, Masao; Takahashi, Hironobu; Kenmoku, Hiromichi; Yagi, Yasuyuki; Matsui, Nobuaki; Akagi, Masaaki

2013-01-01

While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the FcεRI receptor and degranulation by IgE cross-linking were comparable with BMMC. By flow cytometry we observed surface expression of c-Kit prior to that of FcεRI on FLMC, although on BMMC the expression of c-Kit came after FcεRI. The surface expression levels of Sca-1 and c-Kit, a marker of putative mast cell precursors, were slightly different between bone marrow cells and fetal liver cells, suggesting that differentiation stage or cell type are not necessarily equivalent between both lineages. Moreover, this indicates that phenotypically similar mast cells may not have undergone an identical process of differentiation. By comprehensive analysis using the next generation sequencer, the same frequency of gene expression was observed for 98.6% of all transcripts in both cell types. These results indicate that FLMC could represent a new and useful tool for exploring mast cell differentiation, and may help to elucidate the roles of individual proteins in the function of mast cells where gene manipulation can induce embryonic lethality in the mid to late stages of pregnancy. PMID:23573287
Divided Identity: Part-Time Faculty in Public Colleges and Universities

ERIC Educational Resources Information Center

Levin, John S.; Montero Hernandez, Virginia

2014-01-01

This article addresses the identity claims of part-time faculty at three types of higher education institutions. Using culture theory and professional identity theory, the article documents that part-time faculty members across institutions have a divided sense of identity. On the one hand, they perceive themselves as professionals based on their…
Co-infusion of haplo-identical CD19-chimeric antigen receptor T cells and stem cells achieved full donor engraftment in refractory acute lymphoblastic leukemia.

PubMed

Cai, Bo; Guo, Mei; Wang, Yao; Zhang, Yajing; Yang, Jun; Guo, Yelei; Dai, Hanren; Yu, Changlin; Sun, Qiyun; Qiao, Jianhui; Hu, Kaixun; Zuo, Hongli; Dong, Zheng; Zhang, Zechuan; Feng, Mingxing; Li, Bingxia; Sun, Yujing; Liu, Tieqiang; Liu, Zhiqing; Wang, Yi; Huang, Yajing; Yao, Bo; Han, Weidong; Ai, Huisheng

2016-11-25

Elderly patients with relapsed and refractory acute lymphoblastic leukemia (ALL) have poor prognosis. Autologous CD19 chimeric antigen receptor-modified T (CAR-T) cells have potentials to cure patients with B cell ALL; however, safety and efficacy of allogeneic CD19 CAR-T cells are still undetermined. We treated a 71-year-old female with relapsed and refractory ALL who received co-infusion of haplo-identical donor-derived CD19-directed CAR-T cells and mobilized peripheral blood stem cells (PBSC) following induction chemotherapy. Undetectable minimal residual disease by flow cytometry was achieved, and full donor cell engraftment was established. The transient release of cytokines and mild fever were detected. Significantly elevated serum lactate dehydrogenase, alanine transaminase, bilirubin and glutamic-oxalacetic transaminase were observed from days 14 to 18, all of which were reversible after immunosuppressive therapy. Our preliminary results suggest that co-infusion of haplo-identical donor-derived CAR-T cells and mobilized PBSCs may induce full donor engraftment in relapsed and refractory ALL including elderly patients, but complications related to donor cell infusions should still be cautioned. Allogeneic CART-19 for Elderly Relapsed/Refractory CD19+ ALL. NCT02799550.
I want it and I want it now: using a temporal discounting paradigm to examine predictors of consumer impulsivity.

PubMed

Dittmar, Helga; Bond, Rod

2010-11-01

This paper proposes a new model of consumer impulsivity, using type of good, a person's endorsement of materialistic values, and identity deficits as predictors. Traditional decision making and psychological accounts see impulsive behaviour as a general overweighing of short-term gratification (I want that dress now) relative to longer-term concerns, irrespective of consumer good. Our proposal is that consumers' impulsivity (a) differs according to type of good and (b) is linked systematically to a combination of materialistic values and high identity deficits. Beginning with Study 1, three experiments, using a temporal discounting paradigm, show consistently that discount rates are higher for goods that are seen as highly expressive of identity (e.g., clothes) than goods not expressive of identity (e.g., basic body care products). For materialistic consumers, identity deficits predict discount rates for identity-expressive goods (Study 2), and discount rates change for materialistic individuals when their identity deficits are made salient (Study 3). These findings support a conceptualization of consumer impulsivity as identity-seeking behaviour.

Zebrafish pronephros tubulogenesis and epithelial identity maintenance are reliant on the polarity proteins Prkc iota and zeta.

PubMed

Gerlach, Gary F; Wingert, Rebecca A

2014-12-15

The zebrafish pronephros provides an excellent in vivo system to study the mechanisms of vertebrate nephron development. When and how renal progenitors in the zebrafish embryo undergo tubulogenesis to form nephrons is poorly understood, but is known to involve a mesenchymal to epithelial transition (MET) and the acquisition of polarity. Here, we determined the precise timing of these events in pronephros tubulogenesis. As the ternary polarity complex is an essential regulator of epithelial cell polarity across tissues, we performed gene knockdown studies to assess the roles of the related factors atypical protein kinase C iota and zeta (prkcι, prkcζ). We found that prkcι and prkcζ serve partially redundant functions to establish pronephros tubule epithelium polarity. Further, the loss of prkcι or the combined knockdown of prkcι/ζ disrupted proximal tubule morphogenesis and podocyte migration due to cardiac defects that prevented normal fluid flow to the kidney. Surprisingly, tubule cells in prkcι/ζ morphants displayed ectopic expression of the transcription factor pax2a and the podocyte-associated genes wt1a, wt1b, and podxl, suggesting that prkcι/ζ are needed to maintain renal epithelial identity. Knockdown of genes essential for cardiac contractility and vascular flow to the kidney, such as tnnt2a, or elimination of pronephros fluid output through knockdown of the intraflagellar transport gene ift88, was not associated with ectopic pronephros gene expression, thus suggesting a unique role for prkcι/ζ in maintaining tubule epithelial identity separate from the consequence of disruptions to renal fluid flow. Interestingly, knockdown of pax2a, but not wt1a, was sufficient to rescue ectopic tubule gene expression in prkcι/ζ morphants. These data suggest a model in which the redundant activities of prkcι and prkcζ are essential to establish tubule epithelial polarity and also serve to maintain proper epithelial cell type identity in the tubule by inhibiting pax2a expression. These studies provide a valuable foundation for further analysis of MET during nephrogenesis, and have implications for understanding the pathways that affect nephron epithelial cells during kidney disease and regeneration. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
Light-induced c-Fos expression in the mouse suprachiasmatic nucleus: immunoelectron microscopy reveals co-localization in multiple cell types.

PubMed

Castel, M; Belenky, M; Cohen, S; Wagner, S; Schwartz, W J

1997-09-01

Although light is known to regulate the level of c-fos gene expression in the suprachiasmatic nucleus (SCN), the site of an endogenous circadian clock, little is known about the identities of the photically activated cells. We used light-microscopic immunocytochemistry and immunoelectron microscopy to detect c-Fos protein in the SCN of Sabra mice exposed to brief nocturnal light pulses at zeitgeber time 15-16. Stimulation with light pulses that saturated the phase-shifting response of the circadian locomotor rhythm revealed an upper limit to the number of photo-inducible c-Fos cells at about one-fifth of the estimated total SCN cell population. This functionally defined set was morphologically and phenotypically heterogeneous. About 24% could be labelled for vasoactive intestinal polypeptide, 13% for vasopressin-neurophysin, and 7% for glial fibrillary acidic protein. The remaining 56% of c-Fos-positive cells were largely of unknown phenotype, although many were presumptive interneurons, some of which were immunoreactive for nitric oxide synthase.
Differentiation of Shewanella putrefaciens and Shewanella alga on the basis of whole-cell protein profiles, ribotyping, phenotypic characterization, and 16S rRNA gene sequence analysis.

PubMed Central

Vogel, B F; Jørgensen, K; Christensen, H; Olsen, J E; Gram, L

1997-01-01

Seventy-six presumed Shewanella putrefaciens isolates from fish, oil drillings, and clinical specimens, the type strain of Shewanella putrefaciens (ATCC 8071), the type strain of Shewanella alga (IAM 14159), and the type strain of Shewanella hanedai (ATCC 33224) were compared by several typing methods. Numerical analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell protein and ribotyping patterns showed that the strains were separated into two distinct clusters with 56% +/- 10% and 40% +/- 14% similarity for whole-cell protein profiling and ribotyping, respectively. One cluster consisted of 26 isolates with 52 to 55 mol% G + C and included 15 human isolates, mostly clinical specimens, 8 isolates from marine waters, and the type strain of S. alga. This homogeneous cluster of mesophilic, halotolerant strains was by all analyses identical to the recently defined species S. alga (U. Simidu et al., Int. J. Syst. Bacteriol, 40:331-336, 1990). Fifty-two typically psychrotolerant strains formed the other, more heterogeneous major cluster, with 43 to 47 mol% G + C. The type strain of S. putrefaciens was included in this group. The two groups were confirmed by 16S rRNA gene sequence analysis. It is concluded that the isolates must be considered two different species, S. alga and S. putrefaciens, and that most mesophilic isolates formerly identified as S. putrefaciens belong to S. alga. The ecological role and potential pathogenicity of S. alga can be evaluated only if the organism is correctly identified. PMID:9172338
The lack of luminous matter in the poles

NASA Astrophysics Data System (ADS)

Shatsova, R. B.; Anisimova, G. B.

2010-01-01

Many RR Lyral type stars, globular clusters and dwarf galaxus of Local Group are localized in the thin oval envelopes around the holes. 12 ovals over ▵b = 30° are in the zone of Polar Ring of the Galaxy l = (97° and 277°)+/-30°. IRAS sources on λ = 60° and 100 mμ have roughly similar distribution. These small formations partly resemble the cells of large scale of the Universe and maybe have the identical nature. It is possible that the observable picture is formed by the dark matter.
Identification of amino acids important for substrate specificity in sucrose transporters using gene shuffling.

PubMed

Reinders, Anke; Sun, Ye; Karvonen, Kayla L; Ward, John M

2012-08-31

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.
Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling*

PubMed Central

Reinders, Anke; Sun, Ye; Karvonen, Kayla L.; Ward, John M.

2012-01-01

Plant sucrose transporters (SUTs) are H+-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general. PMID:22807445
Fortuitous detection of uniparental isodisomy of chromosome 6.

PubMed Central

Bittencourt, M C; Morris, M A; Chabod, J; Gos, A; Lamy, B; Fellmann, F; Antonarakis, S E; Plouvier, E; Herve, P; Tiberghien, P

1997-01-01

Uniparental isodisomy is defined as the inheritance of two copies of the same parental chromosome and can result in defects when it produces homozygosity for a recessive mutation or in the presence of imprinting. We describe the detection of a chromosome 6 uniparental isodisomy in a 9 year old girl, discovered during a search for an HLA identical sib. HLA typing, erythrocyte phenotyping, and genotypes of microsatellite polymorphisms were compatible with a paternal isodisomy of chromosome 6, with normal biparental origin of the other chromosomes. Paternal cells were not responsive to the patient's cells in mixed lymphocyte cultures. This fortuitous detection of a chromosome 6 isodisomy suggests that cases of chromosome 6 UPD may not be deleterious and may therefore go undetected. PMID:9032654
Formation and differentiation of the avian sclerotome.

PubMed

Christ, Bodo; Huang, Ruijin; Scaal, Martin

2004-08-01

The avian sclerotome forms by epitheliomesenchymal transition of the ventral half-somite. Sclerotome development is characterized by a craniocaudal polarization, resegmentation, and axial identity. Its formation is controlled by signals from the notochord, the neural tube, the lateral plate mesoderm, and the myotome. These signals and crosstalk between somite cells lead to the separation of various subdomains, such as the central and ventral sclerotomes that express Pax1 under the control of Sonic hedgehog and Noggin, and the dorsal and lateral sclerotome that do not express Pax1 and are controlled by Bmp-4. Further subdomains that give rise to specific derivatives are the syndetome, neurotome, meningotome, and arthrotome. The molecular control of subdomain formation and cell type specification is discussed. Copyright 2004 Springer-Verlag
Unreliable patient identification warrants ABO typing at admission to check existing records before transfusion.

PubMed

Ferrera-Tourenc, V; Lassale, B; Chiaroni, J; Dettori, I

2015-06-01

This study describes patient identification errors leading to transfusional near-misses in blood issued by the Alps Mediterranean French Blood Establishment (EFSAM) to Marseille Public Hospitals (APHM) over an 18-month period. The EFSAM consolidates 14 blood banks in southeast France. It supplies 149 hospitals and maintains a centralized database on ABO types used at all area hospitals. As an added precaution against incompatible transfusion, the APHM requires ABO testing at each admission regardless of whether the patient has an ABO record. The study goal was to determine if admission testing was warranted. Discrepancies between ABO type determined by admission testing and records in the centralized database were investigated. The root cause for each discrepancy was classified as specimen collection or patient admission error. Causes of patient admission events were further subclassified as namesake (name similarity) or impersonation (identity fraud). The incidence of ABO discrepancies was 1:2334 including a 1:3329 incidence of patient admission events. Impersonation was the main cause of identity events accounting for 90.3% of cases. The APHM's ABO control policy prevented 19 incompatible transfusions. In relation to the 48,593 packed red cell units transfused, this would have corresponded to a risk of 1:2526. Collecting and storing ABO typing results in a centralized database is an essential public health tool. It allows crosschecking of current test results with past records and avoids redundant testing. However, as patient identification remains unreliable, ABO typing at each admission is still warranted to prevent transfusion errors. Copyright © 2015 Elsevier Masson SAS. All rights reserved.
Diversity in spatial scope of contrast adaptation among mouse retinal ganglion cells

PubMed Central

Khani, Mohammad Hossein

2017-01-01

Retinal ganglion cells adapt to changes in visual contrast by adjusting their response kinetics and sensitivity. While much work has focused on the time scales of these adaptation processes, less is known about the spatial scale of contrast adaptation. For example, do small, localized contrast changes affect a cell’s signal processing across its entire receptive field? Previous investigations have provided conflicting evidence, suggesting that contrast adaptation occurs either locally within subregions of a ganglion cell’s receptive field or globally over the receptive field in its entirety. Here, we investigated the spatial extent of contrast adaptation in ganglion cells of the isolated mouse retina through multielectrode-array recordings. We applied visual stimuli so that ganglion cell receptive fields contained regions where the average contrast level changed periodically as well as regions with constant average contrast level. This allowed us to analyze temporal stimulus integration and sensitivity separately for stimulus regions with and without contrast changes. We found that the spatial scope of contrast adaptation depends strongly on cell identity, with some ganglion cells displaying clear local adaptation, whereas others, in particular large transient ganglion cells, adapted globally to contrast changes. Thus, the spatial scope of contrast adaptation in mouse retinal ganglion cells appears to be cell-type specific. This could reflect differences in mechanisms of contrast adaptation and may contribute to the functional diversity of different ganglion cell types. NEW & NOTEWORTHY Understanding whether adaptation of a neuron in a sensory system can occur locally inside the receptive field or whether it always globally affects the entire receptive field is important for understanding how the neuron processes complex sensory stimuli. For mouse retinal ganglion cells, we here show that both local and global contrast adaptation exist and that this diversity in spatial scope can contribute to the functional diversity of retinal ganglion cell types. PMID:28904106
Adult Olfactory Bulb Interneuron Phenotypes Identified by Targeting Embryonic and Postnatal Neural Progenitors

PubMed Central

Figueres-Oñate, Maria; López-Mascaraque, Laura

2016-01-01

Neurons are generated during embryonic development and in adulthood, although adult neurogenesis is restricted to two main brain regions, the hippocampus and olfactory bulb. The subventricular zone (SVZ) of the lateral ventricles generates neural stem/progenitor cells that continually provide the olfactory bulb (OB) with new granule or periglomerular neurons, cells that arrive from the SVZ via the rostral migratory stream. The continued neurogenesis and the adequate integration of these newly generated interneurons is essential to maintain homeostasis in the olfactory bulb, where the differentiation of these cells into specific neural cell types is strongly influenced by temporal cues. Therefore, identifying the critical features that control the generation of adult OB interneurons at either pre- or post-natal stages is important to understand the dynamic contribution of neural stem cells. Here, we used in utero and neonatal SVZ electroporation along with a transposase-mediated stable integration plasmid, in order to track interneurons and glial lineages in the OB. These plasmids are valuable tools to study the development of OB interneurons from embryonic and post-natal SVZ progenitors. Accordingly, we examined the location and identity of the adult progeny of embryonic and post-natally transfected progenitors by examining neurochemical markers in the adult OB. These data reveal the different cell types in the olfactory bulb that are generated in function of age and different electroporation conditions. PMID:27242400
Flow-cytometric enrichment of Pacific bluefin tuna type A spermatogonia based on light-scattering properties.

PubMed

Ichida, Kensuke; Kise, Kazuyoshi; Morita, Tetsuro; Yazawa, Ryosuke; Takeuchi, Yutaka; Yoshizaki, Goro

2017-10-01

We previously established surrogate broodstock in which the donor germ cells transplanted into the peritoneal cavities of xenogeneic recipients were capable of developing into functional eggs and sperm in teleost fish. In this transplantation system, only the undifferentiated germ cells such as type A spermatogonia (ASG) or a portion of the ASG population were capable of being incorporated into the genital ridges of the recipients and undergo gametogenesis. Therefore, the use of enriched ASGs can be expected to achieve efficient donor-cell incorporation. Here, we established a method of isolation and enrichment of the ASG of Pacific bluefin tuna using flow cytometry. Whole testicular cell suspensions were fractionated by forward and side scatter properties, following which ASGs were enriched in a fraction in which the forward scatter signal was relatively high and side scatter signal was relatively low. The diameter of sorted cells using the fraction was identical to the size of ASGs observed in histological analysis, and these cells also expressed the vasa gene. In addition, we succeeded in applying this method to several maturation stages of Pacific bluefin tuna. Since this method was based on light-scattering characteristics of ASGs, it can potentially be applied to various teleosts. We expect that this method can contribute to the production of seeds of Pacific bluefin tuna using surrogate broodstock. Copyright © 2017 Elsevier Inc. All rights reserved.
Bacterial persistence by RNA endonucleases

PubMed Central

Maisonneuve, Etienne; Shakespeare, Lana J.; Jørgensen, Mikkel Girke; Gerdes, Kenn

2011-01-01

Bacteria form persisters, individual cells that are highly tolerant to different types of antibiotics. Persister cells are genetically identical to nontolerant kin but have entered a dormant state in which they are recalcitrant to the killing activity of the antibiotics. The molecular mechanisms underlying bacterial persistence are unknown. Here, we show that the ubiquitous Lon (Long Form Filament) protease and mRNA endonucleases (mRNases) encoded by toxin-antitoxin (TA) loci are required for persistence in Escherichia coli. Successive deletion of the 10 mRNase-encoding TA loci of E. coli progressively reduced the level of persisters, showing that persistence is a phenotype common to TA loci. In all cases tested, the antitoxins, which control the activities of the mRNases, are Lon substrates. Consistently, cells lacking lon generated a highly reduced level of persisters. Moreover, Lon overproduction dramatically increased the levels of persisters in wild-type cells but not in cells lacking the 10 mRNases. These results support a simple model according to which mRNases encoded by TA loci are activated in a small fraction of growing cells by Lon-mediated degradation of the antitoxins. Activation of the mRNases, in turn, inhibits global cellular translation, and thereby induces dormancy and persistence. Many pathogenic bacteria known to enter dormant states have a plethora of TA genes. Therefore, in the future, the discoveries described here may lead to a mechanistic understanding of the persistence phenomenon in pathogenic bacteria. PMID:21788497
Recruited brain tumor-derived mesenchymal stem cells contribute to brain tumor progression.

PubMed

Behnan, Jinan; Isakson, Pauline; Joel, Mrinal; Cilio, Corrado; Langmoen, Iver A; Vik-Mo, Einar O; Badn, Wiaam

2014-05-01

The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These brain tumor-derived mesenchymal stem cells (BT-MSCs) are defined with the phenotype (Lin-Sca-1+CD9+CD44+CD166+/-) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro and that the non-MSC population is nontumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild-type GL261 inoculated into green fluorescent protein (GFP)-transgenic mice and GL261-GFP cells inoculated into wild-type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD44+CD9+CD166+ both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus, we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients. © 2013 AlphaMed Press.
Streptococcus group B typing: comparison of counter-immunoelectrophoresis with the precipitin method.

PubMed

Kubín, V; Jelínková, J; Franêk, J

1977-07-01

The method of counter-immunoelectrophoresis (CIE) was tested for its applicability to group B streptococcus typing. The results obtained were compared with the typing by the ring precipitin test. Identical antigens and identical hyperimmune typing serum batches had been used in both methods. A large majority of 75 freshly isolated strains were typed identically by both methods. Five strains with a weak antigenic outfit were untypable by the ring precipitin test but were typed by CIE owing to a higher sensitivity of CIE method. Two strains were typable by the precipitin test but not by CIE; an explanation for this phenomenon is lacking. The CIE method in group B typing is specific, rapid, highly sensitive and relatively simple. It requires strict maintenance of standard conditions. The method is economical with respect to manipulation and material, requires small amounts of diagnostic antisera. Potent antisera may be used diluted. Moreover, sera for CIE typing need not be absorbed to remove group B antibodies. CIE method is practicable for group B streptococcus typing, especially in laboratories carrying out routine large scale type identification.
Variation is function: Are single cell differences functionally important?: Testing the hypothesis that single cell variation is required for aggregate function.

PubMed

Dueck, Hannah; Eberwine, James; Kim, Junhyong

2016-02-01

There is a growing appreciation of the extent of transcriptome variation across individual cells of the same cell type. While expression variation may be a byproduct of, for example, dynamic or homeostatic processes, here we consider whether single-cell molecular variation per se might be crucial for population-level function. Under this hypothesis, molecular variation indicates a diversity of hidden functional capacities within an ensemble of identical cells, and this functional diversity facilitates collective behavior that would be inaccessible to a homogenous population. In reviewing this topic, we explore possible functions that might be carried by a heterogeneous ensemble of cells; however, this question has proven difficult to test, both because methods to manipulate molecular variation are limited and because it is complicated to define, and measure, population-level function. We consider several possible methods to further pursue the hypothesis that variation is function through the use of comparative analysis and novel experimental techniques. © 2015 The Authors. BioEssays published by WILEY Periodicals, Inc.
[Gastric lymphoma: still an interdisciplinary challenge].

PubMed

Barth, T F E; Floßbach, L; Möller, P

2013-05-01

Differentiation of chronic gastritis from marginal zone B-cell lymphoma (MZoL) of MALT type is often difficult for the pathologist. Diagnostic tools include CD20 stain to highlight lymphoepithelial lesions, Wotherspoon grading of the infiltrate, and clonality analysis of the B-cells. MZoL may partially transform into a diffuse, large B-cell lymphoma, which the authors have named blastic MZoL. Blastic MZoL may be present with or without small cell MZoL. Without this component, blastic MzoL, while being CD10-negative, is presently difficult to positively diagnose since specific immune markers are still lacking. Blastic MZoL has a very favourable outcome compared to conventional diffuse large B-cell lymphomas (DLBCL). Moreover, there are conventional DLBCL in the stomach, mostly in a setting of a secondary organ involvement. The biology of these gastric DLBCL is identical to their extragastric counterparts. This is also true for primary gastric Burkitt lymphoma and mucosal involvement in B-CLL or mantle cell lymphoma. Unfavourable outcomes are always observed for EBV-triggered lymphoproliferations in immunodeficiency and peripheral T-cell lymphomas which might also arise or be initially diagnosed in the stomach.
Morphogenesis of the vulva and the vulval-uterine connection.

PubMed Central

Gupta, Bhagwati P; Hanna-Rose, Wendy; Sternberg, Paul W

2012-01-01

The C. elegans hermaphrodite vulva is an established model system to study mechanisms of cell fate specification and tissue morphogenesis. The adult vulva is a tubular shaped organ composed of seven concentric toroids that arise from selective fusion between differentiated vulval progeny. The dorsal end of the vulval tubule is connected to the uterus via a multinucleate syncytium utse (uterine-seam) cell. The vulval tubule and utse are formed as a result of changes in morphogenetic processes such as cell polarity, adhesion, and invagination. A number of genes controlling these processes are conserved all the way up to human and function in similar developmental contexts. This makes it possible to extend the findings to other metazoan systems. Gene expression studies in the vulval and uterine cells have revealed the presence of regulatory networks specifying distinct cell fates. Thus, these two cell types serve as a good system to understand how gene networks confer unique cell identities both experimentally and computationally. This chapter focuses on morphogenetic processes during the formation of the vulva and its connection to uterus. PMID:23208727
Morphogenesis of the vulva and the vulval-uterine connection.

PubMed

Gupta, Bhagwati P; Hanna-Rose, Wendy; Sternberg, Paul W

2012-11-30

The C. elegans hermaphrodite vulva is an established model system to study mechanisms of cell fate specification and tissue morphogenesis. The adult vulva is a tubular shaped organ composed of seven concentric toroids that arise from selective fusion between differentiated vulval progeny. The dorsal end of the vulval tubule is connected to the uterus via a multinucleate syncytium utse (uterine-seam) cell. The vulval tubule and utse are formed as a result of changes in morphogenetic processes such as cell polarity, adhesion, and invagination. A number of genes controlling these processes are conserved all the way up to human and function in similar developmental contexts. This makes it possible to extend the findings to other metazoan systems. Gene expression studies in the vulval and uterine cells have revealed the presence of regulatory networks specifying distinct cell fates. Thus, these two cell types serve as a good system to understand how gene networks confer unique cell identities both experimentally and computationally. This chapter focuses on morphogenetic processes during the formation of the vulva and its connection to uterus.
Is the place cell a "supple" engram?

PubMed

Routtenberg, Aryeh

2015-06-01

This short note, which honors Nobelists O'Keefe and the Mosers, asks how the patterning of inputs to a single place cell regulates its firing. Because the combination of inputs to a single CA1 place cell is very large, the generally accepted view is rejected that inputs to a place cell are relatively restricted, near identical repetition upon re-presentation of the environment. The alternative proposed here is that when any 100 excitatory inputs are fired activating a subset combination, which is a large number, selected from the 30,000 synapses, this leads to CA1 cell firing. The selection of the combination of inputs is a very large number it nonetheless leads to the conclusion that even though the same cell dutifully fires when the animal is in an identical location, the inputs that fire the place cell are nonetheless obligatorily non-identical. This CA1 input combinatorial proposal may help us understand the physiological underpinnings of the memory mechanism arising from supple synapses (Routtenberg (2013), Hippocampus 23:202-206). © 2015 Wiley Periodicals, Inc.

The development of sex role stereotypes in the third year: relationships to gender labeling, gender identity, sex-typed toy preference, and family characteristics.

PubMed

Weinraub, M; Clemens, L P; Sockloff, A; Ethridge, T; Gracely, E; Myers, B

1984-08-01

The onset and development of preschoolers' awareness of sex role stereotypes, gender labeling, gender identity, and sex-typed toy preference were explored in 26-, 31-, and 36-month-old children. Gender labeling, gender identity, sex-typed toy preferences, and awareness of adult sex role differences were observed in significantly more 26-month-old children than would have been expected by chance. Verbal gender labeling was observed in a majority of 26-month-olds, while verbal and nonverbal gender identity were observed in a majority of 31-month-olds. Nonverbal gender labeling and awareness of adult sex role differences were observed in a majority of children by 36 months. No evidence of awareness of sex differences in children's toys was found in the majority of children at any age. Awareness of sex role differences in children's toys was not related to awareness of adult sex role differences. Brighter children were more aware of adult sex role differences. Sex-typed toy preference was not related to awareness of sex role differences but was related to gender identity. Predictors of sex role development included the mothers' employment, and the father's personality traits, attitudes toward women, and sex-typed activities in the home. Implications for theories of early sex role development are discussed.
Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules▿ †

PubMed Central

Rahaman, Abdur; Miao, Wei; Turkewitz, Aaron P.

2009-01-01

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal β/γ-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, ΔGRT1 ΔGRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in ΔGRT1 ΔGRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from ΔGRT1 ΔGRT2 cells appear less adhesive than those from the wild type. PMID:19684282
Independent transport and sorting of functionally distinct protein families in Tetrahymena thermophila dense core secretory granules.

PubMed

Rahaman, Abdur; Miao, Wei; Turkewitz, Aaron P

2009-10-01

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal beta/gamma-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, DeltaGRT1 DeltaGRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in DeltaGRT1 DeltaGRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from DeltaGRT1 DeltaGRT2 cells appear less adhesive than those from the wild type.
77 FR 38758 - Proposed Amendment to the Standards of Identity for Distilled Spirits; Comment Period Extension

Federal Register 2010, 2011, 2012, 2013, 2014

2012-06-29

... Identity for Distilled Spirits; Comment Period Extension AGENCY: Alcohol and Tobacco Tax and Trade Bureau... to the Standards of Identity for Distilled Spirits, for an additional 10 days. In Notice No. 127, a... the standards of identity regulations for distilled spirits to include ``Cacha[ccedil]a'' as a type of...
Causes of mortality after haploidentical hematopoietic stem cell transplantation and the comparison with HLA-identical sibling hematopoietic stem cell transplantation.

PubMed

Yan, C H; Xu, L P; Wang, F R; Chen, H; Han, W; Wang, Yu; Wang, J Z; Liu, K Y; Huang, X J

2016-03-01

This study was performed to investigate incidence, causes and factors influencing mortality after haploidentical hematopoietic stem cell transplantation (HSCT) and to compare differences between haploidentical HSCT and HLA-identical sibling HSCT. From January 2000 to June 2011, 1411 patients with acute leukemia or myelodysplastic syndrome were included in this study. Of these patients, 571 received HLA-identical sibling HSCT and 840 received haploidentical HSCT. The cumulative incidence of overall mortality and transplant-related mortality (TRM) after haploidentical HSCT was higher than those after HLA-identical sibling HSCT (38.7% vs. 33.3%, P=0.012 and 27.5% vs. 19.9%, P=0.002), but the incidence of relapse-related mortality (RRM) did not differ between the two groups (15.6% vs. 16.7%, P=0.943). A multivariate analysis suggested that high-risk disease status and haploidentical HSCT correlated with a higher incidence of overall mortality (P<0.0001, hazard ratio=1.911 and P=0.019, hazard ratio=1.249); in addition, in haploidentical HSCT, only high-risk disease status correlated with a higher incidence of overall mortality (P<0.0001, hazard ratio=1.845). Our study suggested that haploidentical HSCT provided a higher incidence of overall mortality and TRM but the same incidence of RRM compared with HLA-identical sibling HSCT. Therefore, HLA-identical sibling HSCT remains the first choice, but haploidentical HSCT is available for patients without an HLA-identical sibling donor.
The effect of missing KIR ligands, activating KIR genotype and haplotype on the outcome of T-cell-replete hematopoietic stem cell transplantation from HLA-identical siblings in Thai patients.

PubMed

Khanuntong, S; Kuptawintu, P; Upaisilpsathaporn, K; Poolchareon, A; Bunworasate, U; Hirankarn, N

2016-06-01

This study was a retrospective analysis of Thai patients undergoing T-replete hematopoietic stem cell transplant from human leukocyte antigen (HLA)-identical sibling donors. We investigated 66 patients, including 40 patients with acute myeloid leukemia (AML), 12 patients with acute lymphoblastic leukemia and 14 patients with chronic myeloid leukemia. Killer cell immunoglobulin-like receptor (KIR) genes and HLA ligands were typed by polymerase chain reaction-sequence specific oligonucleotide probes. We analyzed the effect of the number of missing KIR ligands (Bw4, C1 and C2) on clinical outcomes. A beneficial effect of missing KIR ligand was not observed in both univariate and multivariate analysis. When we analyzed the effect of specific missing KIR ligand on clinical outcomes, there was a trend that patients with missing A11 ligand had lower relapse rate (P = 0.076). Therefore, we also conducted the analysis by including the group with missing KIR ligands of Bw4, C1, C2 and A11. Patients with two or more than two missing KIR ligands had a trend for better clinical outcome including reduced relapse (P = 055) and statistically significant in terms of reduced acute graft-vs-host disease (aGVHD) rate (P = 0.013). In multivariate analysis, patients with two or more than two missing KIR ligands had a statistically significant better clinical outcome in terms of reduced aGVHD rate (HR = 0.155, 95%CI = 0.040-0.605, P = 0.007). The association between clinical outcome with KIR haplotypes, centromeric B haplotype and activating KIR was not observed here. Although the sample size in this study is rather limited, these data can later be subjected to meta-analysis to help reach the conclusion of the usefulness of this additional promising KIR genotyping in various hematopoietic stem cell transplantation types. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Selecting the correct cellular model for assessing of the biological response of collagen-based biomaterials.

PubMed

Davidenko, Natalia; Hamaia, Samir; Bax, Daniel V; Malcor, Jean-Daniel; Schuster, Carlos F; Gullberg, Donald; Farndale, Richard W; Best, Serena M; Cameron, Ruth E

2018-01-01

Accurate evaluation of the biological performance of biomaterials requires the correct assessment of their native-like cell ligation properties. However, cell attachment studies often overlook the details of the substrate-cell binding mechanisms, be they integrin-mediated or non-specific, and ignore the class- and species-specificities of the cell adhesion receptor involved. In this work we have used different collagen (Col) substrates (fibrillar collagens I, II and III and network-forming Col IV), containing different affinity cell-recognition motifs, to establish the influence of the receptor identity and species-specificity on collagen-cell interactive properties. Receptor expression was varied by using cells of different origin, or transfecting collagen-binding integrins into integrin-null cells. These include mouse C2C12 myoblasts transfected with human α1, α2, α10 or α11; human fibrosarcoma HT1080 cells which constitutively express only human α2β1, and rat glioma Rugli cells, with only rat α1β1. Using these lines, the nature of integrin binding sites was studied in order to delineate the bioactivity of different collagen substrates. Integrin ligation was studied on collagen coatings alongside synthetic (GFOGER/GLOGEN) and Toolkit (Col II-28/Col III-7) triple-helical peptides to evaluate (1) their affinity towards different integrins and (2) to confirm the activity of the inserted integrin in the transfected cells. Thin films of dermal and tendon Col I were used to evaluate the influence of the carbodiimide (EDC)-based treatment on the cellular response on Col of different origin. The results showed that the binding properties of transfected C2C12 cells to collagens depend on the identity of inserted integrin. Similar ligation characteristics were observed using α1+ and α10+ cells, but these were distinct from the similar binding features of α2+ and α11+ cells. Recombinant human and rat-α1 I domain binding to collagens and peptides correlated with the cell adhesion results, showing receptor class- and species-specificities. The understanding of the physiologically relevant cell anchorage characteristics of bio-constructs may assist in the selection of (1) the optimum collagen source for cellular supports and (2) the correct cellular model for their biological assessment. This, in turn, may allow reliable prediction of the biological performance of bio-scaffolds in vivo for specific TE applications. Integrins play a vital role in cellular responses to environmental cues during early-stage cell-substrate interaction. We describe physiologically relevant cell anchorage to collagen substrates that present different affinity cell-recognition motifs, to provide experimental tools to assist in understanding integrin binding. Using different cell types and recombinant integrin α1-I-domains, we found that cellular response was highly dependent on collagen type, origin and EDC-crosslinking status, as well as on the integrin class and species of origin. This comprehensive study establishes selectivity amongst the four collagen-binding integrins and species-specific properties that together may influence choice of cell type and receptor in different experimental settings. This work offers key guidance in selecting of the correct cellular model for the biological testing of collagen-based biomaterials. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
[Gastric MALT-type lymphoma. Pathology, pathogenesis, diagnostics and therapy].

PubMed

Eck, M; Fischbach, W

2010-05-01

Helicobacter pylori infection plays a central role in the development of gastric MALT-type (mucosa-associated lymphoid tissue) lymphoma. Infection results in chronic H. pylori gastritis and stimulates together with antigens or autoantigens proliferation of B-lymphocytes which is the basis for the neoplastic transformation. Histology of MALT-type lymphoma is architecturally similar to the physiological MALT. Invasion and destruction of the gastric epithelium with development of so-called lympho-epithelial lesions is the most important diagnostic criterion. Cytologically MALT-lymphoma resembles centrocytes and monocytes. For definitive lymphoma diagnosis and for the differential diagnosis from other small cell lymphomas in the stomach immunohistochemistry can be helpful. The phenotype of MALT-type lymphoma is identical to non-neoplastic B-lymphocytes of the marginal zone (CD20+, CD5-, CD10- and CD23-). Individual therapy is strongly dependent from histological type and lymphoma stadium. Therapy modalities are H. pylori eradication, radiochemotherapy, surgery or a combination of these. Aim of the therapy is the complete lymphoma regression and cure of the disease.
"We ARE Our Instrument!": Forming a Singer Identity

ERIC Educational Resources Information Center

O'Bryan, Jessica

2015-01-01

Preparation for the operatic stage means that for some students their voice will undergo significant transformation during training. For most operatic singers, voice type will determine future roles, the music they perform and potential career trajectory. Voice type becomes a facet of identity and position within the operatic world. This article…
Membranous glomerulopathy with spherules: an uncommon variant with obscure pathogenesis.

PubMed

Kowalewska, Jolanta; Smith, Kelly D; Hudkins, Kelly L; Chang, Anthony; Fogo, Agnes B; Houghton, Donald; Leslie, Deena; Aitchison, John; Nicosia, Roberto F; Alpers, Charles E

2006-06-01

Occasional case reports of membranous glomerulopathy described unique subepithelial accumulations of an unusual type of immune deposit composed of spherular structures. The identity of such structures as nuclear pores has been suggested, but not established. We identified a cohort of patients (n = 14, including 1 patient with disease recurrence in an allograft) who presented with nephrotic syndrome and had renal biopsy specimens with light and immunofluorescence microscopic findings characteristic of membranous glomerulopathy. These patients were distinguished by ultrastructural studies that showed glomerular capillary wall accumulations of subepithelial immune deposits composed of uniform spherular structures, while lacking the typical granular electron-dense deposits seen in membranous glomerulopathy. The molecular identity of these spherular structures as nuclear pores was tested by using immunofluorescence microscopy and immunohistochemistry with mouse monoclonal antinuclear pore antibodies (Covance, Princeton, NJ) and anti-Nuclear Pore-O-Linked Glycoprotein (Affinity BioReagents Inc, Golden, CO) antibodies. Measurement of spherular structures by using high-magnification electron microscopy showed an average diameter of 84.5 nm, which correlated well with accepted diameters of nuclear pores (80 to 120 nm). Immunofluorescence microscopy and immunoperoxidase staining with both antibodies showed characteristic beaded staining of nuclear membranes of multiple cell types within normal control kidney, but no staining of immune-type deposits within glomerular basement membranes. These cases form a rare, but distinctive, morphological subclass of membranous glomerulopathy. The antigenic specificity of immune deposits in these cases remains elusive.
WE-H-BRA-08: A Monte Carlo Cell Nucleus Model for Assessing Cell Survival Probability Based On Particle Track Structure Analysis

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

Lee, B; Georgia Institute of Technology, Atlanta, GA; Wang, C

Purpose: To correlate the damage produced by particles of different types and qualities to cell survival on the basis of nanodosimetric analysis and advanced DNA structures in the cell nucleus. Methods: A Monte Carlo code was developed to simulate subnuclear DNA chromatin fibers (CFs) of 30nm utilizing a mean-free-path approach common to radiation transport. The cell nucleus was modeled as a spherical region containing 6000 chromatin-dense domains (CDs) of 400nm diameter, with additional CFs modeled in a sparser interchromatin region. The Geant4-DNA code was utilized to produce a particle track database representing various particles at different energies and dose quantities.more » These tracks were used to stochastically position the DNA structures based on their mean free path to interaction with CFs. Excitation and ionization events intersecting CFs were analyzed using the DBSCAN clustering algorithm for assessment of the likelihood of producing DSBs. Simulated DSBs were then assessed based on their proximity to one another for a probability of inducing cell death. Results: Variations in energy deposition to chromatin fibers match expectations based on differences in particle track structure. The quality of damage to CFs based on different particle types indicate more severe damage by high-LET radiation than low-LET radiation of identical particles. In addition, the model indicates more severe damage by protons than of alpha particles of same LET, which is consistent with differences in their track structure. Cell survival curves have been produced showing the L-Q behavior of sparsely ionizing radiation. Conclusion: Initial results indicate the feasibility of producing cell survival curves based on the Monte Carlo cell nucleus method. Accurate correlation between simulated DNA damage to cell survival on the basis of nanodosimetric analysis can provide insight into the biological responses to various radiation types. Current efforts are directed at producing cell survival curves for high-LET radiation.« less
Characterization of multi-drug tolerant persister cells in Streptococcus suis.

PubMed

Willenborg, Jörg; Willms, Daniela; Bertram, Ralph; Goethe, Ralph; Valentin-Weigand, Peter

2014-05-12

Persister cells constitute a subpopulation of dormant cells within a microbial population which are genetically identical but phenotypically different to regular cells. Notably, persister cells show an elevated tolerance to antimicrobial agents. Thus, they are considered to represent a microbial 'bet-hedging' strategy and are of particular importance in pathogenic bacteria. We studied the ability of the zoonotic pathogen Streptococcus (S.) suis to form multi-drug tolerant variants and identified persister cells dependent on the initial bacterial growth phase. We observed lower numbers of persisters in exponential phase cultures than in stationary growth phase populations. S. suis persister cells showed a high tolerance to a variety of antibiotics, and the phenotype was not inherited as tested with four passages of S. suis populations. Furthermore, we provide evidence that the persister phenotype is related to expression of genes involved in general metabolic pathways since we found higher numbers of persister cells in a mutant strain defective in the catabolic arginine deiminase system as compared to its parental wild type strain. Finally, we observed persister cell formation also in other S. suis strains and pathogenic streptococcal species. Taken together, this is the first study that reports multi-drug tolerant persister cells in the zoonotic pathogen S. suis.
Induction of an interleukin-1 receptor (IL-1R) on monocytic cells. Evidence that the receptor is not encoded by a T cell-type IL-1R mRNA.

PubMed

Spriggs, M K; Lioubin, P J; Slack, J; Dower, S K; Jonas, U; Cosman, D; Sims, J E; Bauer, J

1990-12-25

Primary human monocytes and the human monocytic cell line THP-1 were induced to express receptors for interleukin-1 alpha (IL-1 alpha) and IL-1 beta. Treatment of primary monocytes with dexamethasone resulted in a 10-fold increase in receptor number over untreated cells, to approximately 2,000 receptors/cell. Treatment of THP-1 cells with phorbol ester followed by prostaglandin E2 and dexamethasone resulted in the expression of approximately 30,000 receptors/cell. Competitive binding assays on THP-1 cells showed that both IL-1 alpha and IL-1 beta bind to the same receptor. The monocyte IL-1R is significantly smaller (63 kDa) than the T cell IL-1R (80 kDa) and is immunologically distinct. However, induction of monocytes and monocytic cell lines leads to the appearance of an abundant mRNA of approximately 5,000 bases which hybridizes to a cDNA probe from the T cell-type IL-1R. Sequence data obtained from a cDNA clone of this mRNA indicate that the message is identical to the T cell IL-1R mRNA throughout the coding region. A smaller mRNA, also homologous to the T cell IL-1R mRNA, accumulated in induced THP-1 cells and has a shorter 3'-untranslated region than the larger. Data are presented which suggest that neither form of this message encodes the 63-kDa IL-1R, but rather that this protein is the product of a separate nonhomologous mRNA.
A subset of high Gleason grade prostate carcinomas contain a large burden of prostate cancer syndecan-1 positive stromal cells.

PubMed

Sharpe, Benjamin; Alghezi, Dhafer A; Cattermole, Claire; Beresford, Mark; Bowen, Rebecca; Mitchard, John; Chalmers, Andrew D

2017-05-01

There is a pressing need to identify prognostic and predictive biomarkers for prostate cancer to aid treatment decisions in both early and advanced disease settings. Syndecan-1, a heparan sulfate proteoglycan, has been previously identified as a potential prognostic biomarker by multiple studies at the tissue and serum level. However, other studies have questioned its utility. Anti-Syndecan-1 immunohistochemistry was carried out on 157 prostate tissue samples (including cancerous, adjacent normal tissue, and non-diseased prostate) from three independent cohorts of patients. A population of Syndecan-1 positive stromal cells was identified and the number and morphological parameters of these cells quantified. The identity of the Syndecan-1-positive stromal cells was assessed by multiplex immunofluorescence using a range of common cell lineage markers. Finally, the burden of Syndecan-1 positive stromal cells was tested for association with clinical parameters. We identified a previously unreported cell type which is marked by Syndecan-1 expression and is found in the stroma of prostate tumors and adjacent normal tissue but not in non-diseased prostate. We call these cells Prostate Cancer Syndecan-1 Positive (PCSP) cells. Immunofluorescence analysis revealed that the PCSP cell population did not co-stain with markers of common prostate epithelial, stromal, or immune cell populations. However, morphological analysis revealed that PCSP cells are often elongated and displayed prominent lamellipodia, suggesting they are an unidentified migratory cell population. Analysis of clinical parameters showed that PCSP cells were found with a frequency of 20-35% of all tumors evaluated, but were not present in non-diseased normal tissue. Interestingly, a subset of primary Gleason 5 prostate tumors had a high burden of PCSP cells. The current study identifies PCSP cells as a novel, potentially migratory cell type, which is marked by Syndecan-1 expression and is found in the stroma of prostate carcinomas, adjacent normal tissue, but not in non-diseased prostate. A subset of poor prognosis high Gleason grade 5 tumors had a particularly high PCSP cell burden, suggesting an association between this unidentified cell type and tumor aggressiveness. © 2017 Wiley Periodicals, Inc.
Development and characterization of a novel human Waldenström Macroglobulinemia cell line (RPCI-WM1; Roswell Park Cancer Institute-Waldenström Macroglobulinemia 1)

PubMed Central

Chitta, Kasyapa S.; Paulus, Aneel; Ailawadhi, Sikander; Foster, Barbara A.; Moser, Michael T.; Starostik, Petr; Masood, Aisha; Sher, Taimur; Miller, Kena C.; Iancu, Dan M.; Conroy, Jeffrey; Nowak, Norma J.; Sait, Sheila N.; Personett, David A.; Coleman, Morton; Furman, Richard R.; Martin, Peter; Ansell, Stephen M.; Lee, Kelvin; Chanan-Khan, Asher A.

2015-01-01

Understanding the biology of Waldenström Macroglobulinemia is hindered by a lack of preclinical models. We report a novel cell line, RPCI-WM1, from a patient treated for WM. The cell line secreted human IgM (hIgM) with k-light chain restriction identical to the primary tumor. The cell line has a modal chromosomal number of 46 and harbors chromosomal changes such as deletion of 6q21, monoallelic deletion of 9p21 (CDKN2A), 13q14 (RB1) and 18q21 (BCL-2) with a consistent amplification of 14q32 (IgH) identical to its founding tumor sample. Clonal relationship was confirmed by identical CDR3 length and single nucleotide polymorphisms as well as a matching IgH sequence of the cell line and founding tumor. Both also harbor a heterozygous, non-synonymous mutation at amino acid 265 in MYD88 gene (L265P). The cell line expresses most of the cell surface markers present on the parent cells. Over all, RPCI-WM1 represents a valuable model to study WM. PMID:22812491
Development and characterization of a novel human Waldenström macroglobulinemia cell line: RPCI-WM1, Roswell Park Cancer Institute - Waldenström Macroglobulinemia 1.

PubMed

Chitta, Kasyapa S; Paulus, Aneel; Ailawadhi, Sikander; Foster, Barbara A; Moser, Michael T; Starostik, Petr; Masood, Aisha; Sher, Taimur; Miller, Kena C; Iancu, Dan M; Conroy, Jeffrey; Nowak, Norma J; Sait, Sheila N; Personett, David A; Coleman, Morton; Furman, Richard R; Martin, Peter; Ansell, Stephen M; Lee, Kelvin; Chanan-Khan, Asher A

2013-02-01

Understanding the biology of Waldenström macroglobulinemia is hindered by a lack of preclinical models. We report a novel cell line, RPCI-WM1, from a patient treated for WM. The cell line secretes human immunoglobulin M (h-IgM) with κ-light chain restriction identical to the primary tumor. The cell line has a modal chromosomal number of 46 and harbors chromosomal changes such as deletion of 6q21, monoallelic deletion of 9p21 (CDKN2A), 13q14 (RB1) and 18q21 (BCL-2), with a consistent amplification of 14q32 (immunoglobulin heavy chain; IgH) identical to its founding tumor sample. The clonal relationship is confirmed by identical CDR3 length and single nucleotide polymorphisms as well as a matching IgH sequence of the cell line and founding tumor. Both also harbor a heterozygous, non-synonymous mutation at amino acid 265 in the MYD88 gene (L265P). The cell line expresses most of the cell surface markers present on the parent cells. Overall, RPCI-WM1 represents a valuable model to study Waldenström macroglobulinemia.
Lifestyle behaviors and ethnic identity among diverse women at high risk for type 2 diabetes.

PubMed

Brown, Susan D; Ehrlich, Samantha F; Kubo, Ai; Tsai, Ai-Lin; Hedderson, Monique M; Quesenberry, Charles P; Ferrara, Assiamira

2016-07-01

Diet and physical activity lifestyle behaviors are modifiable risk factors for type 2 diabetes and are shaped by culture, potentially influencing diabetes health disparities. We examined whether ethnic identity-the strength of attachment to one's ethnic group, and a long-standing focus of psychological research-could help account for variations in lifestyle behaviors within a diverse population at high risk for chronic disease. Using data from the Gestational Diabetes' Effects on Moms trial, this US-based cross-sectional study included 1463 pregnant women (74% from minority ethnic/racial groups; 46% born outside the US) with gestational diabetes (GDM), a common pregnancy complication conferring high risk for type 2 diabetes after delivery. Mixed linear regression models examined whether ethnic identity is associated with lifestyle behaviors after adjusting for demographic, clinical, and acculturative characteristics (e.g., nativity and length of residence in the US). In the overall sample, a one-unit increase in ethnic identity score was significantly associated with 3% greater fiber intake, 4% greater fruit/vegetable intake, 11% greater total activity, and 11% greater walking (p values < 0.01). Within ethnic/racial groups, a one-unit increase in ethnic identity score was significantly associated with 17% greater fiber intake among Filipina women; 5% lower total caloric intake among non-Hispanic White women; and 40% greater total activity, 35% greater walking, and 8% greater total caloric intake among Latina women (p values ≤ 0.03). Results from this large study suggest that ethnic group attachment is associated with some lifestyle behaviors, independent of acculturation indicators, among young women with GDM who are at high risk for type 2 diabetes. Stronger ethnic identity may promote certain choices known to be associated with reduced risk of type 2 diabetes. Prospective research is needed to clarify the temporal nature of associations between ethnic identity and modifiable diabetes risk factors. Copyright © 2016 Elsevier Ltd. All rights reserved.
The use of embryonic cells in the treatment of osteochondral defects of the knee: an ovine in vivo study.

PubMed

Manunta, Andrea Fabio; Zedde, Pietro; Pilicchi, Susanna; Rocca, Stefano; Pool, Roy R; Dattena, Maria; Masala, Gerolamo; Mara, Laura; Casu, Sara; Sanna, Daniela; Manunta, Maria Lucia; Passino, Eraldo Sanna

2016-01-01

the aim of this study was to determine whether local delivery of embryonic stem-like (ESL) cells into osteochondral defects in the femoral condyles of sheep would enhance regeneration of hyaline articular cartilage. male ESL cells embedded in fibrin glue were engrafted into osteochondral defects in the medial condyles (ESL-M) of the left femur in 22 ewes. An identical defect was created in the medial condyle of the contralateral stifle joint and left untreated as a control (empty defect, ED). The ewes were divided into 5 groups. Four sheep each were euthanized at 1, 2, 6, and 12 months from surgery, and 6 ewes were euthanized 24 months post-implantation. To study the effect of varying loads on the long-term regeneration process, an identical defect was also created and ESL cell engraftment performed in the lateral condyle (ESL-L) of the left stifle joint of the animals in the 12- and 24-month groups. The evaluation of regenerated tissue was performed by biomechanical, macroscopic, histological, immunohistochemical (collagen type II) and fluorescent in situ hybridization (FISH) assays. no significant differences were found between treated and control sites in the biomechanical assays at any time point. ESL cell grafts showed significantly greater macroscopic evidence of regeneration as compared to controls at 24 months after surgery; significantly better histological evidence of repair in ESL-M samples versus controls was found throughout the considered period. At 24 months from surgery there was significantly improved integration of graft edges with the host tissue in the ESL-M as compared to the ESL-L samples, demonstrating that load bearing positively affects the long-term regeneration process. ESL cells enhanced the regeneration of hyaline cartilage. FISH confirmed that the regenerative tissue originated from ESL cells. ESL cells are able to self-renew for prolonged periods without differentiation and, most importantly, to differentiate into a large variety of tissues.
The use of embryonic cells in the treatment of osteochondral defects of the knee: an ovine in vivo study

PubMed Central

MANUNTA, ANDREA FABIO; ZEDDE, PIETRO; PILICCHI, SUSANNA; ROCCA, STEFANO; POOL, ROY R.; DATTENA, MARIA; MASALA, GEROLAMO; MARA, LAURA; CASU, SARA; SANNA, DANIELA; MANUNTA, MARIA LUCIA; PASSINO, ERALDO SANNA

2016-01-01

Purpose the aim of this study was to determine whether local delivery of embryonic stem-like (ESL) cells into osteochondral defects in the femoral condyles of sheep would enhance regeneration of hyaline articular cartilage. Methods male ESL cells embedded in fibrin glue were engrafted into osteochondral defects in the medial condyles (ESL-M) of the left femur in 22 ewes. An identical defect was created in the medial condyle of the contralateral stifle joint and left untreated as a control (empty defect, ED). The ewes were divided into 5 groups. Four sheep each were euthanized at 1, 2, 6, and 12 months from surgery, and 6 ewes were euthanized 24 months post-implantation. To study the effect of varying loads on the long-term regeneration process, an identical defect was also created and ESL cell engraftment performed in the lateral condyle (ESL-L) of the left stifle joint of the animals in the 12- and 24-month groups. The evaluation of regenerated tissue was performed by biomechanical, macroscopic, histological, immunohistochemical (collagen type II) and fluorescent in situ hybridization (FISH) assays. Results no significant differences were found between treated and control sites in the biomechanical assays at any time point. ESL cell grafts showed significantly greater macroscopic evidence of regeneration as compared to controls at 24 months after surgery; significantly better histological evidence of repair in ESL-M samples versus controls was found throughout the considered period. At 24 months from surgery there was significantly improved integration of graft edges with the host tissue in the ESL-M as compared to the ESL-L samples, demonstrating that load bearing positively affects the long-term regeneration process. Conclusions ESL cells enhanced the regeneration of hyaline cartilage. FISH confirmed that the regenerative tissue originated from ESL cells. Clinical Relevance ESL cells are able to self-renew for prolonged periods without differentiation and, most importantly, to differentiate into a large variety of tissues. PMID:27602346
British-Pakistani women's perspectives of diabetes self-management: the role of identity.

PubMed

Majeed-Ariss, Rabiya; Jackson, Cath; Knapp, Peter; Cheater, Francine M

2015-09-01

To explore the effects of type 2 diabetes on British-Pakistani women's identity and its relationship with self-management. Type 2 diabetes is more prevalent and has worse outcomes among some ethnic minority groups. This may be due to poorer self-management and an inadequate match of health services to patient needs. The influence that type 2 diabetes has on British-Pakistani women's identity and subsequent self-management has received limited attention. An explorative qualitative study. Face-to-face semi-structured English and Urdu language interviews were conducted with a purposively selected heterogeneous sample of 15 British-Pakistani women with type 2 diabetes. Transcripts were analysed thematically. Four themes emerged: Perceived change in self emphasised how British-Pakistani women underwent a conscious adaptation of identity following diagnosis; Familiarity with ill health reflected women's adjustment to their changed identity over time; Diagnosis improves social support enabled women to accept changes within themselves and Supporting family is a barrier to self-management demonstrated how family roles were an aspect of women's identities that was resilient to change. The over-arching theme Role re-alignment enables successful self-management encapsulated how self-management was a continuous process where achievements needed to be sustained. Inter-generational differences were also noted: first generation women talked about challenges associated with ageing and co-morbidities; second generation women talked about familial and work roles competing with self-management. The complex nature of British-Pakistani women's self-identification requires consideration when planning and delivering healthcare. Culturally competent practice should recognise how generational status influences self-identity and diabetes self-management in ethnically diverse women. Health professionals should remain mindful of effective self-management occurring alongside, and being influenced by, other aspects of life. © 2015 John Wiley & Sons Ltd.

Myogenic tone is impaired at low arterial pressure in mice deficient in the low-voltage-activated CaV 3.1 T-type Ca(2+) channel.

PubMed

Björling, K; Morita, H; Olsen, M F; Prodan, A; Hansen, P B; Lory, P; Holstein-Rathlou, N-H; Jensen, L J

2013-04-01

Using mice deficient in the CaV 3.1 T-type Ca(2+) channel, the aim of the present study was to elucidate the molecular identity of non-L-type channels involved in vascular tone regulation in mesenteric arteries and arterioles. We used immunofluorescence microscopy to localize CaV 3.1 channels, patch clamp electrophysiology to test the effects of a putative T-type channel blocker NNC 55-0396 on whole-cell Ca(2+) currents, pressure myography and Ca(2+) imaging to test diameter and Ca(2+) responses of the applied vasoconstrictors, and Q-PCR to check mRNA expression levels of several Ca(2+) handling proteins in wild-type and CaV 3.1(-/-) mice. Our data indicated that CaV 3.1 channels are important for the maintenance of myogenic tone at low pressures (40-80 mm Hg), whereas they are not involved in high-voltage-activated Ca(2+) currents, Ca(2+) entry or vasoconstriction to high KCl in mesenteric arteries and arterioles. Furthermore, we show that NNC 55-0396 is not a specific T-type channel inhibitor, as it potently blocks L-type and non-L-type high-voltage-activated Ca(2+) currents in mouse mesenteric vascular smooth muscle cell. Our data using mice deficient in the CaV 3.1 T-type channel represent new evidence for the involvement of non-L-type channels in arteriolar tone regulation. We showed that CaV 3.1 channels are important for the myogenic tone at low arterial pressure, which is potentially relevant under resting conditions in vivo. Moreover, CaV 3.1 channels are not involved in Ca(2+) entry and vasoconstriction to large depolarization with, for example, high KCl. Finally, we caution against using NNC 55-0396 as a specific T-type channel blocker in native cells expressing high-voltage-activated Ca(2+) channels. Acta Physiologica © 2013 Scandinavian Physiological Society.
Rapid and highly efficient construction of TALE-based transcriptional regulators and nucleases for genome modification.

PubMed

Li, Lixin; Piatek, Marek J; Atef, Ahmed; Piatek, Agnieszka; Wibowo, Anjar; Fang, Xiaoyun; Sabir, J S M; Zhu, Jian-Kang; Mahfouz, Magdy M

2012-03-01

Transcription activator-like effectors (TALEs) can be used as DNA-targeting modules by engineering their repeat domains to dictate user-selected sequence specificity. TALEs have been shown to function as site-specific transcriptional activators in a variety of cell types and organisms. TALE nucleases (TALENs), generated by fusing the FokI cleavage domain to TALE, have been used to create genomic double-strand breaks. The identity of the TALE repeat variable di-residues, their number, and their order dictate the DNA sequence specificity. Because TALE repeats are nearly identical, their assembly by cloning or even by synthesis is challenging and time consuming. Here, we report the development and use of a rapid and straightforward approach for the construction of designer TALE (dTALE) activators and nucleases with user-selected DNA target specificity. Using our plasmid set of 100 repeat modules, researchers can assemble repeat domains for any 14-nucleotide target sequence in one sequential restriction-ligation cloning step and in only 24 h. We generated several custom dTALEs and dTALENs with new target sequence specificities and validated their function by transient expression in tobacco leaves and in vitro DNA cleavage assays, respectively. Moreover, we developed a web tool, called idTALE, to facilitate the design of dTALENs and the identification of their genomic targets and potential off-targets in the genomes of several model species. Our dTALE repeat assembly approach along with the web tool idTALE will expedite genome-engineering applications in a variety of cell types and organisms including plants.
Sequence and RT-PCR expression analysis of two peroxidases from Arabidopsis thaliana belonging to a novel evolutionary branch of plant peroxidases.

PubMed

Kjaersgård, I V; Jespersen, H M; Rasmussen, S K; Welinder, K G

1997-03-01

cDNA clones encoding two new Arabidopsis thaliana peroxidases, ATP 1a and ATP 2a, have been identified by searching the Arabidopsis database of expressed sequence tags (dbEST). They represent a novel branch of hitherto uncharacterized plant peroxidases which is only 35% identical in amino acid sequence to the well characterized group of basic plant peroxidases represented by the horseradish (Armoracia rusticana) isoperoxidases HRP C, HRP E5 and the similar Arabidopsis isoperoxidases ATP Ca, ATP Cb, and ATP Ea. However ATP 1a is 87% identical in amino acid sequence to a peroxidase encoded by an mRNA isolated from cotton (Gossypium hirsutum). As cotton and Arabidopsis belong to rather diverse families (Malvaceae and Crucifereae, respectively), in contrast with Arabidopsis and horseradish (both Crucifereae), the high degree of sequence identity indicates that this novel type of peroxidase, albeit of unknown function, is likely to be widespread in plant species. The atp 1 and atp 2 types of cDNA sequences were the most redundant among the 28 different isoperoxidases identified among about 200 peroxidase encoding ESTs. Interestingly, 8 out of totally 38 EST sequences coding for ATP 1 showed three identical nucleotide substitutions. This variant form is designated ATP 1b. Similarly, six out of totally 16 EST sequences coding for ATP 2 showed a number of deletions and nucleotide changes. This variant form is designated ATP 2b. The selected EST clones are full-length and contain coding regions of 993 nucleotides for atp 1a, and 984 nucleotides for atp 2a. These regions show 61% DNA sequence identity. The predicted mature proteins ATP 1a, and ATP 2a are 57% identical in sequence and contain the structurally and functionally important residues, characteristic of the plant peroxidase superfamily. However, they do show two differences of importance to peroxidase catalysis: (1) the asparagine residue linked with the active site distal histidine via hydrogen bonding is absent; (2) an N-glycosylation site is located right at the entrance to the heme channel. The reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify mRNAs coding for ATP 1a/b and ATP 2a/b in germinating seeds, seedlings, roots, leaves, stems, flowers and cell suspension culture using elongation factor 1alpha (EF-1alpha) for the first time as a positive control. Both mRNAs were transcribed at levels comparable to EF-1alpha in all plant tissues investigated which were more than two days old, and in cell suspension culture. In addition, the mRNA coding for ATP 1a/b was found in two day old germinating seeds. The abundant transcription of ATP 1a/b and ATP 2a/b is in line with their many entries in dbEST, and indicates essential roles for these novel peroxidases.
Functional conservation of atonal and Math1 in the CNS and PNS

NASA Technical Reports Server (NTRS)

Ben-Arie, N.; Hassan, B. A.; Bermingham, N. A.; Malicki, D. M.; Armstrong, D.; Matzuk, M.; Bellen, H. J.; Zoghbi, H. Y.

2000-01-01

To determine the extent to which atonal and its mouse homolog Math1 exhibit functional conservation, we inserted (beta)-galactosidase (lacZ) into the Math1 locus and analyzed its expression, evaluated consequences of loss of Math1 function, and expressed Math1 in atonal mutant flies. lacZ under the control of Math1 regulatory elements duplicated the previously known expression pattern of Math1 in the CNS (i.e., the neural tube, dorsal spinal cord, brainstem, and cerebellar external granule neurons) but also revealed new sites of expression: PNS mechanoreceptors (inner ear hair cells and Merkel cells) and articular chondrocytes. Expressing Math1 induced ectopic chordotonal organs (CHOs) in wild-type flies and partially rescued CHO loss in atonal mutant embryos. These data demonstrate that both the mouse and fly homologs encode lineage identity information and, more interestingly, that some of the cells dependent on this information serve similar mechanoreceptor functions.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Greene, Carol Ann, E-mail: carol.greene@auckland.ac.nz; Chang, Chuan-Yuan; Fraser, Cameron J.

Cells thought to be stem cells isolated from the cornea of the eye have been shown to exhibit neurogenic potential. We set out to uncover the identity and location of these cells within the cornea and to elucidate their neuronal protein and gene expression profile during the process of switching to a neuron-like cell. Here we report that every cell of the adult human and rat corneal stroma is capable of differentiating into a neuron-like cell when treated with neurogenic differentiation specifying growth factors. Furthermore, the expression of genes regulating neurogenesis and mature neuronal structure and function was increased. Themore » switch from a corneal stromal cell to a neuron-like cell was also shown to occur in vivo in intact corneas of living rats. Our results clearly indicate that lineage specifying growth factors can affect changes in the protein and gene expression profiles of adult cells, suggesting that possibly many adult cell populations can be made to switch into another type of mature cell by simply modifying the growth factor environment. - Highlights: • Adult corneal stromal cells can differentiated into neuron-like cells. • Neuronal specification of the adult stromal cell population is stochastic. • Neuronal specification in an adult cell population can be brought about by growth factors.« less
Nutrient Shielding in Clusters of Cells

PubMed Central

Lavrentovich, Maxim O.; Koschwanez, John H.; Nelson, David R.

2014-01-01

Cellular nutrient consumption is influenced by both the nutrient uptake kinetics of an individual cell and the cells’ spatial arrangement. Large cell clusters or colonies have inhibited growth at the cluster's center due to the shielding of nutrients by the cells closer to the surface. We develop an effective medium theory that predicts a thickness ℓ of the outer shell of cells in the cluster that receives enough nutrient to grow. The cells are treated as partially absorbing identical spherical nutrient sinks, and we identify a dimensionless parameter ν that characterizes the absorption strength of each cell. The parameter ν can vary over many orders of magnitude between different cell types, ranging from bacteria and yeast to human tissue. The thickness ℓ decreases with increasing ν, increasing cell volume fraction ϕ, and decreasing ambient nutrient concentration ψ∞. The theoretical results are compared with numerical simulations and experiments. In the latter studies, colonies of budding yeast, Saccharomyces cerevisiae, are grown on glucose media and imaged under a confocal microscope. We measure the growth inside the colonies via a fluorescent protein reporter and compare the experimental and theoretical results for the thickness ℓ. PMID:23848711
An Atypical Acidophil Cell Line Tumor Showing Focal Differentiation Toward Both Growth Hormone and Prolactin Cells.

PubMed

Naritaka, Heiji; Kameya, Toru; Sato, Yuichi; Furuhata, Shigeru; Okui, Junichi; Kamiguchi, Yuji; Otani, Mitsuhiro; Toya, Shigeo

1995-01-01

We report a case of giant pituitary adenoma in a child. Computerized tomography (CT) scan revealed a suprasellar extension tumor mass with hydrocephalus. There was no clinical evidence of acromegaly, gigantism, and other hormonal symptoms. Endocrinologic studies showed within normal value of serum growth hormone (GH: 4.2 ng/mL) and slightly increased levels of prolactin (PRL: 78 ng/mL) and other pituitary hormone values were within normal range. On suppression test by bromocryptin, both GH and PRL levels were reduced. Histopathological findings revealed that the tumor consisted of predominantly chromophobic and partly eosinophilic adenoma cells. Immunohistochemical staining detected GH and PRL in a small number of distinctly different adenoma cells, respectively. Nonradioactive in situ hybridization (ISH) also showed GH and PRL mRNA expression in identical immunopositive cells. Electron microscopy (EM) demonstrated adenoma cells with moderate or small numbers of two types of dense granules and without fibrous body which are characteristic of sparsely granulated GH-cell adenomas. The adenoma does not fit into any classification but may be an atypical acidophil cell line tumor showing focal differentiation toward both GH and PRL cells.
An improved protocol that induces human embryonic stem cells to differentiate into neural cells in vitro.

PubMed

Zhou, Jun-Mei; Chu, Jian-Xin; Chen, Xue-Jin

2008-01-01

Human embryonic stem (ES) cells have the capacity for self-renewal and are able to differentiate into any cell type. However, obtaining high-efficient neural differentiation from human ES cells remains a challenge. This study describes an improved 4-stage protocol to induce a human ES cell line derived from a Chinese population to differentiate into neural cells. At the first stage, embryonic bodies (EBs) were formed in a chemically-defined neural inducing medium rather than in traditional serum or serum-replacement medium. At the second stage, rosette-like structures were formed. At the third stage, the rosette-like structures were manually selected rather than enzymatically digested to form floating neurospheres. At the fourth stage, the neurospheres were further differentiated into neurons. The results show that, at the second stage, the rate of the formation of rosette-like structures from EBs induced by noggin was 88+/-6.32%, higher than that of retinoic acid 55+/-5.27%. Immunocytochemistry staining was used to confirm the neural identity of the cells. These results show a major improvement in obtaining efficient neural differentiation of human ES cells.
Upright Imaging of Drosophila Egg Chambers

PubMed Central

Manning, Lathiena; Starz-Gaiano, Michelle

2015-01-01

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective. PMID:25867882
Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays

PubMed Central

2011-01-01

Background Engineered nanomaterials display unique properties that may have impact on human health, and thus require a reliable evaluation of their potential toxicity. Here, we performed a standardized in vitro screening of 23 engineered nanomaterials. We thoroughly characterized the physicochemical properties of the nanomaterials and adapted three classical in vitro toxicity assays to eliminate nanomaterial interference. Nanomaterial toxicity was assessed in ten representative cell lines. Results Six nanomaterials induced oxidative cell stress while only a single nanomaterial reduced cellular metabolic activity and none of the particles affected cell viability. Results from heterogeneous and chemically identical particles suggested that surface chemistry, surface coating and chemical composition are likely determinants of nanomaterial toxicity. Individual cell lines differed significantly in their response, dependent on the particle type and the toxicity endpoint measured. Conclusion In vitro toxicity of the analyzed engineered nanomaterials cannot be attributed to a defined physicochemical property. Therefore, the accurate identification of nanomaterial cytotoxicity requires a matrix based on a set of sensitive cell lines and in vitro assays measuring different cytotoxicity endpoints. PMID:21345205
Full Genome Characterisation of Bluetongue Virus Serotype 6 from the Netherlands 2008 and Comparison to Other Field and Vaccine Strains

PubMed Central

Maan, Sushila; Maan, Narender S.; van Rijn, Piet A.; van Gennip, René G. P.; Sanders, Anna; Wright, Isabel M.; Batten, Carrie; Hoffmann, Bernd; Eschbaumer, Michael; Oura, Chris A. L.; Potgieter, Abraham C.; Nomikou, Kyriaki; Mertens, Peter P.C.

2010-01-01

In mid September 2008, clinical signs of bluetongue (particularly coronitis) were observed in cows on three different farms in eastern Netherlands (Luttenberg, Heeten, and Barchem), two of which had been vaccinated with an inactivated BTV-8 vaccine (during May-June 2008). Bluetongue virus (BTV) infection was also detected on a fourth farm (Oldenzaal) in the same area while testing for export. BTV RNA was subsequently identified by real time RT-PCR targeting genome-segment (Seg-) 10, in blood samples from each farm. The virus was isolated from the Heeten sample (IAH “dsRNA virus reference collection” [dsRNA-VRC] isolate number NET2008/05) and typed as BTV-6 by RT-PCR targeting Seg-2. Sequencing confirmed the virus type, showing an identical Seg-2 sequence to that of the South African BTV-6 live-vaccine-strain. Although most of the other genome segments also showed very high levels of identity to the BTV-6 vaccine (99.7 to 100%), Seg-10 showed greatest identity (98.4%) to the BTV-2 vaccine (RSAvvv2/02), indicating that NET2008/05 had acquired a different Seg-10 by reassortment. Although Seg-7 from NET2008/05 was also most closely related to the BTV-6 vaccine (99.7/100% nt/aa identity), the Seg-7 sequence derived from the blood sample of the same animal (NET2008/06) was identical to that of the Netherlands BTV-8 (NET2006/04 and NET2007/01). This indicates that the blood contained two different Seg-7 sequences, one of which (from the BTV-6 vaccine) was selected during virus isolation in cell-culture. The predominance of the BTV-8 Seg-7 in the blood sample suggests that the virus was in the process of reassorting with the northern field strain of BTV-8. Two genome segments of the virus showed significant differences from the BTV-6 vaccine, indicating that they had been acquired by reassortment event with BTV-8, and another unknown parental-strain. However, the route by which BTV-6 and BTV-8 entered northern Europe was not established. PMID:20428242
Artificial cloning of domestic animals

PubMed Central

Keefer, Carol L.

2015-01-01

Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research. PMID:26195770
Artificial cloning of domestic animals.

PubMed

Keefer, Carol L

2015-07-21

Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research.
Fact or factitious? A psychobiological study of authentic and simulated dissociative identity states.

PubMed

Reinders, A A T S; Reinders, A A T Simone; Willemsen, Antoon T M; Vos, Herry P J; den Boer, Johan A; Nijenhuis, Ellert R S

2012-01-01

Dissociative identity disorder (DID) is a disputed psychiatric disorder. Research findings and clinical observations suggest that DID involves an authentic mental disorder related to factors such as traumatization and disrupted attachment. A competing view indicates that DID is due to fantasy proneness, suggestibility, suggestion, and role-playing. Here we examine whether dissociative identity state-dependent psychobiological features in DID can be induced in high or low fantasy prone individuals by instructed and motivated role-playing, and suggestion. DID patients, high fantasy prone and low fantasy prone controls were studied in two different types of identity states (neutral and trauma-related) in an autobiographical memory script-driven (neutral or trauma-related) imagery paradigm. The controls were instructed to enact the two DID identity states. Twenty-nine subjects participated in the study: 11 patients with DID, 10 high fantasy prone DID simulating controls, and 8 low fantasy prone DID simulating controls. Autonomic and subjective reactions were obtained. Differences in psychophysiological and neural activation patterns were found between the DID patients and both high and low fantasy prone controls. That is, the identity states in DID were not convincingly enacted by DID simulating controls. Thus, important differences regarding regional cerebral bloodflow and psychophysiological responses for different types of identity states in patients with DID were upheld after controlling for DID simulation. The findings are at odds with the idea that differences among different types of dissociative identity states in DID can be explained by high fantasy proneness, motivated role-enactment, and suggestion. They indicate that DID does not have a sociocultural (e.g., iatrogenic) origin.
Emergence of Serotonergic Neurons After Spinal Cord Injury in Turtles

PubMed Central

Fabbiani, Gabriela; Rehermann, María I.; Aldecosea, Carina; Trujillo-Cenóz, Omar; Russo, Raúl E.

2018-01-01

Plasticity of neural circuits takes many forms and plays a fundamental role in regulating behavior to changing demands while maintaining stability. For example, during spinal cord development neurotransmitter identity in neurons is dynamically adjusted in response to changes in the activity of spinal networks. It is reasonable to speculate that this type of plasticity might occur also in mature spinal circuits in response to injury. Because serotonergic signaling has a central role in spinal cord functions, we hypothesized that spinal cord injury (SCI) in the fresh water turtle Trachemys scripta elegans may trigger homeostatic changes in serotonergic innervation. To test this possibility we performed immunohistochemistry for serotonin (5-HT) and key molecules involved in the determination of the serotonergic phenotype before and after SCI. We found that as expected, in the acute phase after injury the dense serotonergic innervation was strongly reduced. However, 30 days after SCI the population of serotonergic cells (5-HT+) increased in segments caudal to the lesion site. These cells expressed the neuronal marker HuC/D and the transcription factor Nkx6.1. The new serotonergic neurons did not incorporate the thymidine analog 5-bromo-2′-deoxyuridine (BrdU) and did not express the proliferating cell nuclear antigen (PCNA) indicating that novel serotonergic neurons were not newborn but post-mitotic cells that have changed their neurochemical identity. Switching towards a serotonergic neurotransmitter phenotype may be a spinal cord homeostatic mechanism to compensate for the loss of descending serotonergic neuromodulation, thereby helping the outstanding functional recovery displayed by turtles. The 5-HT1A receptor agonist (±)-8-Hydroxy-2-dipropylaminotetralin hydrobromide (8-OH-DPAT) blocked the increase in 5-HT+ cells suggesting 5-HT1A receptors may trigger the respecification process. PMID:29593503
Cloning of the Gene Encoding a 22-Kilodalton Cell Surface Antigen of Mycobacterium bovis BCG and Analysis of Its Potential for DNA Vaccination against Tuberculosis

PubMed Central

Lefèvre, Philippe; Denis, Olivier; De Wit, Lucas; Tanghe, Audrey; Vandenbussche, Paul; Content, Jean; Huygen, Kris

2000-01-01

Using spleen cells from mice vaccinated with live Mycobacterium bovis BCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687–2693, 1993). These monoclonal antibodies were used to screen an M. bovis BCG genomic library made in phage λgt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosis Lppx protein and that it contained a sequence 94% identical with the M. leprae 38-mer polypeptide 13B3 recognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenous M. tuberculosis challenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenous M. tuberculosis challenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test. PMID:10678905
Change in Ethnic Identity across the College Transition

ERIC Educational Resources Information Center

Tsai, Kim M.; Fuligni, Andrew J.

2012-01-01

This article examined changes in ethnic identity as a function of college type and residential status and whether differences due to college type could be explained by involvement in extracurricular activities and college ethnic composition. Although no changes in ethnic labeling or belonging were found, there was a normative decrease in ethnic…
Relationship between English Learning Motivation Types and Self-Identity Changes among Chinese Students

ERIC Educational Resources Information Center

Yihong, Gao; Yuan, Zhao; Ying, Cheng; Yan, Zhou

2007-01-01

This study investigated the relationship between English learning motivation types and self-identity changes among university students in the People's Republic of China. The sample obtained from a stratified sampling consisted of 2,278 undergraduates from 30 universities in 29 regions. The instrument was a Likert-scale questionnaire which included…
The C-terminal HRET sequence of Kv1.3 regulates gating rather than targeting of Kv1.3 to the plasma membrane.

PubMed

Voros, Orsolya; Szilagyi, Orsolya; Balajthy, András; Somodi, Sándor; Panyi, Gyorgy; Hajdu, Péter

2018-04-12

Kv1.3 channels are expressed in several cell types including immune cells, such as T lymphocytes. The targeting of Kv1.3 to the plasma membrane is essential for T cell clonal expansion and assumed to be guided by the C-terminus of the channel. Using two point mutants of Kv1.3 with remarkably different features compared to the wild-type Kv1.3 (A413V and H399K having fast inactivation kinetics and tetraethylammonium-insensitivity, respectively) we showed that both Kv1.3 channel variants target to the membrane when the C-terminus was truncated right after the conserved HRET sequence and produce currents identical to those with a full-length C-terminus. The truncation before the HRET sequence (NOHRET channels) resulted in reduced membrane-targeting but non-functional phenotypes. NOHRET channels did not display gating currents, and coexpression with wild-type Kv1.3 did not rescue the NOHRET-A413V phenotype, no heteromeric current was observed. Interestingly, mutants of wild-type Kv1.3 lacking HRET(E) (deletion) or substituted with five alanines for the HRET(E) motif expressed current indistinguishable from the wild-type. These results demonstrate that the C-terminal region of Kv1.3 immediately proximal to the S6 helix is required for the activation gating and conduction, whereas the presence of the distal region of the C-terminus is not exclusively required for trafficking of Kv1.3 to the plasma membrane.
Learning, memory and exploratory similarities in genetically identical cloned dogs.

PubMed

Shin, Chi Won; Kim, Geon A; Park, Won Jun; Park, Kwan Yong; Jeon, Jeong Min; Oh, Hyun Ju; Kim, Min Jung; Lee, Byeong Chun

2016-12-30

Somatic cell nuclear transfer allows generation of genetically identical animals using donor cells derived from animals with particular traits. To date, few studies have investigated whether or not these cloned dogs will show identical behavior patterns. To address this question, learning, memory and exploratory patterns were examined using six cloned dogs with identical nuclear genomes. The variance of total incorrect choice number in the Y-maze test among cloned dogs was significantly lower than that of the control dogs. There was also a significant decrease in variance in the level of exploratory activity in the open fields test compared to age-matched control dogs. These results indicate that cloned dogs show similar cognitive and exploratory patterns, suggesting that these behavioral phenotypes are related to the genotypes of the individuals.

[Risk and crisis management by anesthesiologists regarding 'Guidelines for Actions Against Intraoperative Critical Hemorrhage' published by the Japanese Society of Anesthesiologists and the Japan Society of Transfusion Medicine and Cell Therapy].

PubMed

Irita, Kazuo; Yoshimura, Hayashi; Sakaguchi, Yoshiro; Takamatsu, Chihiro; Tokuda, Kentaro

2008-09-01

According to a survey of anesthesia-related critical incidents by the Japanese Society of Anesthesiologists, hemorrhage was the major cause of cardiac arrest developing in the operating room. To deal with critical hemorrhage swiftly, not only cooperation between anesthesiologists and surgeons but also the linkage of operating rooms with transfusion management divisions and the blood center is important. It is desirable for the hospital transfusion committee to prepare hospital regulations on 'actions to be taken to manage critical hemorrhage', and practice the implementation of these guidelines by simulated drills. When critical hemorrhage occurs, a person in charge is appointed, and an emergency is declared (call for manpower and notification of the emergency to the transfusion management divisions). A person in charge comprehensively assesses the hemostatic condition, hemodynamics, laboratory data, and blood product supply system, and consults the operator regarding the continuation of surgery or changing surgical procedures. When time is short, the cross-matching test is omitted, and the ABO-identical blood is used. When a supply of the identical ABO-type blood is not available, compatible blood type is used. The evolving concept of hemostatic resuscitation seems to be important to prevent coagulopathy, which easily develops during massive hemorrhage. Anesthesiologists should be aware of the risk of such an emergency transfusion and procedures to be taken to switch to transfusion of the ABO-identical blood. Establishment of a hospital emergency transfusion system depends on the overall capability of the critical and crisis management systems of the hospital.
Mismatch for the minor histocompatibility antigen HA-2 and GVHD occurrence in HLA-A*0201-positive Tunisian recipients of HSCs.

PubMed

Sellami, Mohamed Hichem; Torjemane, Lamia; Espadas de Arias, Alejandro; Kaabi, Houda; Ladeb, Saloua; Ben Othman, Tarek; Poli, Francesca; Hmida, Slama

2010-01-01

Graft-versus-Host disease (GVHD) has been widely linked to immunogenetic causes such as disparity between the recipient and its HLA geno-identical donor for some Non-HLA antigens called minor histocompatibility antigens (MiHAgs). HA-2 is one of potential human MiHAgs but its effect on the GVHD occurrence remains not clear. In order to examine such association in the Tunisian cohort of HSCs recipients, we performed a retrospective study on patients who received an HLA-identical HSCT between 2000 and 2009. The study was performed on 60 HLA-A2-positive patients who had received a haematopoietic stem cell transplant from an HLA-identical sibling. All patients received cyclosporine A and/or methotrexate for GVHD prophylaxis. HA-2 genotyping assay was performed with SSP-PCR method and HLA-A*0201 positive samples were identified mainly with Luminex HLA-Typing method. Luminex HLA-Typing assay showed that only 53 cases were positives for the HLA-A*0201 allele. Among these cases, only 3 pairs were mismatched for the MiHAg HA-2. Acute GVHD occurred in 01 HA-2-mismatched pair while chronic GVHD was detected in 02 disparate couples. Univariate and multivariate analyses showed that MiHAg HA-2 disparity does not have any significant effect on the occurrence of either acute or chronic GVHD. This last one appeared to be correlated only with the age of patient (adulthood) (p: 0.011, OR: 22.092). Our findings support the previously reported data denying the influence of the HA-2 disparity on the GVHD occurrence after HSCT.
Enhancer of polycomb coordinates multiple signaling pathways to promote both cyst and germline stem cell differentiation in the Drosophila adult testis

PubMed Central

Feng, Lijuan; Shi, Zhen; Chen, Xin

2017-01-01

Stem cells reside in a particular microenvironment known as a niche. The interaction between extrinsic cues originating from the niche and intrinsic factors in stem cells determines their identity and activity. Maintenance of stem cell identity and stem cell self-renewal are known to be controlled by chromatin factors. Herein, we use the Drosophila adult testis which has two adult stem cell lineages, the germline stem cell (GSC) lineage and the cyst stem cell (CySC) lineage, to study how chromatin factors regulate stem cell differentiation. We find that the chromatin factor Enhancer of Polycomb [E(Pc)] acts in the CySC lineage to negatively control transcription of genes associated with multiple signaling pathways, including JAK-STAT and EGF, to promote cellular differentiation in the CySC lineage. E(Pc) also has a non-cell-autonomous role in regulating GSC lineage differentiation. When E(Pc) is specifically inactivated in the CySC lineage, defects occur in both germ cell differentiation and maintenance of germline identity. Furthermore, compromising Tip60 histone acetyltransferase activity in the CySC lineage recapitulates loss-of-function phenotypes of E(Pc), suggesting that Tip60 and E(Pc) act together, consistent with published biochemical data. In summary, our results demonstrate that E(Pc) plays a central role in coordinating differentiation between the two adult stem cell lineages in Drosophila testes. PMID:28196077
Global versus Local Regulatory Roles for Lrp-Related Proteins: Haemophilus influenzae as a Case Study

PubMed Central

Friedberg, Devorah; Midkiff, Michael; Calvo, Joseph M.

2001-01-01

Lrp (leucine-responsive regulatory protein) plays a global regulatory role in Escherichia coli, affecting expression of dozens of operons. Numerous lrp-related genes have been identified in different bacteria and archaea, including asnC, an E. coli gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated with E. coli Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein from Haemophilus influenzae that shares 75% sequence identity with E. coli Lrp (highest sequence identity among 42 sequences compared). A strain of H. influenzae having an lrfB null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies with lrp+ and lrp E. coli strains by others showed 20 differences). The abundance of LrfB in H. influenzae, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per E. coli cell). LrfB expressed in E. coli replaced Lrp as a repressor of the lrp gene but acted only to a limited extent as an activator of the ilvIH operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function. PMID:11395465
Initial blood storage experiment

NASA Technical Reports Server (NTRS)

Surgenor, Douglas MACN.

1988-01-01

The design of the Initial Blood Storage Experiment (IBSE) was based upon a carefully controlled comparison between identical sets of human blood cell suspensions - red cells, white cell, and platelets - one set of which was transported aboard the Columbia on a 6 day 11 hour mission, and the other held on the ground. Both sets were carried inside stainless steel dewars within specially fabricated flight hardware. Individual bags of cell suspensions were randomly assigned with respect to ground vs orbit status, dewar chamber, and specific location within the dewar. To foster optimal preservation, each cell type was held under specific optimal conditions of pH, ionic strength, solute concentration, gas tension, and temperature. An added variable in this initial experiment was provided by the use of three different polymer/plasticizer formulations for the sealed bags which held the blood cells. At termination of the experiment, aliquots of the suspensions, identified only by code, were distributed to be assayed. Assays were selected to constitute a broad survey of cellular properties and thereby maximize the chances of detection of gravitational effects. A total of 74 different outcome measurements were reported for statistical analysis. When the measurements were completed, the results were entered into the IBSE data base, at which time the data were matched with the original blood bag numbers to determine their status with respect to polymer/plasticizer type, orbit status (orbit or ground), and storage position within the experimental hardware. The data were studied by analysis of variance. Initially, type of bag and orbital status were main factors; later more detailed analyses were made on specific issues such as position in the hardware and specific plastic. If the analysis of variance indicated a statistical significance at the 5 percent level the corresponding p-value was reported.
Exploring the link between environmental identity, behaviors and decision making

NASA Astrophysics Data System (ADS)

Freed, Allison

This study was conducted with undergraduate students at a large university to investigate the association between environmental identity, pro-environmental behaviors and environmental decision-making. This study explored how environmental identity as defined by Clayton (2003) influenced the type of pro-environmental behaviors individuals choose to participate in. Environmental decision-making based on Kahneman's (2003, 2011) System 1 and System 2 framework was also assessed in association with environmental identity. A survey including the Environmental Identity Survey (Clayton, 2003), the Environmentally Responsible Behaviors Index (Smith-Sebasto & D'Acosta, 1995), and a Decision Making Questionnaire were administered. After administering the surveys, eight participants were chosen for a 60-minute interview. The quantitative results of the study showed there was a significant relationship between environmental identity and participating in environmental behaviors more often. There was also a significant relation between environmental identity and making the decision to recycle in a fast and automatic way. The interview results showed that participants with both a strong and a weak environmental identity recycled often and thought it was a fast decision. The results of this study show that certain components of environmental identity are important, but other factors like the physical environment and social norms influence the thinking that goes into recycling more than environmental identity alone. This study provides evidence of the importance of social norms and environmental structures in fostering pro-environmental behaviors and influencing the type of thinking that goes into making environmental decisions. Keywords: environmental identity, environmental behaviors, System 1, System 2, recycling.
Relevance of protein-protein interactions on the biological identity of nanoparticles.

PubMed

Vasti, Cecilia; Bonnet, Laura V; Galiano, Mauricio R; Rojas, Ricardo; Giacomelli, Carla E

2018-06-01

Considering that the use of nanoparticles (NPs) as carriers of therapeutic or theranostic agents has increased in the last years, it is mandatory to understand the interaction between NPs and living systems. In contact with biological fluids, the NPs (synthetic identity) are covered with biomolecules that form a protein corona, which defines the biological identity. It is well known that the protein corona formation is mediated by non-specific physical interactions, but protein-protein interactions (PPI), involving specific recognition sites of the polypeptides, are also involved. This work explores the relationship between the synthetic and biological identities of layered double hydroxides nanoparticles (LDH-NPs) and the effect of the protein corona on the cellular response. With such a purpose, the synthetic identity was modified by coating LDH-NPs with either a single protein or a complex mixture of them, followed by the characterization of the protein corona formed in a commonly used cell culture medium. A proteomic approach was used to identify the protein corona molecules and the PPI network was constructed with a novel bioinformatic tool. The coating on LDH-NPs defines the biological identity in such a way that the composition of the protein corona as well as PPI are changed. Electrostatic interactions appear not to be the only driving force regulating the interactions between NPs, proteins and cells since the specific recognition also play a fundamental role. However, the biological identity of LDH-NPs does not affect the interactions with cells that shows negligible cytotoxicity and high internalization levels. Copyright © 2018 Elsevier B.V. All rights reserved.
Interactive effect of warming, nitrogen and phosphorus limitation on phytoplankton cell size

PubMed Central

Peter, Kalista Higini; Sommer, Ulrich

2015-01-01

Cell size is one of the ecologically most important traits of phytoplankton. The cell size variation is frequently related to temperature and nutrient limitation. In order to disentangle the role of both factors, an experiment was conducted to determine the possible interactions of these factors. Baltic Sea water containing the natural plankton community was used. We performed a factorial combined experiment of temperature, type of nutrient limitation (N vs. P), and strength of nutrient limitation. The type of nutrient limitation was manipulated by altering the N:P ratio of the medium (balanced, N and P limitation) and strength by the dilution rate (0% and 50%) of the semicontinuous cultures. The negative effect of temperature on cell size was strongest under N limitation, intermediate under P limitation, and weakest when N and P were supplied at balanced ratios. However, temperature also influenced the intensity of nutrient imitation, because at higher temperature there was a tendency for dissolved nutrient concentrations to be lower, while the C:N or C:P ratio being higher…higher at identical dilution rates and medium composition. Analyzing the response of cell size to C:N ratios (as index of N limitation) and C:P ratios (as index of P limitation) indicated a clear dominance of the nutrient effect over the direct temperature effect, although the temperature effect was also significant. PMID:25798219
Developmental lineage priming in Dictyostelium by heterogeneous Ras activation.

PubMed

Chattwood, Alex; Nagayama, Koki; Bolourani, Parvin; Harkin, Lauren; Kamjoo, Marzieh; Weeks, Gerald; Thompson, Christopher R L

2013-11-26

In cell culture, genetically identical cells often exhibit heterogeneous behavior, with only 'lineage primed' cells responding to differentiation inducing signals. It has recently been proposed that such heterogeneity exists during normal embryonic development to allow position independent patterning based on 'salt and pepper' differentiation and sorting out. However, the molecular basis of lineage priming and how it leads to reproducible cell type proportioning are poorly understood. To address this, we employed a novel forward genetic approach in the model organism Dictyostelium discoideum. These studies reveal that the Ras-GTPase regulator gefE is required for normal lineage priming and salt and pepper differentiation. This is because Ras-GTPase activity sets the intrinsic response threshold to lineage specific differentiation signals. Importantly, we show that although gefE expression is uniform, transcription of its target, rasD, is both heterogeneous and dynamic, thus providing a novel mechanism for heterogeneity generation and position-independent differentiation. DOI: http://dx.doi.org/10.7554/eLife.01067.001.
Partial nephrectomy for renal tumors: lack of correlation between margin status and local recurrence.

PubMed

Antic, Tatjana; Taxy, Jerome B

2015-05-01

To evaluate the relationship between a positive resection margin in partial nephrectomy (PN) and local recurrence. From January 2005 through December 2012, there were 473 PNs in 466 patients at the University of Chicago. A positive margin was defined as tumor extending to the inked specimen edge, either the parenchymal interface or the peripheral fibroadipose tissue. A local recurrence was defined as an ipsilateral tumor of identical histologic type. Renal cell carcinoma (RCC) accounted for 406 tumors: 243 clear cell RCCs (CRCCs), 77 papillary RCCs (PRCCs), and 47 chromophobe RCCs (CHRCCs). Sixty-one RCCs had positive margins: 43 CRCCs, six PRCCs, nine CHRCCs, and three miscellaneous cell types. Of the 61 positive margins, four CRCCs (all originally multifocal) had a local recurrence, two of which occurred in the same patient. One translocation RCC also recurred. Six cases with negative resection margins had a recurrence. A literature review of 3,803 cases, including our study, shows positive margins in 173, of which 13 recurred; however, 39 with negative margins also recurred. A positive margin in PN seldom correlates with a local recurrence. However, protection from recurrence is not ensured by a negative margin. Copyright© by the American Society for Clinical Pathology.
Establishment and characterization of a human papillomavirus type 16-positive tonsillar carcinoma xenograft in BALB/c nude mice.

PubMed

Letsolo, Boitelo T; Faust, Helena; Ekblad, Lars; Wennerberg, Johan; Forslund, Ola

2016-03-01

Among head and neck cancers, human papillomavirus type 16 (HPV16) is associated with tonsillar carcinomas. Despite this, no HPV16-positive tonsillar cancer cell line has been established in nude mice. Fresh tonsillar carcinoma biopsies were obtained from 23 patients and implanted subcutaneously into nude mice (BALB/c, nu/nu). After 7 months, one xenograft was established. The primary tumor harbored 2.7 copies (95% confidence interval = 2.4-2.9) of HPV16/cell and displayed 99.9% (7904/7906) nucleotide identity to HPV16 (EU118173.1). The xenograft showed increased methylation in two E2-binding sites of the HPV16 genome. Both episomal and integrated HPV16 were detected in the original tumor and in 14 xenografts from the second passage. From this passage, a viral load of 6.4 copies/cell (range = 4.6-9.6) and 3.7 (range = 1.0-5.5) E7-mRNA transcripts/HPV16-genome were detected. This xenograft represents the first established HPV16-positive tonsillar tumor in nude mice and could provide an experimental system of HPV16-positive tonsillar cancers. © 2015 Wiley Periodicals, Inc.
Efficient Analysis of Systems Biology Markup Language Models of Cellular Populations Using Arrays.

PubMed

Watanabe, Leandro; Myers, Chris J

2016-08-19

The Systems Biology Markup Language (SBML) has been widely used for modeling biological systems. Although SBML has been successful in representing a wide variety of biochemical models, the core standard lacks the structure for representing large complex regular systems in a standard way, such as whole-cell and cellular population models. These models require a large number of variables to represent certain aspects of these types of models, such as the chromosome in the whole-cell model and the many identical cell models in a cellular population. While SBML core is not designed to handle these types of models efficiently, the proposed SBML arrays package can represent such regular structures more easily. However, in order to take full advantage of the package, analysis needs to be aware of the arrays structure. When expanding the array constructs within a model, some of the advantages of using arrays are lost. This paper describes a more efficient way to simulate arrayed models. To illustrate the proposed method, this paper uses a population of repressilator and genetic toggle switch circuits as examples. Results show that there are memory benefits using this approach with a modest cost in runtime.
Comparative cell cycle transcriptomics reveals synchronization of developmental transcription factor networks in cancer cells

PubMed Central

Johard, Helena; Mahdessian, Diana; Fedr, Radek; Marks, Carolyn; Medalová, Jiřina; Souček, Karel; Lundberg, Emma; Linnarsson, Sten; Bryja, Vítězslav; Sekyrova, Petra; Altun, Mikael; Andäng, Michael

2017-01-01

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development. PMID:29228002
Human leukocyte antigen typing using buccal swabs as accurate and non-invasive substitute for venipuncture in children at risk for celiac disease.

PubMed

Adriaanse, Marlou P M; Vreugdenhil, Anita C E; Vastmans, Véronique; Groeneveld, Lisette; Molenbroeck, Stefan; Schott, Dina A; Voorter, Christina E M; Tilanus, Marcel G J

2016-10-01

Human leukocyte antigen (HLA) typing is an important step in the diagnostic algorithm for celiac disease (CD) and is also used for screening purposes. Collection of blood is invasive and accompanied with emotional impact especially in children. Genetic technological progress now enables HLA typing from buccal cell samples. This study evaluated the reliability and feasibility of HLA typing for CD-associated HLA polymorphisms using buccal swabs as routine test in high-risk individuals. Blood and buccal swabs of 77 children and adolescents with high risk for CD were prospectively collected in this cohort study. Buccal swab collection was performed either by the investigator at the outpatient clinic or by the patient or its parents at home. To evaluate the possibility of self-administration, three families performed the test at home. DNA was extracted using an adapted QIAamp method. Quantity, quality, and purity of DNA were recorded. HLA-DRB1, HLA-DQA1, and HLA-DQB1 typing was examined on buccal cell-derived and blood-derived DNA at low and, if necessary, high resolution level, using sequence-specific oligonucleotide and sequence-based typing, respectively. DNA isolation using buccal swabs yielded a good quality and sufficient quantity of DNA to perform HLA-DQ typing in all individuals. HLA typing results on buccal cell-derived DNA were identical to typing on blood-derived DNA, also for the self-administered samples. Introduction of the buccal swab test for HLA typing of CD risk in routine diagnostics can omit the current venipuncture and enables self-administration at home. Therefore, the buccal swab test is beneficial for individuals with a clinical suspicion for CD, as well as for screening purposes in high-risk populations. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.
28 CFR 80.14 - Disclosure.

Code of Federal Regulations, 2010 CFR

2010-07-01

... the identity of the requesting issuer or domestic concern, the identity of the foreign country in which the proposed conduct is to take place, the general nature and circumstances of the proposed... release shall not disclose either the identity of any foreign sales agents or other types of identifying...
Akt Suppression of TGFβ Signaling Contributes to the Maintenance of Vascular Identity in Embryonic Stem Cell-Derived Endothelial Cells

PubMed Central

Israely, Edo; Ginsberg, Michael; Nolan, Daniel; Ding, Bi-Sen; James, Daylon; Elemento, Olivier; Rafii, Shahin; Rabbany, Sina Y

2016-01-01

The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (EC) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells, and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs, and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFβ-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs, and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFβ-mediated processes that would ordinarily usher these cells to alternate fates. PMID:23963623
Akt suppression of TGFβ signaling contributes to the maintenance of vascular identity in embryonic stem cell-derived endothelial cells.

PubMed

Israely, Edo; Ginsberg, Michael; Nolan, Daniel; Ding, Bi-Sen; James, Daylon; Elemento, Olivier; Rafii, Shahin; Rabbany, Sina Y

2014-01-01

The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (ECs) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFβ-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFβ-mediated processes that would ordinarily usher these cells to alternate fates. © AlphaMed Press.
"We Are a Chinese School": Constructing School Identity from the Lived Experiences of Expatriate and Chinese Teaching Faculty in a Type C International School in Shanghai, China

ERIC Educational Resources Information Center

Poole, Adam

2018-01-01

This study explores school identity by analysing the perceptions of Chinese and expatriate teachers in a Type C, non-traditional international school in Shanghai, China. The purpose of this study was to build on Hayden's (2016) work by offering a detailed description of this type of school which continues to be under researched. A mixed-methods…
Bilingual Children's Literature as a Tool Reflecting Non-Reconciled and Reconciled Identities in the Ethiopian Community in Israel

ERIC Educational Resources Information Center

Kalnisky, Esther; Baratz, Lea

2018-01-01

This study investigates the manner in which new and veteran Ethiopian immigrant students in Israel perceive their identity by investigating their attitudes towards children's books written in both Hebrew and Amharic. Two major types of identity were revealed: (1) a non-reconciled identity that seeks to minimise the visibility of one's ethnic…
Molecular definition of the identity and activation of natural killer cells.

PubMed

Bezman, Natalie A; Kim, Charles C; Sun, Joseph C; Min-Oo, Gundula; Hendricks, Deborah W; Kamimura, Yosuke; Best, J Adam; Goldrath, Ananda W; Lanier, Lewis L

2012-10-01

Using whole-genome microarray data sets of the Immunological Genome Project, we demonstrate a closer transcriptional relationship between NK cells and T cells than between any other leukocytes, distinguished by their shared expression of genes encoding molecules with similar signaling functions. Whereas resting NK cells are known to share expression of a few genes with cytotoxic CD8(+) T cells, our transcriptome-wide analysis demonstrates that the commonalities extend to hundreds of genes, many encoding molecules with unknown functions. Resting NK cells demonstrate a 'preprimed' state compared with naive T cells, which allows NK cells to respond more rapidly to viral infection. Collectively, our data provide a global context for known and previously unknown molecular aspects of NK cell identity and function by delineating the genome-wide repertoire of gene expression of NK cells in various states.

Auxin acts as a local morphogenetic trigger to specify lateral root founder cells

PubMed Central

Dubrovsky, Joseph G.; Sauer, Michael; Napsucialy-Mendivil, Selene; Ivanchenko, Maria G.; Friml, Jiří; Shishkova, Svetlana; Celenza, John; Benková, Eva

2008-01-01

Plants exhibit an exceptional adaptability to different environmental conditions. To a large extent, this adaptability depends on their ability to initiate and form new organs throughout their entire postembryonic life. Plant shoot and root systems unceasingly branch and form axillary shoots or lateral roots, respectively. The first event in the formation of a new organ is specification of founder cells. Several plant hormones, prominent among them auxin, have been implicated in the acquisition of founder cell identity by differentiated cells, but the mechanisms underlying this process are largely elusive. Here, we show that auxin and its local accumulation in root pericycle cells is a necessary and sufficient signal to respecify these cells into lateral root founder cells. Analysis of the alf4–1 mutant suggests that specification of founder cells and the subsequent activation of cell division leading to primordium formation represent two genetically separable events. Time-lapse experiments show that the activation of an auxin response is the earliest detectable event in founder cell specification. Accordingly, local activation of auxin response correlates absolutely with the acquisition of founder cell identity and precedes the actual formation of a lateral root primordium through patterned cell division. Local production and subsequent accumulation of auxin in single pericycle cells induced by Cre-Lox-based activation of auxin synthesis converts them into founder cells. Thus, auxin is the local instructive signal that is sufficient for acquisition of founder cell identity and can be considered a morphogenetic trigger in postembryonic plant organogenesis. PMID:18559858
Broken identity: the impact of the Holocaust on identity in Romanian and Polish Jews.

PubMed

Prot, Katarzyna

2008-01-01

The paper is based on interviews conducted with Holocaust survivors in Poland (30 interviews) and Romania (55 interviews). It describes how the Holocaust affected survivor identity. Two aspects of identity are analyzed the sense of personal identity and social identity. Each affects the other but they are largely independent and the trauma of the Holocaust impacted each of them differently. Personal identity seems to be unrelated to either the type of trauma or the survivor's social situation. There are no significant differences in that aspect between Polish and Romanian survivors. Social identity is more related to the survivors' social situation prior to and after the trauma. The sense of identity, both personal and social, is dynamic and changes over time.
Attitudes toward homosexuality among young adults: connections to gender role identity, gender-typed activities, and religiosity.

PubMed

Harbaugh, Evan; Lindsey, Eric W

2015-01-01

Individual differences in attitudes toward homosexuality have been linked to numerous personality and demographic variables. This study investigated the influence that gender role identity, involvement in gender-typed activities, and religiosity plays in this relationship. The sample included 194 undergraduate students from a Northeastern university. Analyses revealed that both males and females who held a more masculine gender role identity and individual commitment to religion scored higher on measures of homophobia and heteronormativity, whereas there was no association between spiritual meaning in life and attitudes toward homosexuality. Among males, but not females, more masculine gender identity and less spiritual meaning in life was associated with greater homophobia. The importance of the findings for research on the origins of attitudes toward individuals with a homosexual orientation are discussed, as well as the potential directions for future research on connections between gender role identity, religious affiliation, and attitudes toward gays and lesbians.
Expression of alpha and beta subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the Wv or Sld loci.

PubMed

Schulte, B A; Steel, K P

1994-07-01

Mice homozygous for mutations at the viable dominant spotting (Wv) and Steel-dickie (Sld) loci exhibit a similar phenotype which includes deafness. The auditory dysfunction derives from failure of the stria vascularis to develop normally and to generate a high positive endocochlear potential (EP). Because strial function is driven by Na,K-ATPase its expression was investigated in inner ears of Wv/Wv and Sld/Sld mice and their wild-type littermates by immunostaining with antisera against four of the enzyme's subunit isoforms. Wild-type mice from two different genetic backgrounds showed an identical distribution of subunit isoforms among inner ear transport cells. Several epithelial cell types coexpressed the alpha 1 and beta 1 subunits. Vestibular dark cells showed no reactivity for beta 1 but expressed abundant beta 2, whereas, strial marginal cells stained strongly for both beta isoforms. The only qualitative difference between mutant and wild-type mice was the absence of beta 1 subunit in marginal cells of the mutant's stria. However, it is unlikely that this difference accounts for failure of mutants to generate a high EP because the beta 1 subunit is not present in the stria vascularis of either rats or gerbils with normal EP values. Strong immunostaining for Na,K-ATPase in lateral wall fibrocytes of normal mice along with diminished immunoreactivity in the mutants supports the concept that these strategically located transport fibrocytes actively resorb K+ leaked across Reissner's membrane into scala vestibuli or effluxed from hair cells and nerves into scala tympani. It is further speculated that the resorbed K+ normally is siphoned down its concentration gradient into the intrastrial space through gap junctions between fibrocytes and strial basal and intermediate cells where it is recycled back to endolymph via marginal cells. Thus, failure of mutants to generate a positive EP could be explained by the absence of intermediate cells which may form the final link in the conduit for moving K+ from perilymph to the intrastrial compartment.
Biophysics and dynamics of natural and engineered stem cell microenvironments.

PubMed

Keung, Albert J; Healy, Kevin E; Kumar, Sanjay; Schaffer, David V

2010-01-01

Stem cells are defined by their ability to self-renew and to differentiate into one or more mature lineages, and they reside within natural niches in many types of adult and embryonic tissues that present them with complex signals to regulate these two hallmark properties. The diverse nature of these in vivo microenvironments raises important questions about the microenvironmental cues regulating stem cell plasticity, and the stem cell field has built a strong foundation of knowledge on the biochemical identities and regulatory effects of the soluble, cellular, and extracellular matrix factors surrounding stem cells through the isolation and culture of stem cells in vitro within microenvironments that, in effect, emulate the properties of the natural niche. Recent work, however, has expanded the field's perspective to include biophysical and dynamic characteristics of the microenvironment. These include biomechanical characteristics such as elastic modulus, shear force, and cyclic strain; architectural properties such as geometry, topography, and dimensionality; and dynamic structures and ligand profiles. We will review how these microenvironmental characteristics have been shown to regulate stem cell fate and discuss future research directions that may help expand our current understanding of stem cell biology and aid its application to regenerative medicine.
Feasibility studies of oncornavirus production in microcarrier cultures.

PubMed

Manousos, M; Ahmed, M; Torchio, C; Wolff, J; Shibley, G; Stephens, R; Mayyasi, S

1980-06-01

Studies conducted with virus-infected monolayer cell cultures have demonstrated the feasibility of producing several tumor-associated viruses in microcarrier (mc) cultures (Sephadex G50 beads treated with DEAE-chloride). The efficiency of cell adherence to mc varied with the cell type, the pH of the growth medium, and the stirring force applied to keep the mc in suspension. Most cells attached firmly to the mc and could not be removed easily with routine trypsinization procedures. Techniques using Enzar-T and Pronase were effective in detaching cells from mc in 10 to 15 min while retaining 95% cell viability. After detachment, Ficoll gradients were used for rapid and complete separation of viable cell suspensions from the mc. Retrovirus production in large volumes of mc cultures was investigated with periodic harvesting of growth fluids. Physical, biochemical, and biological properties of the Mason-Pfizer monkey virus and the RD114 virus recovered from the mc cultures were identical to those produced in conventional cultures. The utilization of mc has several applications in research and short-term cultures, but the as-yet-unsolved technical problems met were found to be serious limitations when attempting mass cell culturing on a long-term basis.
AN ELECTRON MICROSCOPE STUDY OF MATURE AND DIFFERENTIATING PANETH CELLS IN THE RAT, ESPECIALLY OF THEIR ENDOPLASMIC RETICULUM AND LYSOSOMES

PubMed Central

Behnke, O.; Moe, H.

1964-01-01

In an electron microsope study, the morphology of mature Paneth cells from the small intestine of adult rats is compared with that of differentiating Paneth cells from young rats 2 to 4 weeks old. All mature cells exhibit a marked polarity similar to that of other exocrine gland cells and contain a well developed endoplasmic reticulum, an elaborate Golgi complex, and numerous large secretory granules; they also possess an abundance of lysosomes. The most conspicuous occurrence in the process of differentiation is the development of the endoplasmic reticulum. The most immature Paneth cells possess an endoplasmic reticulum of the vesicular type, which, during maturation, is replaced by the characteristic lamellated ergastoplasm of the mature cell. At a certain stage of differentiation the cavities of the developing cisternae show numerous communications with the perinuclear space, suggesting an outgrowth of the ergastoplasm from the nuclear envelope. Furthermore, the cavities and the perinuclear space at this particular stage contain a material which shows a remarkable intrinsic periodicity. An identical periodicity was exhibited by material contained in Golgi cisternae and secretory granules. Lysosomes are also present in the differentiating cells. PMID:14206428
Mechanical positioning of multiple nuclei in muscle cells.

PubMed

Manhart, Angelika; Windner, Stefanie; Baylies, Mary; Mogilner, Alex

2018-06-01

Many types of large cells have multiple nuclei. In skeletal muscle fibers, the nuclei are distributed along the cell to maximize their internuclear distances. This myonuclear positioning is crucial for cell function. Although microtubules, microtubule associated proteins, and motors have been implicated, mechanisms responsible for myonuclear positioning remain unclear. We used a combination of rough interacting particle and detailed agent-based modeling to examine computationally the hypothesis that a force balance generated by microtubules positions the muscle nuclei. Rather than assuming the nature and identity of the forces, we simulated various types of forces between the pairs of nuclei and between the nuclei and cell boundary to position the myonuclei according to the laws of mechanics. We started with a large number of potential interacting particle models and computationally screened these models for their ability to fit biological data on nuclear positions in hundreds of Drosophila larval muscle cells. This reverse engineering approach resulted in a small number of feasible models, the one with the best fit suggests that the nuclei repel each other and the cell boundary with forces that decrease with distance. The model makes nontrivial predictions about the increased nuclear density near the cell poles, the zigzag patterns of the nuclear positions in wider cells, and about correlations between the cell width and elongated nuclear shapes, all of which we confirm by image analysis of the biological data. We support the predictions of the interacting particle model with simulations of an agent-based mechanical model. Taken together, our data suggest that microtubules growing from nuclear envelopes push on the neighboring nuclei and the cell boundaries, which is sufficient to establish the nearly-uniform nuclear spreading observed in muscle fibers.
The 5-HT{sub 2A} serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

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

Sonier, Brigitte; Arseneault, Madeleine; Institut National de la Recherche Scientifique-Institut Armand-Frappier, Montreal, Que.

2006-05-19

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT{sub 2A} serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTTmore » proliferation assay. We have demonstrated that the 5-HT{sub 2A} receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT{sub 2A} receptor present in this cell line is identical to the 5-HT{sub 2A} receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT{sub 2A} receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT{sub 2A} receptor subtype, which is fully expressed in this cell line.« less
Lethal H1N1 influenza A virus infection alters the murine alveolar type II cell surfactant lipidome.

PubMed

Woods, Parker S; Doolittle, Lauren M; Rosas, Lucia E; Joseph, Lisa M; Calomeni, Edward P; Davis, Ian C

2016-12-01

Alveolar type II (ATII) epithelial cells are the primary site of influenza virus replication in the distal lung. Development of acute respiratory distress syndrome in influenza-infected mice correlates with significant alterations in ATII cell function. However, the impact of infection on ATII cell surfactant lipid metabolism has not been explored. C57BL/6 mice were inoculated intranasally with influenza A/WSN/33 (H1N1) virus (10,000 plaque-forming units/mouse) or mock-infected with virus diluent. ATII cells were isolated by a standard lung digestion protocol at 2 and 6 days postinfection. Levels of 77 surfactant lipid-related compounds of known identity in each ATII cell sample were measured by ultra-high-performance liquid chromatography-mass spectrometry. In other mice, bronchoalveolar lavage fluid was collected to measure lipid and protein content using commercial assay kits. Relative to mock-infected animals, ATII cells from influenza-infected mice contained reduced levels of major surfactant phospholipids (phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine) but increased levels of minor phospholipids (phosphatidylserine, phosphatidylinositol, and sphingomyelin), cholesterol, and diacylglycerol. These changes were accompanied by reductions in cytidine 5'-diphosphocholine and 5'-diphosphoethanolamine (liponucleotide precursors for ATII cell phosphatidylcholine and phosphatidylethanolamine synthesis, respectively). ATII cell lamellar bodies were ultrastructurally abnormal after infection. Changes in ATII cell phospholipids were reflected in the composition of bronchoalveolar lavage fluid, which contained reduced amounts of phosphatidylcholine and phosphatidylglycerol but increased amounts of sphingomyelin, cholesterol, and protein. Influenza infection significantly alters ATII cell surfactant lipid metabolism, which may contribute to surfactant dysfunction and development of acute respiratory distress syndrome in influenza-infected mice. Copyright © 2016 the American Physiological Society.
Lethal H1N1 influenza A virus infection alters the murine alveolar type II cell surfactant lipidome

PubMed Central

Woods, Parker S.; Doolittle, Lauren M.; Rosas, Lucia E.; Joseph, Lisa M.; Calomeni, Edward P.

2016-01-01

Alveolar type II (ATII) epithelial cells are the primary site of influenza virus replication in the distal lung. Development of acute respiratory distress syndrome in influenza-infected mice correlates with significant alterations in ATII cell function. However, the impact of infection on ATII cell surfactant lipid metabolism has not been explored. C57BL/6 mice were inoculated intranasally with influenza A/WSN/33 (H1N1) virus (10,000 plaque-forming units/mouse) or mock-infected with virus diluent. ATII cells were isolated by a standard lung digestion protocol at 2 and 6 days postinfection. Levels of 77 surfactant lipid-related compounds of known identity in each ATII cell sample were measured by ultra-high-performance liquid chromatography-mass spectrometry. In other mice, bronchoalveolar lavage fluid was collected to measure lipid and protein content using commercial assay kits. Relative to mock-infected animals, ATII cells from influenza-infected mice contained reduced levels of major surfactant phospholipids (phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine) but increased levels of minor phospholipids (phosphatidylserine, phosphatidylinositol, and sphingomyelin), cholesterol, and diacylglycerol. These changes were accompanied by reductions in cytidine 5′-diphosphocholine and 5′-diphosphoethanolamine (liponucleotide precursors for ATII cell phosphatidylcholine and phosphatidylethanolamine synthesis, respectively). ATII cell lamellar bodies were ultrastructurally abnormal after infection. Changes in ATII cell phospholipids were reflected in the composition of bronchoalveolar lavage fluid, which contained reduced amounts of phosphatidylcholine and phosphatidylglycerol but increased amounts of sphingomyelin, cholesterol, and protein. Influenza infection significantly alters ATII cell surfactant lipid metabolism, which may contribute to surfactant dysfunction and development of acute respiratory distress syndrome in influenza-infected mice. PMID:27836900
Short tandem repeat profiling: part of an overall strategy for reducing the frequency of cell misidentification.

PubMed

Nims, Raymond W; Sykes, Greg; Cottrill, Karin; Ikonomi, Pranvera; Elmore, Eugene

2010-12-01

The role of cell authentication in biomedical science has received considerable attention, especially within the past decade. This quality control attribute is now beginning to be given the emphasis it deserves by granting agencies and by scientific journals. Short tandem repeat (STR) profiling, one of a few DNA profiling technologies now available, is being proposed for routine identification (authentication) of human cell lines, stem cells, and tissues. The advantage of this technique over methods such as isoenzyme analysis, karyotyping, human leukocyte antigen typing, etc., is that STR profiling can establish identity to the individual level, provided that the appropriate number and types of loci are evaluated. To best employ this technology, a standardized protocol and a data-driven, quality-controlled, and publically searchable database will be necessary. This public STR database (currently under development) will enable investigators to rapidly authenticate human-based cultures to the individual from whom the cells were sourced. Use of similar approaches for non-human animal cells will require developing other suitable loci sets. While implementing STR analysis on a more routine basis should significantly reduce the frequency of cell misidentification, additional technologies may be needed as part of an overall authentication paradigm. For instance, isoenzyme analysis, PCR-based DNA amplification, and sequence-based barcoding methods enable rapid confirmation of a cell line's species of origin while screening against cross-contaminations, especially when the cells present are not recognized by the species-specific STR method. Karyotyping may also be needed as a supporting tool during establishment of an STR database. Finally, good cell culture practices must always remain a major component of any effort to reduce the frequency of cell misidentification.
New Century, New Identities: Building on a Typology of Nonheterosexual College Men

ERIC Educational Resources Information Center

Dilley, Patrick

2010-01-01

Drawing on recent ethnographic studies of nonheterosexual youth, nonheterosexual identity development, and online collegiate identity management, this article outlines an extension of prior typological work concerning nonheterosexual male college students proposed by the author. Two new types ("Twitter Twinks" and "Lads Without Labels") are…
Exogenous ACE2 Expression Allows Refractory Cell Lines To Support Severe Acute Respiratory Syndrome Coronavirus Replication

PubMed Central

Mossel, Eric C.; Huang, Cheng; Narayanan, Krishna; Makino, Shinji; Tesh, Robert B.; Peters, C. J.

2005-01-01

Of 30 cell lines and primary cells examined, productive severe acute respiratory syndrome coronavirus (Urbani strain) (SARS-CoV) infection after low-multiplicity inoculation was detected in only six: three African green monkey kidney epithelial cell lines (Vero, Vero E6, and MA104), a human colon epithelial line (CaCo-2), a porcine kidney epithelial line [PK(15)], and mink lung epithelial cells (Mv 1 Lu). SARS-CoV produced a lytic infection in Vero, Vero E6, and MA104 cells, but there was no visible cytopathic effect in Caco-2, Mv 1 Lu, or PK(15) cells. Multistep growth kinetics were identical in Vero E6 and MA104 cells, with maximum titer reached 24 h postinoculation (hpi). Virus titer was maximal 96 hpi in CaCo-2 cells, and virus was continually produced from infected CaCo-2 cells for at least 6 weeks after infection. CaCo-2 was the only human cell type of 13 tested that supported efficient SARS-CoV replication. Expression of the SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2), resulted in SARS-CoV replication in all refractory cell lines examined. Titers achieved were variable and dependent upon the method of ACE2 expression. PMID:15731278
GMP-compliant human adipose tissue-derived mesenchymal stem cells for cellular therapy.

PubMed

Aghayan, Hamid-Reza; Goodarzi, Parisa; Arjmand, Babak

2015-01-01

Stem cells, which can be derived from different sources, demonstrate promising therapeutic evidences for cellular therapies. Among various types of stem cell, mesenchymal stem cells are one of the most common stem cells that are used in cellular therapy. Human subcutaneous adipose tissue provides an easy accessible source of mesenchymal stem cells with some considerable advantages. Accordingly, various preclinical and clinical investigations have shown enormous potential of adipose-derived stromal cells in regenerative medicine. Consequently, increasing clinical applications of these cells has elucidated the importance of safety concerns regarding clinical transplantation. Therefore, clinical-grade preparation of adipose-derived stromal cells in accordance with current good manufacturing practice guidelines is an essential part of their clinical applications to ensure the safety, quality, characteristics, and identity of cell products. Additionally, GMP-compliant cell manufacturing involves several issues to provide a quality assurance system during translation from the basic stem cell sciences into clinical investigations and applications. On the other hand, advanced cellular therapy requires extensive validation, process control, and documentation. It also evidently elucidates the critical importance of production methods and probable risks. Therefore, implementation of a quality management and assurance system in accordance with GMP guidelines can greatly reduce these risks particularly in the higher-risk category or "more than minimally manipulated" products.
Nitrosative damage to free and zinc-bound cysteine thiols underlies nitric oxide toxicity in wild-type Borrelia burgdorferi

PubMed Central

Bourret, Travis J; Boylan, Julie A; Lawrence, Kevin A; Gherardini, Frank C

2011-01-01

Borrelia burgdorferi encounters potentially harmful reactive nitrogen species (RNS) throughout its infective cycle. In this study, diethylamine NONOate (DEA/NO) was used to characterize the lethal effects of RNS on B. burgdorferi. RNS produce a variety of DNA lesions in a broad spectrum of microbial pathogens; however, levels of the DNA deamination product, deoxyinosine, and the numbers of apurinic/apyrimidinic (AP) sites were identical in DNA isolated from untreated and DEA/NO-treated B. burgdorferi cells. Strains with mutations in the nucleotide excision repair (NER) pathway genes uvrC or uvrB treated with DEA/NO had significantly higher spontaneous mutation frequencies, increased numbers of AP sites in DNA and reduced survival compared with wild-type controls. Polyunsaturated fatty acids in B. burgdorferi cell membranes, which are susceptible to peroxidation by reactive oxygen species (ROS), were not sensitive to RNS-mediated lipid peroxidation. However, treatment of B. burgdorferi cells with DEA/NO resulted in nitrosative damage to several proteins, including the zinc-dependent glycolytic enzyme fructose-1,6-bisphosphate aldolase (BB0445), the Borrelia oxidative stress regulator (BosR) and neutrophil-activating protein (NapA). Collectively, these data suggested that nitrosative damage to proteins harbouring free or zinc-bound cysteine thiols, rather than DNA or membrane lipids underlies RNS toxicity in wild-type B. burgdorferi. PMID:21564333
Regeneration of asymmetric somatic hybrid plants from the fusion of two types of wheat with Russian wildrye.

PubMed

Li, Cuiling; Xia, Guangmin; Xiang, Fengning; Zhou, Chuanen; Cheng, Aixia

2004-12-01

Two types of protoplasts of wheat (Triticum aestivum L. cv. Jinan 177) were used in fusion experiments--cha9, with a high division frequency, and 176, with a high regeneration frequency. The fusion combination of either cha9 or 176 protoplasts with Russian wildrye protoplasts failed to produce regenerated calli. When a mixture of cha9 and 176 protoplasts were fused with those of Russian wildrye, 14 fusion-derived calli were produced, of which seven differentiated into green plants and two differentiated into albinos. The morphology of all hybrid plants strongly resembled that of the parental wheat type. The hybrid nature of the cell lines was confirmed by cytological, isozyme, random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analyses. GISH analysis revealed that only chromosome fragments of Russian wildrye were transferred to the wheat chromosomes of hybrid calli and plants. Simple sequence repeat (SSR) analysis of the chloroplast genome of the hybrids with seven pairs of wheat-specific chloroplast microsatellite primers indicated that all of the cell lines had band patterns identical to wheat. Our results show that highly asymmetric somatic hybrid calli and plants can be produced via symmetric fusion in a triparental fusion system. The dominant effect of two wheat cell lines on the exclusion of Russian wildrye chromosomes is discussed.
Neurogenin3 restricts serotonergic neuron differentiation to the hindbrain.

PubMed

Carcagno, Abel L; Di Bella, Daniela J; Goulding, Martyn; Guillemot, Francois; Lanuza, Guillermo M

2014-11-12

The development of the nervous system is critically dependent on the production of functionally diverse neuronal cell types at their correct locations. In the embryonic neural tube, dorsoventral signaling has emerged as a fundamental mechanism for generating neuronal diversity. In contrast, far less is known about how different neuronal cell types are organized along the rostrocaudal axis. In the developing mouse and chick neural tube, hindbrain serotonergic neurons and spinal glutamatergic V3 interneurons are produced from ventral p3 progenitors, which possess a common transcriptional identity but are confined to distinct anterior-posterior territories. In this study, we show that the expression of the transcription factor Neurogenin3 (Neurog3) in the spinal cord controls the correct specification of p3-derived neurons. Gain- and loss-of-function manipulations in the chick and mouse embryo show that Neurog3 switches ventral progenitors from a serotonergic to V3 differentiation program by repressing Ascl1 in spinal p3 progenitors through a mechanism dependent on Hes proteins. In this way, Neurog3 establishes the posterior boundary of the serotonergic system by actively suppressing serotonergic specification in the spinal cord. These results explain how equivalent p3 progenitors within the hindbrain and the spinal cord produce functionally distinct neuron cell types. Copyright © 2014 the authors 0270-6474/14/3415223-11$15.00/0.
The Development of Sex Role Stereotypes in the Third Year: Relationships to Gender Labeling, Gender Identity, Sex-Typed Toy Preference, and Family Characteristics.

ERIC Educational Resources Information Center

Weinraub, Marsha; And Others

1984-01-01

The onset and development of preschoolers' awareness of sex role stereotypes, gender labeling, gender identity, and sex-typed toy preference were explored in 26-, 31-, and 36-month-old children. Family characteristics that affect early sex role development also were investigated. (Author/RH)
On the identity of Mastacanthus Suffrian, 1852 and Sternoglosus Suffrian, 1866 and key to world genera of Pachybrachina (Chrysomelidae: Cryptocephalinae: Cryptocephalini)

USDA-ARS?s Scientific Manuscript database

Pachybrachina includes 8 genera worldwide. The identity of the two Neotropical genera Mastacanthus Suffrian and Sternoglosus Suffrian is established. Redescription of the type species of both genera and an illustrated key to world genera are provided. Type species of Sternoglosus and the lectotype f...

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