Cell type-specific genetic and optogenetic tools reveal hippocampal CA2 circuits.

Science.gov (United States)

Kohara, Keigo; Pignatelli, Michele; Rivest, Alexander J; Jung, Hae-Yoon; Kitamura, Takashi; Suh, Junghyup; Frank, Dominic; Kajikawa, Koichiro; Mise, Nathan; Obata, Yuichi; Wickersham, Ian R; Tonegawa, Susumu

2014-02-01

The formation and recall of episodic memory requires precise information processing by the entorhinal-hippocampal network. For several decades, the trisynaptic circuit entorhinal cortex layer II (ECII)→dentate gyrus→CA3→CA1 and the monosynaptic circuit ECIII→CA1 have been considered the primary substrates of the network responsible for learning and memory. Circuits linked to another hippocampal region, CA2, have only recently come to light. Using highly cell type-specific transgenic mouse lines, optogenetics and patch-clamp recordings, we found that dentate gyrus cells, long believed to not project to CA2, send functional monosynaptic inputs to CA2 pyramidal cells through abundant longitudinal projections. CA2 innervated CA1 to complete an alternate trisynaptic circuit, but, unlike CA3, projected preferentially to the deep, rather than to the superficial, sublayer of CA1. Furthermore, contrary to existing knowledge, ECIII did not project to CA2. Our results allow a deeper understanding of the biology of learning and memory.

Modification of surface/neuron interfaces for neural cell-type specific responses: a review

International Nuclear Information System (INIS)

Chen, Cen; Kong, Xiangdong; Lee, In-Seop

2016-01-01

Surface/neuron interfaces have played an important role in neural repair including neural prostheses and tissue engineered scaffolds. This comprehensive literature review covers recent studies on the modification of surface/neuron interfaces. These interfaces are identified in cases both where the surfaces of substrates or scaffolds were in direct contact with cells and where the surfaces were modified to facilitate cell adhesion and controlling cell-type specific responses. Different sources of cells for neural repair are described, such as pheochromocytoma neuronal-like cell, neural stem cell (NSC), embryonic stem cell (ESC), mesenchymal stem cell (MSC) and induced pluripotent stem cell (iPS). Commonly modified methods are discussed including patterned surfaces at micro- or nano-scale, surface modification with conducting coatings, and functionalized surfaces with immobilized bioactive molecules. These approaches to control cell-type specific responses have enormous potential implications in neural repair. (paper)

Cell Type-Specific Contributions to the TSC Neuropathology

Science.gov (United States)

2017-08-01

AWARD NUMBER: W81XWH-16-1-0415 TITLE: Cell Type-Specific Contributions to the TSC Neuropathology PRINCIPAL INVESTIGATOR: Gabriella D’Arcangelo...AND SUBTITLE Cell Type-Specific Contributions to the TSC Neuropathology 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-16-1-0415 5c. PROGRAM...how heterozygous and homozygous Tsc2 mutations affect the development of mutant excitatory neurons as well as other surrounding brain cells , in vivo

Cell-type-specific gene delivery into neuronal cells in vitro and in vivo

International Nuclear Information System (INIS)

Parveen, Zahida; Mukhtar, Muhammad; Rafi, Mohammed; Wenger, David A.; Siddiqui, Khwaja M.; Siler, Catherine A.; Dietzschold, Bernhard; Pomerantz, Roger J.; Schnell, Matthias J.; Dornburg, Ralph

2003-01-01

The avian retroviruses reticuloendotheliosis virus strain A (REV-A) and spleen necrosis virus (SNV) are not naturally infectious in human cells. However, REV-A-derived viral vectors efficiently infect human cells when they are pseudotyped with envelope proteins displaying targeting ligands specific for human cell-surface receptors. Here we report that vectors containing the gag region of REV-A and pol of SNV can be pseudotyped with the envelope protein of vesicular stomatitis virus (VSV) and the glycoproteins of different rabies virus (RV) strains. Vectors pseudotyped with the envelope protein of the highly neurotropic RV strain CVS-N2c facilitated cell type-specific gene delivery into mouse and human neurons, but did not infect other human cell types. Moreover, when such vector particles were injected into the brain of newborn mice, only neuronal cells were infected in vivo. Cell-type-specific gene delivery into neurons may present quite specific gene therapy approaches for many degenerative diseases of the brain

[Molecular biology of renal cancer: bases for genetic directed therapy in advanced disease].

Science.gov (United States)

Maroto Rey, José Pablo; Cillán Narvaez, Elena

2013-06-01

There has been expansion of therapeutic options in the management of metastatic renal cell carcinoma due to a better knowledge of the molecular biology of kidney cancers. There are different tumors grouped under the term renal cell carcinoma, being clear cell cancer the most frequent and accounting for 80% of kidney tumors. Mutations in the Von Hippel-Lindau gene can be identified in up to 80% of sporadic clear cell cancer, linking a genetically inheritable disease where vascular tumors are frequent, with renal cell cancer. Other histologic types present specific alterations in molecular pathways, like c-MET in papillary type I tumors, and Fumarase Hydratase in papillary type II tumors. Identification of the molecular alteration for a specific tumor may offer an opportunity for treatment selection based on biomarkers, and, in the future, for developing an engineering designed genetic treatment.

Spatial separation of photosynthesis and ethanol production by cell type-specific metabolic engineering of filamentous cyanobacteria.

Science.gov (United States)

Ehira, Shigeki; Takeuchi, Takuto; Higo, Akiyoshi

2018-02-01

Cyanobacteria, which perform oxygenic photosynthesis, have drawn attention as hosts for the direct production of biofuels and commodity chemicals from CO 2 and H 2 O using light energy. Although cyanobacteria capable of producing diverse chemicals have been generated by metabolic engineering, anaerobic non-photosynthetic culture conditions are often necessary for their production. In this study, we conducted cell type-specific metabolic engineering of the filamentous cyanobacterium Anabaena sp. PCC 7120, which forms a terminally differentiated cell called a heterocyst with a semi-regular spacing of 10-15 cells. Because heterocysts are specialized cells for nitrogen fixation, the intracellular oxygen level of heterocysts is maintained very low even when adjacent cells perform oxygenic photosynthesis. Pyruvate decarboxylase of Zymomonas mobilis and alcohol dehydrogenase of Synechocystis sp. PCC 6803 were exclusively expressed in heterocysts. Ethanol production was concomitant with nitrogen fixation in genetically engineered Anabaena sp. PCC 7120. Engineering of carbon metabolism in heterocysts improved ethanol production, and strain ET14, with an extra copy of the invB gene expressed from a heterocyst-specific promoter, produced 130.9 mg L -1 of ethanol after 9 days. ET14 produced 1681.9 mg L -1 of ethanol by increasing the CO 2 supply. Ethanol production per heterocyst cell was approximately threefold higher than that per cell of unicellular cyanobacterium. This study demonstrates the potential of heterocysts for anaerobic production of biofuels and commodity chemicals under oxygenic photosynthetic conditions.

Analysis of antigen-specific B-cell memory directly ex vivo.

Science.gov (United States)

McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.

Freedom of expression: cell-type-specific gene profiling.

Science.gov (United States)

Otsuki, Leo; Cheetham, Seth W; Brand, Andrea H

2014-01-01

Cell fate and behavior are results of differential gene regulation, making techniques to profile gene expression in specific cell types highly desirable. Many methods now enable investigation at the DNA, RNA and protein level. This review introduces the most recent and popular techniques, and discusses key issues influencing the choice between these such as ease, cost and applicability of information gained. Interdisciplinary collaborations will no doubt contribute further advances, including not just in single cell type but single-cell expression profiling. © 2014 Wiley Periodicals, Inc.

Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

Science.gov (United States)

Lu, Chenggang; Fuller, Margaret T

2015-12-01

Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s). In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC), a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs), spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus.

Science.gov (United States)

Smith, Emily M; Lajoie, Bryan R; Jain, Gaurav; Dekker, Job

2016-01-07

Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation. Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Cell type-specific translational repression of Cyclin B during meiosis in males.

Science.gov (United States)

Baker, Catherine Craig; Gim, Byung Soo; Fuller, Margaret T

2015-10-01

The unique cell cycle dynamics of meiosis are controlled by layers of regulation imposed on core mitotic cell cycle machinery components by the program of germ cell development. Although the mechanisms that regulate Cdk1/Cyclin B activity in meiosis in oocytes have been well studied, little is known about the trans-acting factors responsible for developmental control of these factors in male gametogenesis. During meiotic prophase in Drosophila males, transcript for the core cell cycle protein Cyclin B1 (CycB) is expressed in spermatocytes, but the protein does not accumulate in spermatocytes until just before the meiotic divisions. Here, we show that two interacting proteins, Rbp4 and Fest, expressed at the onset of spermatocyte differentiation under control of the developmental program of male gametogenesis, function to direct cell type- and stage-specific repression of translation of the core G2/M cell cycle component cycB during the specialized cell cycle of male meiosis. Binding of Fest to Rbp4 requires a 31-amino acid region within Rbp4. Rbp4 and Fest are required for translational repression of cycB in immature spermatocytes, with Rbp4 binding sequences in a cell type-specific shortened form of the cycB 3' UTR. Finally, we show that Fest is required for proper execution of meiosis I. © 2015. Published by The Company of Biologists Ltd.

Recruitment of Mediator Complex by Cell Type and Stage-Specific Factors Required for Tissue-Specific TAF Dependent Gene Activation in an Adult Stem Cell Lineage.

Directory of Open Access Journals (Sweden)

Chenggang Lu

2015-12-01

Full Text Available Onset of terminal differentiation in adult stem cell lineages is commonly marked by robust activation of new transcriptional programs required to make the appropriate differentiated cell type(s. In the Drosophila male germ line stem cell lineage, the switch from proliferating spermatogonia to spermatocyte is accompanied by one of the most dramatic transcriptional changes in the fly, as over 1000 new transcripts turn on in preparation for meiosis and spermatid differentiation. Here we show that function of the coactivator complex Mediator is required for activation of hundreds of new transcripts in the spermatocyte program. Mediator appears to act in a sequential hierarchy, with the testis activating Complex (tMAC, a cell type specific form of the Mip/dREAM general repressor, required to recruit Mediator subunits to the chromatin, and Mediator function required to recruit the testis TAFs (tTAFs, spermatocyte specific homologs of subunits of TFIID. Mediator, tMAC and the tTAFs co-regulate expression of a major set of spermatid differentiation genes. The Mediator subunit Med22 binds the tMAC component Topi when the two are coexpressed in S2 cells, suggesting direct recruitment. Loss of Med22 function in spermatocytes causes meiosis I maturation arrest male infertility, similar to loss of function of the tMAC subunits or the tTAFs. Our results illuminate how cell type specific versions of the Mip/dREAM complex and the general transcription machinery cooperate to drive selective gene activation during differentiation in stem cell lineages.

Cell-type specific four-component hydrogel.

Directory of Open Access Journals (Sweden)

Timo Aberle

Full Text Available In the field of regenerative medicine we aim to develop implant matrices for specific tissue needs. By combining two per se, cell-permissive gel systems with enzymatic crosslinkers (gelatin/transglutaminase and fibrinogen/thrombin to generate a blend (technical term: quattroGel, an unexpected cell-selectivity evolved. QuattroGels were porous and formed cavities in the cell diameter range, possessed gelation kinetics in the minute range, viscoelastic properties and a mechanical strength appropriate for general cell adhesion, and restricted diffusion. Cell proliferation of endothelial cells, chondrocytes and fibroblasts was essentially unaffected. In contrast, on quattroGels neither endothelial cells formed vascular tubes nor did primary neurons extend neurites in significant amounts. Only chondrocytes differentiated properly as judged by collagen isoform expression. The biophysical quattroGel characteristics appeared to leave distinct cell processes such as mitosis unaffected and favored differentiation of sessile cells, but hampered differentiation of migratory cells. This cell-type selectivity is of interest e.g. during articular cartilage or invertebral disc repair, where pathological innervation and angiogenesis represent adverse events in tissue engineering.

ON Bipolar Cells in Macaque Retina: Type-Specific Synaptic Connectivity with Special Reference to OFF Counterparts

Science.gov (United States)

Tsukamoto, Yoshihiko; Omi, Naoko

2016-01-01

To date, 12 macaque bipolar cell types have been described. This list includes all morphology types first outlined by Polyak (1941) using the Golgi method in the primate retina and subsequently identified by other researchers using electron microscopy (EM) combined with the Golgi method, serial section transmission EM (SSTEM), and immunohistochemical imaging. We used SSTEM for the rod-dense perifoveal area of macaque retina, reconfirmed ON (cone) bipolar cells to be classified as invaginating midget bipolar (IMB), diffuse bipolar (DB)4, DB5, DB6, giant bipolar (GB), and blue bipolar (BB) types, and clarified their type-specific connectivity. DB4 cells made reciprocal synapses with a kind of ON-OFF lateral amacrine cell, similar to OFF DB2 cells. GB cells contacted rods and cones, similar to OFF DB3b cells. Retinal circuits formed by GB and DB3b cells are thought to substantiate the psychophysical finding of fast rod signals in mesopic vision. DB6 cell output synapses were directed to ON midget ganglion (MG) cells at 70% of ribbon contacts, similar to OFF DB1 cells that directed 60% of ribbon contacts to OFF MG cells. IMB cells contacted medium- or long-wavelength sensitive (M/L-) cones but not short-wavelength sensitive (S-) cones, while BB cells contacted S-cones but not M/L-cones. However, IMB and BB dendrites had similar morphological architectures, and a BB cell contacting a single S-cone resembled an IMB cell. Thus, both IMB and BB may be the ON bipolar counterparts of the OFF flat midget bipolar (FMB) type, likewise DB4 of DB2, DB5 of DB3a, DB6 of DB1, and GB of DB3b OFF bipolar type. The ON DB plus GB, and OFF DB cells predominantly contacted M/L-cones and their outputs were directed mainly to parasol ganglion (PG) cells but also moderately to MG cells. BB cells directed S-cone-driven outputs almost exclusively to small bistratified ganglion (SBG) cells. Some FMB cells predominantly contacted S-cones and their outputs were directed to OFF MG cells. Thus, two

Cell type-specific characterization of nuclear DNA contents within complex tissues and organs

Directory of Open Access Journals (Sweden)

Lambert Georgina M

2005-10-01

Full Text Available Abstract Background Eukaryotic organisms are defined by the presence of a nucleus, which encloses the chromosomal DNA, and is characterized by its DNA content (C-value. Complex eukaryotic organisms contain organs and tissues that comprise interspersions of different cell types, within which polysomaty, endoreduplication, and cell cycle arrest is frequently observed. Little is known about the distribution of C-values across different cell types within these organs and tissues. Results We have developed, and describe here, a method to precisely define the C-value status within any specific cell type within complex organs and tissues of plants. We illustrate the application of this method to Arabidopsis thaliana, specifically focusing on the different cell types found within the root. Conclusion The method accurately and conveniently charts C-value within specific cell types, and provides novel insight into developmental processes. The method is, in principle, applicable to any transformable organism, including mammals, within which cell type specificity of regulation of endoreduplication, of polysomaty, and of cell cycle arrest is suspected.

Direct Genesis of Functional Rodent and Human Schwann Cells from Skin Mesenchymal Precursors

Directory of Open Access Journals (Sweden)

Matthew P. Krause

2014-07-01

Full Text Available Recent reports of directed reprogramming have raised questions about the stability of cell lineages. Here, we have addressed this issue, focusing upon skin-derived precursors (SKPs, a dermally derived precursor cell. We show by lineage tracing that murine SKPs from dorsal skin originate from mesenchymal and not neural crest-derived cells. These mesenchymally derived SKPs can, without genetic manipulation, generate functional Schwann cells, a neural crest cell type, and are highly similar at the transcriptional level to Schwann cells isolated from the peripheral nerve. This is not a mouse-specific phenomenon, since human SKPs that are highly similar at the transcriptome level can be made from neural crest-derived facial and mesodermally derived foreskin dermis and the foreskin SKPs can make myelinating Schwann cells. Thus, nonneural crest-derived mesenchymal precursors can differentiate into bona fide peripheral glia in the absence of genetic manipulation, suggesting that developmentally defined lineage boundaries are more flexible than widely thought.

Brain-specific enhancers for cell-based therapy

Science.gov (United States)

Visel, Axel; Rubenstein, John L.R.; Chen, Ying-Jiun; Pennacchio, Len A.; Vogt, Daniel; Nicholas, Cory; Kriegstein, Arnold

2018-04-24

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

CXCR3 Directs Antigen-Specific Effector CD4+ T Cell Migration to the Lung During Parainfluenza Virus Infection

DEFF Research Database (Denmark)

Kohlmeier, Jacob E; Cookenham, Tres; Miller, Shannon C

2009-01-01

effector CD4(+) T cell migration to the lungs. To assess the role of CCR5 and CXCR3 in vivo, we directly compared the migration of Ag-specific wild-type and chemokine receptor-deficient effector T cells in mixed bone marrow chimeric mice during a parainfluenza virus infection. CXCR3-deficient effector CD4......(+) T cells were 5- to 10-fold less efficient at migrating to the lung compared with wild-type cells, whereas CCR5-deficient effector T cells were not impaired in their migration to the lung. In contrast to its role in trafficking, CXCR3 had no impact on effector CD4(+) T cell proliferation, phenotype......, or function in any of the tissues examined. These findings demonstrate that CXCR3 controls virus-specific effector CD4(+) T cell migration in vivo, and suggest that blocking CXCR3-mediated recruitment may limit T cell-induced immunopathology during respiratory virus infections....

Cancer Patient T Cells Genetically Targeted to Prostate-Specific Membrane Antigen Specifically Lyse Prostate Cancer Cells and Release Cytokines in Response to Prostate-Specific Membrane Antigen

Directory of Open Access Journals (Sweden)

Michael C. Gong

1999-06-01

Full Text Available The expression of immunoglobulin-based artificial receptors in normal T lymphocytes provides a means to target lymphocytes to cell surface antigens independently of major histocompatibility complex restriction. Such artificial receptors have been previously shown to confer antigen-specific tumoricidal properties in murine T cells. We constructed a novel ζ chain fusion receptor specific for prostate-specific membrane antigen (PSMA termed Pz-1. PSMA is a cell-surface glycoprotein expressed on prostate cancer cells and the neovascular endothelium of multiple carcinomas. We show that primary T cells harvested from five of five patients with different stages of prostate cancer and transduced with the Pz-1 receptor readily lyse prostate cancer cells. Having established a culture system using fibroblasts that express PSMA, we next show that T cells expressing the Pz-1 receptor release cytokines in response to cell-bound PSMA. Furthermore, we show that the cytokine release is greatly augmented by B7.1-mediated costimulation. Thus, our findings support the feasibility of adoptive cell therapy by using genetically engineered T cells in prostate cancer patients and suggest that both CD4+ and CD8+ T lymphocyte functions can be synergistically targeted against tumor cells.

Digital sorting of complex tissues for cell type-specific gene expression profiles.

Science.gov (United States)

Zhong, Yi; Wan, Ying-Wooi; Pang, Kaifang; Chow, Lionel M L; Liu, Zhandong

2013-03-07

Cellular heterogeneity is present in almost all gene expression profiles. However, transcriptome analysis of tissue specimens often ignores the cellular heterogeneity present in these samples. Standard deconvolution algorithms require prior knowledge of the cell type frequencies within a tissue or their in vitro expression profiles. Furthermore, these algorithms tend to report biased estimations. Here, we describe a Digital Sorting Algorithm (DSA) for extracting cell-type specific gene expression profiles from mixed tissue samples that is unbiased and does not require prior knowledge of cell type frequencies. The results suggest that DSA is a specific and sensitivity algorithm in gene expression profile deconvolution and will be useful in studying individual cell types of complex tissues.

Epigenetic regulation of normal human mammary cell type-specific miRNAs

Energy Technology Data Exchange (ETDEWEB)

Vrba, Lukas [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Inst. of Plant Molecular Biology, Ceske Budejovice (Czech Republic). Biology Centre ASCR; Garbe, James C. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Center; Stampfer, Martha R. [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center; Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Life Sciences Center; Futscher, Bernard W. [Univ. of Arizona, Tucson, AZ (United States). Arizona Cancer Center and Dept. of Pharmacology & Toxicology

2011-08-26

Epigenetic mechanisms are important regulators of cell type–specific genes, including miRNAs. In order to identify cell type-specific miRNAs regulated by epigenetic mechanisms, we undertook a global analysis of miRNA expression and epigenetic states in three isogenic pairs of human mammary epithelial cells (HMEC) and human mammary fibroblasts (HMF), which represent two differentiated cell types typically present within a given organ, each with a distinct phenotype and a distinct epigenotype. While miRNA expression and epigenetic states showed strong interindividual concordance within a given cell type, almost 10% of the expressed miRNA showed a cell type–specific pattern of expression that was linked to the epigenetic state of their promoter. The tissue-specific miRNA genes were epigenetically repressed in nonexpressing cells by DNA methylation (38%) and H3K27me3 (58%), with only a small set of miRNAs (21%) showing a dual epigenetic repression where both DNA methylation and H3K27me3 were present at their promoters, such as MIR10A and MIR10B. Individual miRNA clusters of closely related miRNA gene families can each display cell type–specific repression by the same or complementary epigenetic mechanisms, such as the MIR200 family, and MIR205, where fibroblasts repress MIR200C/141 by DNA methylation, MIR200A/200B/429 by H3K27me3, and MIR205 by both DNA methylation and H3K27me3. Since deregulation of many of the epigenetically regulated miRNAs that we identified have been linked to disease processes such as cancer, it is predicted that compromise of the epigenetic control mechanisms is important for this process. Overall, these results highlight the importance of epigenetic regulation in the control of normal cell type–specific miRNA expression.

Directed genetic modification of African horse sickness virus by reverse genetics

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Elaine Vermaak

2015-07-01

Full Text Available African horse sickness virus (AHSV, a member of the Orbivirus genus in the family Reoviridae, is an arthropod-transmitted pathogen that causes a devastating disease in horses with a mortality rate greater than 90%. Fundamental research on AHSV and the development of safe, efficacious vaccines could benefit greatly from an uncomplicated genetic modification method to generate recombinant AHSV. We demonstrate that infectious AHSV can be recovered by transfection of permissive mammalian cells with transcripts derived in vitro from purified AHSV core particles. These findings were expanded to establish a genetic modification system for AHSV that is based on transfection of the cells with a mixture of purified core transcripts and a synthetic T7 transcript. This approach was applied successfully to recover a directed cross-serotype reassortant AHSV and to introduce a marker sequence into the viral genome. The ability to manipulate the AHSV genome and engineer specific mutants will increase understanding of AHSV replication and pathogenicity, as well as provide a tool for generating designer vaccine strains.

Discovery of cell-type specific DNA motif grammar in cis-regulatory elements using random Forest.

Science.gov (United States)

Wang, Xin; Lin, Peijie; Ho, Joshua W K

2018-01-19

It has been observed that many transcription factors (TFs) can bind to different genomic loci depending on the cell type in which a TF is expressed in, even though the individual TF usually binds to the same core motif in different cell types. How a TF can bind to the genome in such a highly cell-type specific manner, is a critical research question. One hypothesis is that a TF requires co-binding of different TFs in different cell types. If this is the case, it may be possible to observe different combinations of TF motifs - a motif grammar - located at the TF binding sites in different cell types. In this study, we develop a bioinformatics method to systematically identify DNA motifs in TF binding sites across multiple cell types based on published ChIP-seq data, and address two questions: (1) can we build a machine learning classifier to predict cell-type specificity based on motif combinations alone, and (2) can we extract meaningful cell-type specific motif grammars from this classifier model. We present a Random Forest (RF) based approach to build a multi-class classifier to predict the cell-type specificity of a TF binding site given its motif content. We applied this RF classifier to two published ChIP-seq datasets of TF (TCF7L2 and MAX) across multiple cell types. Using cross-validation, we show that motif combinations alone are indeed predictive of cell types. Furthermore, we present a rule mining approach to extract the most discriminatory rules in the RF classifier, thus allowing us to discover the underlying cell-type specific motif grammar. Our bioinformatics analysis supports the hypothesis that combinatorial TF motif patterns are cell-type specific.

Genetic associations for pathogen-specific clinical mastitis and patterns of peaks in somatic cell count

NARCIS (Netherlands)

Haas, de Y.; Barkema, H.W.; Schukken, Y.H.; Veerkamp, R.F.

2003-01-01

Genetic associations were estimated between pathogen-specific cases of clinical mastitis (CM), lactational average somatic cell score (LACSCS), and patterns of peaks in somatic cell count (SCC) which were based on deviations from the typical lactation curve for SCC. The dataset contained test-day

ERE environment- and cell type-specific transcriptional effects of estrogen in normal endometrial cells.

Science.gov (United States)

Lascombe, I; Sallot, M; Vuillermoz, C; Weisz, A; Adessi, G L; Jouvenot, M

1998-04-30

Our previous results have suggested a repression of E2 (17beta-estradiol) effect on the c-fos gene of cultured guinea-pig endometrial cells. To investigate this repression, the expression of three human c-fos gene recombinants, pFC1-BL (-2250/+41), pFC2-BL (-1400/+41) and pFC2E (-1300/-1050 and -230/+41), known to be E2-responsive in Hela cells, was studied in stromal (SC) and glandular epithelial cells (GEC). In both cellular types, pFC1-BL was not induced by E2, even in the presence of growth factors or co-transfected estrogen receptor. The pattern of pFC2-BL and pFC2E expression was strikingly different and depended on the cellular type: pFC2-BL and pFC2E induction was restricted to the glandular epithelial cells and did not occur in the SCs. We argue for a repression of E2 action which is dependent on the estrogen-responsive cis-acting element (ERE) environment and also cell type-specific involving DNA/protein and/or protein/protein interactions with cellular type-specific factors.

Correlation and regression analyses of genetic effects for different types of cells in mammals under radiation and chemical treatment

International Nuclear Information System (INIS)

Slutskaya, N.G.; Mosseh, I.B.

2006-01-01

Data about genetic mutations under radiation and chemical treatment for different types of cells have been analyzed with correlation and regression analyses. Linear correlation between different genetic effects in sex cells and somatic cells have found. The results may be extrapolated on sex cells of human and mammals. (authors)

Engineering kidney cells: reprogramming and directed differentiation to renal tissues.

Science.gov (United States)

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.

Genetic counseling and the ethical issues around direct to consumer genetic testing.

Science.gov (United States)

Hawkins, Alice K; Ho, Anita

2012-06-01

Over the last several years, direct to consumer(DTC) genetic testing has received increasing attention in the public, healthcare and academic realms. DTC genetic testing companies face considerable criticism and scepticism,particularly from the medical and genetic counseling community. This raises the question of what specific aspects of DTC genetic testing provoke concerns, and conversely,promises, for genetic counselors. This paper addresses this question by exploring DTC genetic testing through an ethic allens. By considering the fundamental ethical approaches influencing genetic counseling (the ethic of care and principle-based ethics) we highlight the specific ethical concerns raised by DTC genetic testing companies. Ultimately,when considering the ethics of DTC testing in a genetic counseling context, we should think of it as a balancing act. We need careful and detailed consideration of the risks and troubling aspects of such testing, as well as the potentially beneficial direct and indirect impacts of the increased availability of DTC genetic testing. As a result it is essential that genetic counselors stay informed and involved in the ongoing debate about DTC genetic testing and DTC companies. Doing so will ensure that the ethical theories and principles fundamental to the profession of genetic counseling are promoted not just in traditional counseling sessions,but also on a broader level. Ultimately this will help ensure that the public enjoys the benefits of an increasingly genetic based healthcare system.

Identification of Cell Type-Specific Differences in Erythropoietin Receptor Signaling in Primary Erythroid and Lung Cancer Cells.

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Ruth Merkle

2016-08-01

Full Text Available Lung cancer, with its most prevalent form non-small-cell lung carcinoma (NSCLC, is one of the leading causes of cancer-related deaths worldwide, and is commonly treated with chemotherapeutic drugs such as cisplatin. Lung cancer patients frequently suffer from chemotherapy-induced anemia, which can be treated with erythropoietin (EPO. However, studies have indicated that EPO not only promotes erythropoiesis in hematopoietic cells, but may also enhance survival of NSCLC cells. Here, we verified that the NSCLC cell line H838 expresses functional erythropoietin receptors (EPOR and that treatment with EPO reduces cisplatin-induced apoptosis. To pinpoint differences in EPO-induced survival signaling in erythroid progenitor cells (CFU-E, colony forming unit-erythroid and H838 cells, we combined mathematical modeling with a method for feature selection, the L1 regularization. Utilizing an example model and simulated data, we demonstrated that this approach enables the accurate identification and quantification of cell type-specific parameters. We applied our strategy to quantitative time-resolved data of EPO-induced JAK/STAT signaling generated by quantitative immunoblotting, mass spectrometry and quantitative real-time PCR (qRT-PCR in CFU-E and H838 cells as well as H838 cells overexpressing human EPOR (H838-HA-hEPOR. The established parsimonious mathematical model was able to simultaneously describe the data sets of CFU-E, H838 and H838-HA-hEPOR cells. Seven cell type-specific parameters were identified that included for example parameters for nuclear translocation of STAT5 and target gene induction. Cell type-specific differences in target gene induction were experimentally validated by qRT-PCR experiments. The systematic identification of pathway differences and sensitivities of EPOR signaling in CFU-E and H838 cells revealed potential targets for intervention to selectively inhibit EPO-induced signaling in the tumor cells but leave the responses in

Invariant natural killer T-cell control of type 1 diabetes: a dendritic cell genetic decision of a silver bullet or Russian roulette.

Science.gov (United States)

Driver, John P; Scheuplein, Felix; Chen, Yi-Guang; Grier, Alexandra E; Wilson, S Brian; Serreze, David V

2010-02-01

In part, activation of invariant natural killer T (iNKT)-cells with the superagonist alpha-galactosylceramide (alpha-GalCer) inhibits the development of T-cell-mediated autoimmune type 1 diabetes in NOD mice by inducing the downstream differentiation of antigen-presenting dendritic cells (DCs) to an immunotolerogenic state. However, in other systems iNKT-cell activation has an adjuvant-like effect that enhances rather than suppresses various immunological responses. Thus, we tested whether in some circumstances genetic variation would enable activated iNKT-cells to support rather than inhibit type 1 diabetes development. We tested whether iNKT-conditioned DCs in NOD mice and a major histocompatibility complex-matched C57BL/6 (B6) background congenic stock differed in capacity to inhibit type 1 diabetes induced by the adoptive transfer of pathogenic AI4 CD8 T-cells. Unlike those of NOD origin, iNKT-conditioned DCs in the B6 background stock matured to a state that actually supported rather than inhibited AI4 T-cell-induced type 1 diabetes. The induction of a differing activity pattern of T-cell costimulatory molecules varying in capacity to override programmed death-ligand-1 inhibitory effects contributes to the respective ability of iNKT-conditioned DCs in NOD and B6 background mice to inhibit or support type 1 diabetes development. Genetic differences inherent to both iNKT-cells and DCs contribute to their varying interactions in NOD and B6.H2(g7) mice. This great variability in the interactions between iNKT-cells and DCs in two inbred mouse strains should raise a cautionary note about considering manipulation of this axis as a potential type 1 diabetes prevention therapy in genetically heterogeneous humans.

Appendiceal goblet cell carcinoids and adenocarcinomas ex-goblet cell carcinoid are genetically distinct from primary colorectal-type adenocarcinoma of the appendix

DEFF Research Database (Denmark)

Jesinghaus, Moritz; Konukiewitz, Björn; Foersch, Sebastian

2018-01-01

The appendix gives rise to goblet cell carcinoids, which represent special carcinomas with distinct biological and histological features. Their genetic background and molecular relationship to colorectal adenocarcinoma is largely unknown. We therefore performed a next-generation sequencing analysis...... a morphomolecular entity, histologically and genetically distinct from appendiceal colorectal-type adenocarcinomas and its colorectal counterparts. Altered Wnt-signaling associated genes, apart from APC, may act as potential drivers of these neoplasms. The absence of KRAS/NRAS mutations might render some....../adenocarcinoma ex-goblet cell carcinoid (n=2, respectively). Mutations in colorectal cancer-related genes (eg, TP53, KRAS, APC) were rare to absent in both, goblet cell carcinoids and adenocarcinomas ex-goblet cell carcinoid, but frequent in primary colorectal-type adenocarcinomas of the appendix. Additional large...

Proliferation requirements of cytomegalovirus-specific, effector-type human CD8+ T cells

NARCIS (Netherlands)

van Leeuwen, Ester M.; Gamadia, Laila E.; Baars, Paul A.; Remmerswaal, Ester B.; ten Berge, Ineke J.; van Lier, René A.

2002-01-01

Two prototypic types of virus-specific CD8(+) T cells can be found in latently infected individuals: CD45R0(+)CD27(+)CCR7(-) effector-memory, and CD45RA(+)CD27(-)CCR7(-) effector-type cells. It has recently been implied that CD45RA(+)CD27(-)CCR7(-) T cells are terminally differentiated effector

Cell-type-specific responses of RT4 neural cell lines to dibutyryl-cAMP: branch determination versus maturation

International Nuclear Information System (INIS)

Droms, K.; Sueoka, N.

1987-01-01

This report describes the induction of cell-type-specific maturation, by dibutyryl-cAMP and testololactone, of neuronal and glial properties in a family of cell lines derived from a rat peripheral neurotumor, RT4. This maturation allows further understanding of the process of determination because of the close lineage relationship between the cell types of the RT4 family. The RT4 family is characterized by the spontaneous conversion of one of the cell types, RT4-AC (stem-cell type), to any of three derivative cell types, RT4-B, RT4-D, or RT4-E, with a frequency of about 10(-5). The RT4-AC cells express some properties characteristic of both neuronal and glial cells. Of these neural properties expressed by RT4-AC cells, only the neuronal properties are expressed by the RT4-B and RT4-E cells, and only the glial properties are expressed by the RT4-D cells. This in vitro cell-type conversion of RT4-AC to three derivative cell types is a branch point for the coordinate regulation of several properties and seems to resemble determination in vivo. In our standard culture conditions, several other neuronal and glial properties are not expressed by these cell types. However, addition of dibutyryl-cAMP induces expression of additional properties, in a cell-type-specific manner: formation of long cellular processes in the RT4-B8 and RT4-E5 cell lines and expression of high-affinity uptake of gamma-aminobutyric acid, by a glial-cell-specific mechanism, in the RT4-D6-2 cell line. These new properties are maximally expressed 2-3 days after addition of dibutyryl-cAMP

Long-term in vitro, cell-type-specific genome-wide reprogramming of gene expression

International Nuclear Information System (INIS)

Hakelien, Anne-Mari; Gaustad, Kristine G.; Taranger, Christel K.; Skalhegg, Bjorn S.; Kuentziger, Thomas; Collas, Philippe

2005-01-01

We demonstrate a cell extract-based, genome-wide and heritable reprogramming of gene expression in vitro. Kidney epithelial 293T cells have previously been shown to take on T cell properties following a brief treatment with an extract of Jurkat T cells. We show here that 293T cells exposed for 1 h to a Jurkat cell extract undergo genome-wide, target cell-type-specific and long-lasting transcriptional changes. Microarray analyses indicate that on any given week after extract treatment, ∼2500 genes are upregulated >3-fold, of which ∼900 are also expressed in Jurkat cells. Concomitantly, ∼1500 genes are downregulated or repressed, of which ∼500 are also downregulated in Jurkat cells. Gene expression changes persist for over 30 passages (∼80 population doublings) in culture. Target cell-type specificity of these changes is shown by the lack of activation or repression of Jurkat-specific genes by extracts of 293T cells or carcinoma cells. Quantitative RT-PCR analysis confirms the long-term transcriptional activation of genes involved in key T cell functions. Additionally, growth of cells in suspended aggregates, expression of CD3 and CD28 T cell surface markers, and interleukin-2 secretion by 293T cells treated with extract of adult peripheral blood T cells illustrate a functional nuclear reprogramming. Therefore, target cell-type-specific and heritable changes in gene expression, and alterations in cell function, can be promoted by extracts derived from transformed cells as well as from adult primary cells

GLYCAN-DIRECTED CAR-T CELLS.

Science.gov (United States)

Steentoft, Catharina; Migliorini, Denis; King, Tiffany R; Mandel, Ulla; June, Carl H; Posey, Avery D

2018-01-23

Cancer immunotherapy is rapidly advancing in the treatment of a variety of hematopoietic cancers, including pediatric acute lymphoblastic leukemia and diffuse large B cell lymphoma, with chimeric antigen receptor (CAR)-T cells. CARs are genetically encoded artificial T cell receptors that combine the antigen specificity of an antibody with the machinery of T cell activation. However, implementation of CAR technology in the treatment of solid tumors has been progressing much slower. Solid tumors are characterized by a number of challenges that need to be overcome, including cellular heterogeneity, immunosuppressive tumor microenvironment (TME), and, in particular, few known cancer-specific targets. Post-translational modifications that differentially occur in malignant cells generate valid cell surface, cancer-specific targets for CAR-T cells. We previously demonstrated that CAR-T cells targeting an aberrant O-glycosylation of MUC1, a common cancer marker associated with changes in cell adhesion, tumor growth, and poor prognosis, could control malignant growth in mouse models. Here, we discuss the field of glycan-directed CAR-T cells and review the different classes of antibodies specific for glycan-targeting, including the generation of high affinity O-glycopeptide antibodies. Finally, we discuss historic and recently investigated glycan targets for CAR-T cells and provide our perspective on how targeting the tumor glycoproteome and/or glycome will improve CAR-T immunotherapy. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency.

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Marina E Tourlakis

2015-06-01

Full Text Available Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15(Ink4b and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis

In Vivo Senescence in the Sbds-Deficient Murine Pancreas: Cell-Type Specific Consequences of Translation Insufficiency

Science.gov (United States)

Tourlakis, Marina E.; Zhang, Siyi; Ball, Heather L.; Gandhi, Rikesh; Liu, Hongrui; Zhong, Jian; Yuan, Julie S.; Guidos, Cynthia J.; Durie, Peter R.; Rommens, Johanna M.

2015-01-01

Genetic models of ribosome dysfunction show selective organ failure, highlighting a gap in our understanding of cell-type specific responses to translation insufficiency. Translation defects underlie a growing list of inherited and acquired cancer-predisposition syndromes referred to as ribosomopathies. We sought to identify molecular mechanisms underlying organ failure in a recessive ribosomopathy, with particular emphasis on the pancreas, an organ with a high and reiterative requirement for protein synthesis. Biallelic loss of function mutations in SBDS are associated with the ribosomopathy Shwachman-Diamond syndrome, which is typified by pancreatic dysfunction, bone marrow failure, skeletal abnormalities and neurological phenotypes. Targeted disruption of Sbds in the murine pancreas resulted in p53 stabilization early in the postnatal period, specifically in acinar cells. Decreased Myc expression was observed and atrophy of the adult SDS pancreas could be explained by the senescence of acinar cells, characterized by induction of Tgfβ, p15Ink4b and components of the senescence-associated secretory program. This is the first report of senescence, a tumour suppression mechanism, in association with SDS or in response to a ribosomopathy. Genetic ablation of p53 largely resolved digestive enzyme synthesis and acinar compartment hypoplasia, but resulted in decreased cell size, a hallmark of decreased translation capacity. Moreover, p53 ablation resulted in expression of acinar dedifferentiation markers and extensive apoptosis. Our findings indicate a protective role for p53 and senescence in response to Sbds ablation in the pancreas. In contrast to the pancreas, the Tgfβ molecular signature was not detected in fetal bone marrow, liver or brain of mouse models with constitutive Sbds ablation. Nevertheless, as observed with the adult pancreas phenotype, disease phenotypes of embryonic tissues, including marked neuronal cell death due to apoptosis, were determined to

Prespacer processing and specific integration in a Type I-A CRISPR system

Science.gov (United States)

Rollie, Clare; Graham, Shirley; Rouillon, Christophe

2018-01-01

Abstract The CRISPR–Cas system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. Adaptation is dependent on the Cas1 and Cas2 proteins along with varying accessory proteins. Here we analyse the process in Sulfolobus solfataricus, showing that while Cas1 and Cas2 catalyze spacer integration in vitro, host factors are required for specificity. Specific integration also requires at least 400 bp of the leader sequence, and is dependent on the presence of hydrolysable ATP, suggestive of an active process that may involve DNA remodelling. Specific spacer integration is associated with processing of prespacer 3′ ends in a PAM-dependent manner. This is reflected in PAM-dependent processing of prespacer 3′ ends in vitro in the presence of cell lysate or the Cas4 nuclease, in a reaction consistent with PAM-directed binding and protection of prespacer DNA. These results highlight the diverse interplay between CRISPR–Cas elements and host proteins across CRISPR types. PMID:29228332

Genetic ablation of root cap cells in Arabidopsis

OpenAIRE

Tsugeki, Ryuji; Fedoroff, Nina V.

1999-01-01

The root cap is increasingly appreciated as a complex and dynamic plant organ. Root caps sense and transmit environmental signals, synthesize and secrete small molecules and macromolecules, and in some species shed metabolically active cells. However, it is not known whether root caps are essential for normal shoot and root development. We report the identification of a root cap-specific promoter and describe its use to genetically ablate root caps by directing root cap-specific expression of...

PINK1 is required for timely cell-type specific mitochondrial clearance during Drosophila midgut metamorphosis.

Science.gov (United States)

Liu, Yan; Lin, Jingjing; Zhang, Minjie; Chen, Kai; Yang, Shengxi; Wang, Qun; Yang, Hongqin; Xie, Shusen; Zhou, Yongjian; Zhang, Xi; Chen, Fei; Yang, Yufeng

2016-11-15

Mitophagy is the selective degradation of mitochondria by autophagy, which is an important mitochondrial quality and quantity control process. During Drosophila metamorphosis, the degradation of midgut involves a large change in length and organization, which is mediated by autophagy. Here we noticed a cell-type specific mitochondrial clearance process that occurs in enterocytes (ECs), while most mitochondria remain in intestinal stem cells (ISCs) during metamorphosis. Although PINK1/PARKIN represent the canonical pathway for the elimination of impaired mitochondria in varied pathological conditions, their roles in developmental processes or normal physiological conditions have been less studied. To examine the potential contribution of PINK1 in developmental processes, we monitored the dynamic expression pattern of PINK1 in the midgut development by taking advantage of a newly CRISPR/Cas9 generated knock-in fly strain expressing PINK1-mCherry fusion protein that presumably recapitulates the endogenous expression pattern of PINK1. We disclosed a spatiotemporal correlation between the expression pattern of PINK1 and the mitochondrial clearance or persistence in ECs or ISCs respectively. By mosaic genetic analysis, we then demonstrated that PINK1 and PARKIN function epistatically to mediate the specific timely removal of mitochondria, and are involved in global autophagy in ECs during Drosophila midgut metamorphosis, with kinase-dead PINK1 exerting dominant negative effects. Taken together, our studies concluded that the PINK1/PARKIN is crucial for timely cell-type specific mitophagy under physiological conditions and demonstrated again that Drosophila midgut metamorphosis might serve as an elegant in vivo model to study autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

Homophilic and Heterophilic Interactions of Type II Cadherins Identify Specificity Groups Underlying Cell-Adhesive Behavior

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Julia Brasch

2018-05-01

Full Text Available Summary: Type II cadherins are cell-cell adhesion proteins critical for tissue patterning and neuronal targeting but whose molecular binding code remains poorly understood. Here, we delineate binding preferences for type II cadherin cell-adhesive regions, revealing extensive heterophilic interactions between specific pairs, in addition to homophilic interactions. Three distinct specificity groups emerge from our analysis with members that share highly similar heterophilic binding patterns and favor binding to one another. Structures of adhesive fragments from each specificity group confirm near-identical dimer topology conserved throughout the family, allowing interface residues whose conservation corresponds to specificity preferences to be identified. We show that targeted mutation of these residues converts binding preferences between specificity groups in biophysical and co-culture assays. Our results provide a detailed understanding of the type II cadherin interaction map and a basis for defining their role in tissue patterning and for the emerging importance of their heterophilic interactions in neural connectivity. : Type II cadherins are a family of vertebrate cell adhesion proteins expressed primarily in the CNS. Brasch et al. measure binding between adhesive fragments, revealing homophilic and extensive selective heterophilic binding with specificities that define groups of similar cadherins. Structures reveal common adhesive dimers, with residues governing cell-adhesive specificity. Keywords: cell adhesion, crystal structure, hemophilic specificity, heterophilic specificity, neural patterning, synaptic targeting, cadherin

Dual-specificity anti-sigma factor reinforces control of cell-type specific gene expression in Bacillus subtilis.

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Mónica Serrano

2015-04-01

Full Text Available Gene expression during spore development in Bacillus subtilis is controlled by cell type-specific RNA polymerase sigma factors. σFand σE control early stages of development in the forespore and the mother cell, respectively. When, at an intermediate stage in development, the mother cell engulfs the forespore, σF is replaced by σG and σE is replaced by σK. The anti-sigma factor CsfB is produced under the control of σF and binds to and inhibits the auto-regulatory σG, but not σF. A position in region 2.1, occupied by an asparagine in σG and by a glutamate in οF, is sufficient for CsfB discrimination of the two sigmas, and allows it to delay the early to late switch in forespore gene expression. We now show that following engulfment completion, csfB is switched on in the mother cell under the control of σK and that CsfB binds to and inhibits σE but not σK, possibly to facilitate the switch from early to late gene expression. We show that a position in region 2.3 occupied by a conserved asparagine in σE and by a conserved glutamate in σK suffices for discrimination by CsfB. We also show that CsfB prevents activation of σG in the mother cell and the premature σG-dependent activation of σK. Thus, CsfB establishes negative feedback loops that curtail the activity of σE and prevent the ectopic activation of σG in the mother cell. The capacity of CsfB to directly block σE activity may also explain how CsfB plays a role as one of the several mechanisms that prevent σE activation in the forespore. Thus the capacity of CsfB to differentiate between the highly similar σF/σG and σE/σK pairs allows it to rinforce the cell-type specificity of these sigma factors and the transition from early to late development in B. subtilis, and possibly in all sporeformers that encode a CsfB orthologue.

Preimplantation genetic diagnosis guided by single-cell genomics

Science.gov (United States)

2013-01-01

Preimplantation genetic diagnosis (PGD) aims to help couples with heritable genetic disorders to avoid the birth of diseased offspring or the recurrence of loss of conception. Following in vitro fertilization, one or a few cells are biopsied from each human preimplantation embryo for genetic testing, allowing diagnosis and selection of healthy embryos for uterine transfer. Although classical methods, including single-cell PCR and fluorescent in situ hybridization, enable PGD for many genetic disorders, they have limitations. They often require family-specific designs and can be labor intensive, resulting in long waiting lists. Furthermore, certain types of genetic anomalies are not easy to diagnose using these classical approaches, and healthy offspring carrying the parental mutant allele(s) can result. Recently, state-of-the-art methods for single-cell genomics have flourished, which may overcome the limitations associated with classical PGD, and these underpin the development of generic assays for PGD that enable selection of embryos not only for the familial genetic disorder in question, but also for various other genetic aberrations and traits at once. Here, we discuss the latest single-cell genomics methodologies based on DNA microarrays, single-nucleotide polymorphism arrays or next-generation sequence analysis. We focus on their strengths, their validation status, their weaknesses and the challenges for implementing them in PGD. PMID:23998893

Evolution of sexes from an ancestral mating-type specification pathway.

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Sa Geng

2014-07-01

Full Text Available Male and female sexes have evolved repeatedly in eukaryotes but the origins of dimorphic sexes and their relationship to mating types in unicellular species are not understood. Volvocine algae include isogamous species such as Chlamydomonas reinhardtii, with two equal-sized mating types, and oogamous multicellular species such as Volvox carteri with sperm-producing males and egg-producing females. Theoretical work predicts genetic linkage of a gamete cell-size regulatory gene(s to an ancestral mating-type locus as a possible step in the evolution of dimorphic gametes, but this idea has not been tested. Here we show that, contrary to predictions, a single conserved mating locus (MT gene in volvocine algae-MID, which encodes a RWP-RK domain transcription factor-evolved from its ancestral role in C. reinhardtii as a mating-type specifier, to become a determinant of sperm and egg development in V. carteri. Transgenic female V. carteri expressing male MID produced functional sperm packets during sexual development. Transgenic male V. carteri with RNA interference (RNAi-mediated knockdowns of VcMID produced functional eggs, or self-fertile hermaphrodites. Post-transcriptional controls were found to regulate cell-type-limited expression and nuclear localization of VcMid protein that restricted its activity to nuclei of developing male germ cells and sperm. Crosses with sex-reversed strains uncoupled sex determination from sex chromosome identity and revealed gender-specific roles for male and female mating locus genes in sexual development, gamete fitness and reproductive success. Our data show genetic continuity between the mating-type specification and sex determination pathways of volvocine algae, and reveal evidence for gender-specific adaptations in the male and female mating locus haplotypes of Volvox. These findings will enable a deeper understanding of how a master regulator of mating-type determination in an ancestral unicellular species was

Germ cell toxicity: significance in genetic and fertility effects of radiation and chemicals

International Nuclear Information System (INIS)

Oakberg, E.F.

1983-01-01

The response of the male and female to radiation and chemicals is different. Any loss of oocytes in the female cannot be replaced, and if severe enough, will result in a shortening of the reproductive span. In the male, a temporary sterile period may be induced owing to destruction of the differentiating spermatogonia, but the stem cells are the most resistant spermatogonial type, are capable of repopulating the seminiferous epithelium, and fertility usually returns. The response of both the male and female changes with development of the embryonic to the adult gonad, and with differentiation and maturation in the adult. The primordial germ cells, early oocytes, and differentiating spermatogonia of the adult male are unusually sensitive to the cytotoxic action of noxious agents, but each agent elicits a specific response owing to the intricate biochemical and physiological changes associated with development and maturation of the gametes. The relationship of germ cell killing to fertility is direct, and long-term fertility effects can be predicted from histological analysis of the gonads. The relationship to genetic effects, on the other hand, is indirect, and acts primarily by limiting the cell stages available for testing, by affecting the distribution of mitotically active stem cells among the different stages of the mitotic cycle, and thereby, changing both the type and frequency of genetic effects observed. 100 references, 38 figures, 7 tables

Type-Directed Partial Evaluation

DEFF Research Database (Denmark)

Danvy, Olivier

1998-01-01

Type-directed partial evaluation uses a normalization function to achieve partial evaluation. These lecture notes review its background, foundations, practice, and applications. Of specific interest is the modular technique of offline and online type-directed partial evaluation in Standard ML...

Type-Directed Partial Evaluation

DEFF Research Database (Denmark)

Danvy, Olivier

1998-01-01

Type-directed partial evaluation uses a normalization function to achieve partial evaluation. These lecture notes review its background, foundations, practice, and applications. Of specific interest is the modular technique of offline and online type-directed partial evaluation in Standard ML of ...

Cell-Type-Specific Splicing of Piezo2 Regulates Mechanotransduction

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Marcin Szczot

2017-12-01

Full Text Available Summary: Piezo2 is a mechanically activated ion channel required for touch discrimination, vibration detection, and proprioception. Here, we discovered that Piezo2 is extensively spliced, producing different Piezo2 isoforms with distinct properties. Sensory neurons from both mice and humans express a large repertoire of Piezo2 variants, whereas non-neuronal tissues express predominantly a single isoform. Notably, even within sensory ganglia, we demonstrate the splicing of Piezo2 to be cell type specific. Biophysical characterization revealed substantial differences in ion permeability, sensitivity to calcium modulation, and inactivation kinetics among Piezo2 splice variants. Together, our results describe, at the molecular level, a potential mechanism by which transduction is tuned, permitting the detection of a variety of mechanosensory stimuli. : Szczot et al. find that the mechanoreceptor Piezo2 is extensively alternatively spliced, generating multiple distinct isoforms. Their findings indicate that these splice products have specific tissue and cell type expression patterns and exhibit differences in receptor properties. Keywords: Piezo, touch, sensation, ion-channel, splicing

Enterovirus strain and type-specific differences in growth kinetics and virus-induced cell destruction in human pancreatic duct epithelial HPDE cells.

Science.gov (United States)

Smura, Teemu; Natri, Olli; Ylipaasto, Petri; Hellman, Marika; Al-Hello, Haider; Piemonti, Lorenzo; Roivainen, Merja

2015-12-02

Enterovirus infections have been suspected to be involved in the development of type 1 diabetes. However, the pathogenetic mechanism of enterovirus-induced type 1 diabetes is not known. Pancreatic ductal cells are closely associated with pancreatic islets. Therefore, enterovirus infections in ductal cells may also affect beta-cells and be involved in the induction of type 1 diabetes. The aim of this study was to assess the ability of different enterovirus strains to infect, replicate and produce cytopathic effect in human pancreatic ductal cells. Furthermore, the viral factors that affect these capabilities were studied. The pancreatic ductal cells were highly susceptible to enterovirus infections. Both viral growth and cytolysis were detected for several enterovirus serotypes. However, the viral growth and capability to induce cytopathic effect (cpe) did not correlate completely. Some of the virus strains replicated in ductal cells without apparent cpe. Furthermore, there were strain-specific differences in the growth kinetics and the ability to cause cpe within some serotypes. Viral adaptation experiments were carried out to study the potential genetic determinants behind these phenotypic differences. The blind-passage of non-lytic CV-B6-Schmitt strain in HPDE-cells resulted in lytic phenotype and increased progeny production. This was associated with the substitution of a single amino acid (K257E) in the virus capsid protein VP1 and the viral ability to use decay accelerating factor (DAF) as a receptor. This study demonstrates considerable plasticity in the cell tropism, receptor usage and cytolytic properties of enteroviruses and underlines the strong effect of single or few amino acid substitutions in cell tropism and lytic capabilities of a given enterovirus. Since ductal cells are anatomically close to pancreatic islets, the capability of enteroviruses to infect and destroy pancreatic ductal cells may also implicate in respect to enterovirus induced type 1

Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition.

Science.gov (United States)

Gervin, Kristina; Page, Christian Magnus; Aass, Hans Christian D; Jansen, Michelle A; Fjeldstad, Heidi Elisabeth; Andreassen, Bettina Kulle; Duijts, Liesbeth; van Meurs, Joyce B; van Zelm, Menno C; Jaddoe, Vincent W; Nordeng, Hedvig; Knudsen, Gunn Peggy; Magnus, Per; Nystad, Wenche; Staff, Anne Cathrine; Felix, Janine F; Lyle, Robert

2016-09-01

Epigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused by cellular heterogeneity is a major concern. This can be adjusted for using reference data consisting of DNA methylation signatures in cell types isolated from blood. However, the most commonly used reference data set is based on blood samples from adult males and is not representative of the cell type composition in neonatal cord blood. The aim of this study was to generate a reference data set from cord blood to enable correct adjustment of the cell type composition in samples collected at birth. The purity of the isolated cell types was very high for all samples (>97.1%), and clustering analyses showed distinct grouping of the cell types according to hematopoietic lineage. We explored whether this cord blood and the adult peripheral blood reference data sets impact the estimation of cell type composition in cord blood samples from an independent birth cohort (MoBa, n = 1092). This revealed significant differences for all cell types. Importantly, comparison of the cell type estimates against matched cell counts both in the cord blood reference samples (n = 11) and in another independent birth cohort (Generation R, n = 195), demonstrated moderate to high correlation of the data. This is the first cord blood reference data set with a comprehensive examination of the downstream application of the data through validation of estimated cell types against matched cell counts.

Improving sensitivity of linear regression-based cell type-specific differential expression deconvolution with per-gene vs. global significance threshold.

Science.gov (United States)

Glass, Edmund R; Dozmorov, Mikhail G

2016-10-06

The goal of many human disease-oriented studies is to detect molecular mechanisms different between healthy controls and patients. Yet, commonly used gene expression measurements from blood samples suffer from variability of cell composition. This variability hinders the detection of differentially expressed genes and is often ignored. Combined with cell counts, heterogeneous gene expression may provide deeper insights into the gene expression differences on the cell type-specific level. Published computational methods use linear regression to estimate cell type-specific differential expression, and a global cutoff to judge significance, such as False Discovery Rate (FDR). Yet, they do not consider many artifacts hidden in high-dimensional gene expression data that may negatively affect linear regression. In this paper we quantify the parameter space affecting the performance of linear regression (sensitivity of cell type-specific differential expression detection) on a per-gene basis. We evaluated the effect of sample sizes, cell type-specific proportion variability, and mean squared error on sensitivity of cell type-specific differential expression detection using linear regression. Each parameter affected variability of cell type-specific expression estimates and, subsequently, the sensitivity of differential expression detection. We provide the R package, LRCDE, which performs linear regression-based cell type-specific differential expression (deconvolution) detection on a gene-by-gene basis. Accounting for variability around cell type-specific gene expression estimates, it computes per-gene t-statistics of differential detection, p-values, t-statistic-based sensitivity, group-specific mean squared error, and several gene-specific diagnostic metrics. The sensitivity of linear regression-based cell type-specific differential expression detection differed for each gene as a function of mean squared error, per group sample sizes, and variability of the proportions

Cell type-specific responses to salinity - the epidermal bladder cell transcriptome of Mesembryanthemum crystallinum.

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Oh, Dong-Ha; Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar; Lee, Sang-Yeol; Bohnert, Hans J; Dassanayake, Maheshi

2015-08-01

Mesembryanthemum crystallinum (ice plant) exhibits extreme tolerance to salt. Epidermal bladder cells (EBCs), developing on the surface of aerial tissues and specialized in sodium sequestration and other protective functions, are critical for the plant's stress adaptation. We present the first transcriptome analysis of EBCs isolated from intact plants, to investigate cell type-specific responses during plant salt adaptation. We developed a de novo assembled, nonredundant EBC reference transcriptome. Using RNAseq, we compared the expression patterns of the EBC-specific transcriptome between control and salt-treated plants. The EBC reference transcriptome consists of 37 341 transcript-contigs, of which 7% showed significantly different expression between salt-treated and control samples. We identified significant changes in ion transport, metabolism related to energy generation and osmolyte accumulation, stress signalling, and organelle functions, as well as a number of lineage-specific genes of unknown function, in response to salt treatment. The salinity-induced EBC transcriptome includes active transcript clusters, refuting the view of EBCs as passive storage compartments in the whole-plant stress response. EBC transcriptomes, differing from those of whole plants or leaf tissue, exemplify the importance of cell type-specific resolution in understanding stress adaptive mechanisms. No claim to original US government works. New Phytologist © 2015 New Phytologist Trust.

Cell type-specific termination of transcription by transposable element sequences.

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Conley, Andrew B; Jordan, I King

2012-09-30

Transposable elements (TEs) encode sequences necessary for their own transposition, including signals required for the termination of transcription. TE sequences within the introns of human genes show an antisense orientation bias, which has been proposed to reflect selection against TE sequences in the sense orientation owing to their ability to terminate the transcription of host gene transcripts. While there is evidence in support of this model for some elements, the extent to which TE sequences actually terminate transcription of human gene across the genome remains an open question. Using high-throughput sequencing data, we have characterized over 9,000 distinct TE-derived sequences that provide transcription termination sites for 5,747 human genes across eight different cell types. Rarefaction curve analysis suggests that there may be twice as many TE-derived termination sites (TE-TTS) genome-wide among all human cell types. The local chromatin environment for these TE-TTS is similar to that seen for 3' UTR canonical TTS and distinct from the chromatin environment of other intragenic TE sequences. However, those TE-TTS located within the introns of human genes were found to be far more cell type-specific than the canonical TTS. TE-TTS were much more likely to be found in the sense orientation than other intragenic TE sequences of the same TE family and TE-TTS in the sense orientation terminate transcription more efficiently than those found in the antisense orientation. Alu sequences were found to provide a large number of relatively weak TTS, whereas LTR elements provided a smaller number of much stronger TTS. TE sequences provide numerous termination sites to human genes, and TE-derived TTS are particularly cell type-specific. Thus, TE sequences provide a powerful mechanism for the diversification of transcriptional profiles between cell types and among evolutionary lineages, since most TE-TTS are evolutionarily young. The extent of transcription

Cardiac Glycoside Glucoevatromonoside Induces Cancer Type-Specific Cell Death

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Naira F. Z. Schneider

2018-03-01

Full Text Available Cardiac glycosides (CGs are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC and high-affinity binding to the Na+/K+-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.

Capacity of lung stroma to educate dendritic cells inhibiting mycobacteria-specific T-cell response depends upon genetic susceptibility to tuberculosis.

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Kapina, Marina A; Rubakova, Elvira I; Majorov, Konstantin B; Logunova, Nadezhda N; Apt, Alexander S

2013-01-01

The balance between activation and inhibition of local immune responses in affected tissues during prolonged chronic infections is important for host protection. There is ample evidence that regulatory, tolerogenic dendritic cells (DC) are developed and present in tissues and inhibit overwhelming inflammatory reactions. Also, it was firmly established that stromal microenvironment of many organs is able to induce development of immature regulatory DC (DCreg), an essential element of a general immune regulatory network. However, direct experimental data demonstrating inhibition of immune responses by stroma-instructed immature DCreg in infectious models are scarce, and virtually nothing is known about functioning of this axis of immunity during tuberculosis (TB) infection. In this study, we demonstrate that lung stromal cells are capable of supporting the development in culture of immature CD11b(+)CD11c(low)CD103(-) DCreg from lineage-negative (lin(-)) bone marrow precursors. DCreg developed on lung stroma isolated from mice of genetically TB-hyper-susceptible I/St and relatively resistant B6 inbred strains inhibited proliferative response of mycobacteria-specific CD4(+) T-cell lines a dose-dependent manner. Importantly, the inhibitory activity of B6 DCreg was substantially higher than that of I/St Dcreg. Moreover, when the donors of stromal cells were chronically infected with virulent mycobacteria, the capacity to instruct inhibitory DCreg was retained in B6, but further diminished in I/St stromal cells. DCreg-provided suppression was mediated by a few soluble mediators, including PGE2, NO and IL-10. The content of CD4(+)Foxp3(+) Treg cells in the mediastinal, lung-draining lymph nodes at the advanced stages of chronic infection did not change in I/St, but increased 2-fold in B6 mice, and lung pathology was much more pronounced in the former mice. Taken together, these data provide genetic evidence that the capacity to maintain populations of regulatory cells

Capacity of lung stroma to educate dendritic cells inhibiting mycobacteria-specific T-cell response depends upon genetic susceptibility to tuberculosis.

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Marina A Kapina

Full Text Available The balance between activation and inhibition of local immune responses in affected tissues during prolonged chronic infections is important for host protection. There is ample evidence that regulatory, tolerogenic dendritic cells (DC are developed and present in tissues and inhibit overwhelming inflammatory reactions. Also, it was firmly established that stromal microenvironment of many organs is able to induce development of immature regulatory DC (DCreg, an essential element of a general immune regulatory network. However, direct experimental data demonstrating inhibition of immune responses by stroma-instructed immature DCreg in infectious models are scarce, and virtually nothing is known about functioning of this axis of immunity during tuberculosis (TB infection. In this study, we demonstrate that lung stromal cells are capable of supporting the development in culture of immature CD11b(+CD11c(lowCD103(- DCreg from lineage-negative (lin(- bone marrow precursors. DCreg developed on lung stroma isolated from mice of genetically TB-hyper-susceptible I/St and relatively resistant B6 inbred strains inhibited proliferative response of mycobacteria-specific CD4(+ T-cell lines a dose-dependent manner. Importantly, the inhibitory activity of B6 DCreg was substantially higher than that of I/St Dcreg. Moreover, when the donors of stromal cells were chronically infected with virulent mycobacteria, the capacity to instruct inhibitory DCreg was retained in B6, but further diminished in I/St stromal cells. DCreg-provided suppression was mediated by a few soluble mediators, including PGE2, NO and IL-10. The content of CD4(+Foxp3(+ Treg cells in the mediastinal, lung-draining lymph nodes at the advanced stages of chronic infection did not change in I/St, but increased 2-fold in B6 mice, and lung pathology was much more pronounced in the former mice. Taken together, these data provide genetic evidence that the capacity to maintain populations of regulatory

Facilitating Neuron-Specific Genetic Manipulations in Drosophila melanogaster Using a Split GAL4 Repressor.

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Dolan, Michael-John; Luan, Haojiang; Shropshire, William C; Sutcliffe, Ben; Cocanougher, Benjamin; Scott, Robert L; Frechter, Shahar; Zlatic, Marta; Jefferis, Gregory S X E; White, Benjamin H

2017-06-01

Efforts to map neural circuits have been galvanized by the development of genetic technologies that permit the manipulation of targeted sets of neurons in the brains of freely behaving animals. The success of these efforts relies on the experimenter's ability to target arbitrarily small subsets of neurons for manipulation, but such specificity of targeting cannot routinely be achieved using existing methods. In Drosophila melanogaster , a widely-used technique for refined cell type-specific manipulation is the Split GAL4 system, which augments the targeting specificity of the binary GAL4-UAS (Upstream Activating Sequence) system by making GAL4 transcriptional activity contingent upon two enhancers, rather than one. To permit more refined targeting, we introduce here the "Killer Zipper" (KZip + ), a suppressor that makes Split GAL4 targeting contingent upon a third enhancer. KZip + acts by disrupting both the formation and activity of Split GAL4 heterodimers, and we show how this added layer of control can be used to selectively remove unwanted cells from a Split GAL4 expression pattern or to subtract neurons of interest from a pattern to determine their requirement in generating a given phenotype. To facilitate application of the KZip + technology, we have developed a versatile set of LexA op -KZip + fly lines that can be used directly with the large number of LexA driver lines with known expression patterns. KZip + significantly sharpens the precision of neuronal genetic control available in Drosophila and may be extended to other organisms where Split GAL4-like systems are used. Copyright © 2017 Dolan et al.

Cell Type-specific Intrinsic Perithreshold Oscillations in Hippocampal GABAergic Interneurons.

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Kang, Young-Jin; Lewis, Hannah Elisabeth Smashey; Young, Mason William; Govindaiah, Gubbi; Greenfield, Lazar John; Garcia-Rill, Edgar; Lee, Sang-Hun

2018-04-15

The hippocampus plays a critical role in learning, memory, and spatial processing through coordinated network activity including theta and gamma oscillations. Recent evidence suggests that hippocampal subregions (e.g., CA1) can generate these oscillations at the network level, at least in part, through GABAergic interneurons. However, it is unclear whether specific GABAergic interneurons generate intrinsic theta and/or gamma oscillations at the single-cell level. Since major types of CA1 interneurons (i.e., parvalbumin-positive basket cells (PVBCs), cannabinoid type 1 receptor-positive basket cells (CB 1 BCs), Schaffer collateral-associated cells (SCAs), neurogliaform cells and ivy cells) are thought to play key roles in network theta and gamma oscillations in the hippocampus, we tested the hypothesis that these cells generate intrinsic perithreshold oscillations at the single-cell level. We performed whole-cell patch-clamp recordings from GABAergic interneurons in the CA1 region of the mouse hippocampus in the presence of synaptic blockers to identify intrinsic perithreshold membrane potential oscillations. The majority of PVBCs (83%), but not the other interneuron subtypes, produced intrinsic perithreshold gamma oscillations if the membrane potential remained above -45 mV. In contrast, CB 1 BCs, SCAs, neurogliaform cells, ivy cells, and the remaining PVBCs (17%) produced intrinsic theta, but not gamma, oscillations. These oscillations were prevented by blockers of persistent sodium current. These data demonstrate that the major types of hippocampal interneurons produce distinct frequency bands of intrinsic perithreshold membrane oscillations. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

Analysis of cell-type-specific gene expression during mouse spermatogenesis

DEFF Research Database (Denmark)

Almstrup, Kristian; Nielsen, John E; Hansen, Martin Asser

2004-01-01

In rodents, changes in gene expression during spermatogenesis can be monitored by sampling testis from each day during postnatal development. However, changes in gene expression at the tissue level can reflect changes in the concentration of an mRNA in a specific cell type, changes in volume of s...

Cell type-specific termination of transcription by transposable element sequences

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Conley Andrew B

2012-09-01

Full Text Available Abstract Background Transposable elements (TEs encode sequences necessary for their own transposition, including signals required for the termination of transcription. TE sequences within the introns of human genes show an antisense orientation bias, which has been proposed to reflect selection against TE sequences in the sense orientation owing to their ability to terminate the transcription of host gene transcripts. While there is evidence in support of this model for some elements, the extent to which TE sequences actually terminate transcription of human gene across the genome remains an open question. Results Using high-throughput sequencing data, we have characterized over 9,000 distinct TE-derived sequences that provide transcription termination sites for 5,747 human genes across eight different cell types. Rarefaction curve analysis suggests that there may be twice as many TE-derived termination sites (TE-TTS genome-wide among all human cell types. The local chromatin environment for these TE-TTS is similar to that seen for 3′ UTR canonical TTS and distinct from the chromatin environment of other intragenic TE sequences. However, those TE-TTS located within the introns of human genes were found to be far more cell type-specific than the canonical TTS. TE-TTS were much more likely to be found in the sense orientation than other intragenic TE sequences of the same TE family and TE-TTS in the sense orientation terminate transcription more efficiently than those found in the antisense orientation. Alu sequences were found to provide a large number of relatively weak TTS, whereas LTR elements provided a smaller number of much stronger TTS. Conclusions TE sequences provide numerous termination sites to human genes, and TE-derived TTS are particularly cell type-specific. Thus, TE sequences provide a powerful mechanism for the diversification of transcriptional profiles between cell types and among evolutionary lineages, since most TE-TTS are

Layer- and Cell Type-Specific Modulation of Excitatory Neuronal Activity in the Neocortex

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Gabriele Radnikow

2018-01-01

Full Text Available From an anatomical point of view the neocortex is subdivided into up to six layers depending on the cortical area. This subdivision has been described already by Meynert and Brodmann in the late 19/early 20. century and is mainly based on cytoarchitectonic features such as the size and location of the pyramidal cell bodies. Hence, cortical lamination is originally an anatomical concept based on the distribution of excitatory neuron. However, it has become apparent in recent years that apart from the layer-specific differences in morphological features, many functional properties of neurons are also dependent on cortical layer or cell type. Such functional differences include changes in neuronal excitability and synaptic activity by neuromodulatory transmitters. Many of these neuromodulators are released from axonal afferents from subcortical brain regions while others are released intrinsically. In this review we aim to describe layer- and cell-type specific differences in the effects of neuromodulator receptors in excitatory neurons in layers 2–6 of different cortical areas. We will focus on the neuromodulator systems using adenosine, acetylcholine, dopamine, and orexin/hypocretin as examples because these neuromodulator systems show important differences in receptor type and distribution, mode of release and functional mechanisms and effects. We try to summarize how layer- and cell type-specific neuromodulation may affect synaptic signaling in cortical microcircuits.

Genetic susceptibility to type 2 diabetes and obesity

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Grarup, Niels; Sandholt, Camilla H; Hansen, Torben

2014-01-01

During the past 7 years, genome-wide association studies have shed light on the contribution of common genomic variants to the genetic architecture of type 2 diabetes, obesity and related intermediate phenotypes. The discoveries have firmly established more than 175 genomic loci associated...... with these phenotypes. Despite the tight correlation between type 2 diabetes and obesity, these conditions do not appear to share a common genetic background, since they have few genetic risk loci in common. The recent genetic discoveries do however highlight specific details of the interplay between the pathogenesis...... progress as regards the concepts, methodologies and derived outcomes of studies of the genetics of type 2 diabetes and obesity, and discuss avenues to be investigated in the future within this research field....

The Genetic Deletion of 6q21 and PRDM1 and Clinical Implications in Extranodal NK/T Cell Lymphoma, Nasal Type

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Li Liang

2015-01-01

Full Text Available 6q21 genetic deletion has been frequently detected in extranodal NK/T cell lymphoma, nasal type (EN-NK/T-NT, and PRDM1 is considered as candidate gene. However, direct detection of PRDM1 deletion has not been well documented. We investigated genetic alterations of 6q21 and PRDM1 in 43 cases of EN-NK/T-NT and cell lines by FISH. PRDM1 expression was evaluated by immunohistochemistry and Western blot. The correlation between genetic alteration and PRDM1 expression and the significance in clinic-pathologic were analyzed. Heterozygous deletion of 6q21 and/or PRDM1 was observed in 24 of 43 cases (55.81% of EN-NK/T-NT including 16 cases (37.21% for 6q21 deletion and 19 cases (44.19% for PRDM1 deletion. Similarly, heterozygous codeletion of 6q21 and PRDM1 was identified in NK92 and NKL cells. The heterozygous deletion of 6q21 and/or PRDM1 was correlated with PRDM1 expression. However, genetic deletion of 6q21 and/or PRDM1 was not correlated with clinicopathological features of EN-NK/T-NT, while PRDM1 expression showed positive effect on the outcome of patients as those as disease site, B symptom, and clinical stage. Thus, heterozygous deletion of 6q21 and/or PRDM1 was frequently detected in EN-NK/T-NT and correlated with downregulation of PRDM1. But the prognostic role of genetic deletion needs to be further evaluated in larger cohort.

Novel Genes Involved in Controlling Specification of Drosophila FMRFamide Neuropeptide Cells.

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Bivik, Caroline; Bahrampour, Shahrzad; Ulvklo, Carina; Nilsson, Patrik; Angel, Anna; Fransson, Fredrik; Lundin, Erika; Renhorn, Jakob; Thor, Stefan

2015-08-01

The expression of neuropeptides is often extremely restricted in the nervous system, making them powerful markers for addressing cell specification . In the developing Drosophila ventral nerve cord, only six cells, the Ap4 neurons, of some 10,000 neurons, express the neuropeptide FMRFamide (FMRFa). Each Ap4/FMRFa neuron is the last-born cell generated by an identifiable and well-studied progenitor cell, neuroblast 5-6 (NB5-6T). The restricted expression of FMRFa and the wealth of information regarding its gene regulation and Ap4 neuron specification makes FMRFa a valuable readout for addressing many aspects of neural development, i.e., spatial and temporal patterning cues, cell cycle control, cell specification, axon transport, and retrograde signaling. To this end, we have conducted a forward genetic screen utilizing an Ap4-specific FMRFa-eGFP transgenic reporter as our readout. A total of 9781 EMS-mutated chromosomes were screened for perturbations in FMRFa-eGFP expression, and 611 mutants were identified. Seventy-nine of the strongest mutants were mapped down to the affected gene by deficiency mapping or whole-genome sequencing. We isolated novel alleles for previously known FMRFa regulators, confirming the validity of the screen. In addition, we identified novel essential genes, including several with previously undefined functions in neural development. Our identification of genes affecting most major steps required for successful terminal differentiation of Ap4 neurons provides a comprehensive view of the genetic flow controlling the generation of highly unique neuronal cell types in the developing nervous system. Copyright © 2015 by the Genetics Society of America.

Testicular germ cell tumors: Molecular genetic and clinicomorphological aspects

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M. V. Nemtsova

2015-03-01

Full Text Available Testicular tumors are the most common form of solid cancer in young men. According to the 2004 WHO classification, testicular germ cell tumors (TGCT may present with different histological types. Embryonic cells of varying grade may be a source of TGCT and the occurrence of this type of tumors is directly related to the formation of a pool of male sex cells and gametogenesis. The paper gives information on mo- lecular stages for the process of formation of male sex cells in health, as well as ways of their impairments leading to TGCT. An investigation of the profiles of gene expression and the spectrum of molecular damages revealed genes responsible for a predisposition to the sporadic and hereditary forms of TGCT. The paper presents the current molecular genetic and clinicomorphological characteristics of TGCT. 

In vivo tracking of genetically engineered, anti-HER2/neu directed natural killer cells to HER2/neu positive mammary tumors with magnetic resonance imaging

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Daldrup-Link, Heike E.; Meier, Reinhardt; Metz, Stephan; Settles, Marcus; Rummeny, Ernst J.; Rudelius, Martina; Piontek, Guido; Schlegel, Juergen; Piert, Morand; Uherek, Christoph; Wels, Winfried

2005-01-01

The purpose of this study is to optimize labeling of the human natural killer (NK) cell line NK-92 with iron-oxide-based contrast agents and to monitor the in vivo distribution of genetically engineered NK-92 cells, which are directed against HER2/neu receptors, to HER2/neu positive mammary tumors with magnetic resonance (MR) imaging. Parental NK-92 cells and genetically modified HER2/neu specific NK-92-scFv(FRP5)-zeta cells, expressing a chimeric antigen receptor specific to the tumor-associated ErbB2 (HER2/neu) antigen, were labeled with ferumoxides and ferucarbotran using simple incubation, lipofection and electroporation techniques. Labeling efficiency was evaluated by MR imaging, Prussian blue stains and spectrometry. Subsequently, ferucarbotran-labeled NK-92-scFv(FRP5)-zeta (n=3) or parental NK-92 cells were intravenously injected into the tail vein of six mice with HER2/neu-positive NIH 3T3 mammary tumors, implanted in the mammary fat pad. The accumulation of the cells in the tumors was monitored by MR imaging before and 12 and 24 h after cell injection (p.i.). MR data were correlated with histopathology. Both the parental NK-92 and the genetically modified NK-92-scFv(FRP5)-zeta cells could be labeled with ferucarbotran and ferumoxides by lipofection and electroporation, but not by simple incubation. The intracellular cytoplasmatic iron-oxide uptake was significantly higher after labeling with ferucarbotran than ferumoxides (P 6 NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, MR showed a progressive signal decline in HER2/neu-positive mammary tumors at 12 and 24 h (p.i.). Conversely, injection of 5 x 10 6 parental NK-92 control cells, not directed against HER2/neu receptors, did not cause significant signal intensity changes of the tumors. Histopathology confirmed an accumulation of the former, but not the latter cells in tumor tissue. The human natural killer cell line NK-92 can be efficiently labeled with clinically applicable iron-oxide contrast

In vivo tracking of genetically engineered, anti-HER2/neu directed natural killer cells to HER2/neu positive mammary tumors with magnetic resonance imaging

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Daldrup-Link, Heike E. [UCSF Medical Center, Department of Radiology, San Francisco, CA (United States); Meier, Reinhardt; Metz, Stephan; Settles, Marcus; Rummeny, Ernst J. [Technical University Munich, Department of Radiology, Munich (Germany); Rudelius, Martina; Piontek, Guido; Schlegel, Juergen [Technical University Munich, Institute of Pathology, Division of Neuropathology, Munich (Germany); Piert, Morand [Technical University Munich, Department of Nuclear Medicine, Munich (Germany); Uherek, Christoph; Wels, Winfried [University of Frankfurt, Georg Speyer House, Frankfurt (Germany)

2005-01-01

The purpose of this study is to optimize labeling of the human natural killer (NK) cell line NK-92 with iron-oxide-based contrast agents and to monitor the in vivo distribution of genetically engineered NK-92 cells, which are directed against HER2/neu receptors, to HER2/neu positive mammary tumors with magnetic resonance (MR) imaging. Parental NK-92 cells and genetically modified HER2/neu specific NK-92-scFv(FRP5)-zeta cells, expressing a chimeric antigen receptor specific to the tumor-associated ErbB2 (HER2/neu) antigen, were labeled with ferumoxides and ferucarbotran using simple incubation, lipofection and electroporation techniques. Labeling efficiency was evaluated by MR imaging, Prussian blue stains and spectrometry. Subsequently, ferucarbotran-labeled NK-92-scFv(FRP5)-zeta (n=3) or parental NK-92 cells were intravenously injected into the tail vein of six mice with HER2/neu-positive NIH 3T3 mammary tumors, implanted in the mammary fat pad. The accumulation of the cells in the tumors was monitored by MR imaging before and 12 and 24 h after cell injection (p.i.). MR data were correlated with histopathology. Both the parental NK-92 and the genetically modified NK-92-scFv(FRP5)-zeta cells could be labeled with ferucarbotran and ferumoxides by lipofection and electroporation, but not by simple incubation. The intracellular cytoplasmatic iron-oxide uptake was significantly higher after labeling with ferucarbotran than ferumoxides (P<0.05). After intravenous injection of 5 x 10{sup 6} NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, MR showed a progressive signal decline in HER2/neu-positive mammary tumors at 12 and 24 h (p.i.). Conversely, injection of 5 x 10{sup 6} parental NK-92 control cells, not directed against HER2/neu receptors, did not cause significant signal intensity changes of the tumors. Histopathology confirmed an accumulation of the former, but not the latter cells in tumor tissue. The human natural killer cell line NK-92 can be efficiently

Direct visualization of antigen-specific T cells: HTLV-1 Tax11-19- specific CD8(+) T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients.

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Greten, T F; Slansky, J E; Kubota, R; Soldan, S S; Jaffee, E M; Leist, T P; Pardoll, D M; Jacobson, S; Schneck, J P

1998-06-23

Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11-19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2-Ig complex to directly visualize HTLV-1 Tax11-19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8(+) lymphocytes specific for the HTLV-1 Tax11-19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11-19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11-19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11-19-specific CD8(+) T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-alpha and gamma-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11-19-specific CD8(+) T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.

Cell-Type Specific Determinants of NRAMP1 Expression in Professional Phagocytes

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Mathieu F. M. Cellier

2013-01-01

Full Text Available The Natural resistance-associated macrophage protein 1 (Nramp1 or Solute carrier 11 member 1, Slc11a1 transports divalent metals across the membrane of late endosomes and lysosomes in professional phagocytes. Nramp1 represents an ancient eukaryotic cell-autonomous defense whereas the gene duplication that yielded Nramp1 and Nramp2 predated the origin of Sarcopterygians (lobe-finned fishes and tetrapods. SLC11A1 genetic polymorphisms associated with human resistance to tuberculosis consist of potential regulatory variants. Herein, current knowledge of the regulation of SLC11A1 gene expression is reviewed and comprehensive analysis of ENCODE data available for hematopoietic cell-types suggests a hypothesis for the regulation of SLC11A1 expression during myeloid development and phagocyte functional polarization. SLC11A1 is part of a 34.6 kb CTCF-insulated locus scattered with predicted regulatory elements: a 3' enhancer, a large 5' enhancer domain and four elements spread around the transcription start site (TSS, including several C/EBP and PU.1 sites. SLC11A1 locus ends appear mobilized by ETS-related factors early during myelopoiesis; activation of both 5' and 3' enhancers in myelo-monocytic cells correlate with transcription factor binding at the TSS. Characterizing the corresponding cis/trans determinants functionally will establish the mechanisms involved and possibly reveal genetic variation that impacts susceptibility to infectious or immune diseases.

Exploring the genetics and non-cell autonomous mechanisms underlying ALS/FTLD.

Science.gov (United States)

Chen, Hongbo; Kankel, Mark W; Su, Susan C; Han, Steve W S; Ofengeim, Dimitry

2018-03-01

Although amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, was first described in 1874, a flurry of genetic discoveries in the last 10 years has markedly increased our understanding of this disease. These findings have not only enhanced our knowledge of mechanisms leading to ALS, but also have revealed that ALS shares many genetic causes with another neurodegenerative disease, frontotemporal lobar dementia (FTLD). In this review, we survey how recent genetic studies have bridged our mechanistic understanding of these two related diseases and how the genetics behind ALS and FTLD point to complex disorders, implicating non-neuronal cell types in disease pathophysiology. The involvement of non-neuronal cell types is consistent with a non-cell autonomous component in these diseases. This is further supported by studies that identified a critical role of immune-associated genes within ALS/FTLD and other neurodegenerative disorders. The molecular functions of these genes support an emerging concept that various non-autonomous functions are involved in neurodegeneration. Further insights into such a mechanism(s) will ultimately lead to a better understanding of potential routes of therapeutic intervention. Facts ALS and FTLD are severe neurodegenerative disorders on the same disease spectrum. Multiple cellular processes including dysregulation of RNA homeostasis, imbalance of proteostasis, contribute to ALS/FTLD pathogenesis. Aberrant function in non-neuronal cell types, including microglia, contributes to ALS/FTLD. Strong neuroimmune and neuroinflammatory components are associated with ALS/FTLD patients. Open Questions Why can patients with similar mutations have different disease manifestations, i.e., why do C9ORF72 mutations lead to motor neuron loss in some patients while others exhibit loss of neurons in the frontotemporal lobe? Do ALS causal mutations result in microglial dysfunction and contribute to ALS/FTLD pathology? How do microglia

Common themes and cell type specific variations of higher order chromatin arrangements in the mouse

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Cremer Thomas

2005-12-01

Full Text Available Abstract Background Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals. Results All mouse cell types showed non-random correlations of radial chromosome territory positions with gene density as well as with chromosome size. The distribution of chromosome territories and pericentromeric heterochromatin changed during differentiation, leading to distinct cell type specific distribution patterns. We exclude a strict dependence of these differences on nuclear shape. Positional differences in mouse cell nuclei were less pronounced compared to human cell nuclei in agreement with smaller differences in chromosome size and gene density. Notably, the position of chromosome territories relative to each other was very variable. Conclusion Chromosome territory arrangements according to chromosome size and gene density provide common, evolutionary conserved themes in both, human and mouse cell types. Our findings are incompatible with a previously reported model of parental genome separation.

Simplification of genotyping techniques of the ABO blood type experiment and exploration of population genetics.

Science.gov (United States)

Hu, Jian; Zhou, Yi-ren; Ding, Jia-lin; Wang, Zhi-yuan; Liu, Ling; Wang, Ye-kai; Lou, Hui-ling; Qiao, Shou-yi; Wu, Yan-hua

2017-05-20

The ABO blood type is one of the most common and widely used genetic traits in humans. Three glycosyltransferase-encoding gene alleles, I A , I B and i, produce three red blood cell surface antigens, by which the ABO blood type is classified. By using the ABO blood type experiment as an ideal case for genetics teaching, we can easily introduce to the students several genetic concepts, including multiple alleles, gene interaction, single nucleotide polymorphism (SNP) and gene evolution. Herein we have innovated and integrated our ABO blood type genetics experiments. First, in the section of Molecular Genetics, a new method of ABO blood genotyping was established: specific primers based on SNP sites were designed to distinguish three alleles through quantitative real-time PCR. Next, the experimental teaching method of Gene Evolution was innovated in the Population Genetics section: a gene-evolution software was developed to simulate the evolutionary tendency of the ABO genotype encoding alleles under diverse conditions. Our reform aims to extend the contents of genetics experiments, to provide additional teaching approaches, and to improve the learning efficiency of our students eventually.

Optimizing Staining Protocols for Laser Microdissection of Specific Cell Types from the Testis Including Carcinoma In Situ

DEFF Research Database (Denmark)

Sonne, Si Brask; Dalgaard, Marlene D; Nielsen, John Erik

2009-01-01

Microarray and RT-PCR based methods are important tools for analysis of gene expression; however, in tissues containing many different cells types, such as the testis, characterization of gene expression in specific cell types can be severely hampered by noise from other cells. The laser microdis......Microarray and RT-PCR based methods are important tools for analysis of gene expression; however, in tissues containing many different cells types, such as the testis, characterization of gene expression in specific cell types can be severely hampered by noise from other cells. The laser...... protocols, and present two staining protocols for frozen sections, one for fast and specific staining of fetal germ cells, testicular carcinoma in situ cells, and other cells with embryonic stem cell-like properties that express the alkaline phosphatase, and one for specific staining of lipid droplet...

Re-analysis of public genetic data reveals a rare X-chromosomal variant associated with type 2 diabetes

DEFF Research Database (Denmark)

Bonàs-Guarch, Sílvia; Guindo-Martínez, Marta; Miguel-Escalada, Irene

2018-01-01

The reanalysis of existing GWAS data represents a powerful and cost-effective opportunity to gain insights into the genetics of complex diseases. By reanalyzing publicly available type 2 diabetes (T2D) genome-wide association studies (GWAS) data for 70,127 subjects, we identify seven novel...... available data using novel genetic resources and analytical approaches....... associated with the expression of Angiotensin II Receptor type 2 gene (AGTR2), a modulator of insulin sensitivity, and exhibits allelic specific activity in muscle cells. Beyond providing insights into the genetics and pathophysiology of T2D, these results also underscore the value of reanalyzing publicly...

Enteroendocrine cell types revisited

DEFF Research Database (Denmark)

Engelstoft, Maja S; Egerod, Kristoffer Lihme; Lund, Mari L

2013-01-01

The GI-tract is profoundly involved in the control of metabolism through peptide hormones secreted from enteroendocrine cells scattered throughout the gut mucosa. A large number of recently generated transgenic reporter mice have allowed for direct characterization of biochemical and cell...... biological properties of these previously highly elusive enteroendocrine cells. In particular the surprisingly broad co-expression of six functionally related hormones in the intestinal enteroendocrine cells indicates that it should be possible to control not only the hormone secretion but also the type...... and number of enteroendocrine cells. However, this will require a more deep understanding of the factors controlling differentiation, gene expression and specification of the enteroendocrine cells during their weekly renewal from progenitor cells in the crypts of the mucosa....

Cell-type-specific expression of NFIX in the developing and adult cerebellum.

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Fraser, James; Essebier, Alexandra; Gronostajski, Richard M; Boden, Mikael; Wainwright, Brandon J; Harvey, Tracey J; Piper, Michael

2017-07-01

Transcription factors from the nuclear factor one (NFI) family have been shown to play a central role in regulating neural progenitor cell differentiation within the embryonic and post-natal brain. NFIA and NFIB, for instance, promote the differentiation and functional maturation of granule neurons within the cerebellum. Mice lacking Nfix exhibit delays in the development of neuronal and glial lineages within the cerebellum, but the cell-type-specific expression of this transcription factor remains undefined. Here, we examined the expression of NFIX, together with various cell-type-specific markers, within the developing and adult cerebellum using both chromogenic immunohistochemistry and co-immunofluorescence labelling and confocal microscopy. In embryos, NFIX was expressed by progenitor cells within the rhombic lip and ventricular zone. After birth, progenitor cells within the external granule layer, as well as migrating and mature granule neurons, expressed NFIX. Within the adult cerebellum, NFIX displayed a broad expression profile, and was evident within granule cells, Bergmann glia, and interneurons, but not within Purkinje neurons. Furthermore, transcriptomic profiling of cerebellar granule neuron progenitor cells showed that multiple splice variants of Nfix are expressed within this germinal zone of the post-natal brain. Collectively, these data suggest that NFIX plays a role in regulating progenitor cell biology within the embryonic and post-natal cerebellum, as well as an ongoing role within multiple neuronal and glial populations within the adult cerebellum.

Genetic algorithms and supernovae type Ia analysis

International Nuclear Information System (INIS)

Bogdanos, Charalampos; Nesseris, Savvas

2009-01-01

We introduce genetic algorithms as a means to analyze supernovae type Ia data and extract model-independent constraints on the evolution of the Dark Energy equation of state w(z) ≡ P DE /ρ DE . Specifically, we will give a brief introduction to the genetic algorithms along with some simple examples to illustrate their advantages and finally we will apply them to the supernovae type Ia data. We find that genetic algorithms can lead to results in line with already established parametric and non-parametric reconstruction methods and could be used as a complementary way of treating SNIa data. As a non-parametric method, genetic algorithms provide a model-independent way to analyze data and can minimize bias due to premature choice of a dark energy model

Cytotoxic T-lymphocyte clones, established by stimulation with the HLA-A2 binding p5365-73 wild type peptide loaded on dendritic cells In vitro, specifically recognize and lyse HLA-A2 tumour cells overexpressing the p53 protein

DEFF Research Database (Denmark)

Barfoed, Annette Malene; Petersen, T R; Kirkin, A F

2000-01-01

of recognizing p53 derived wild type (self) peptides. Furthermore, the capacity of R9V specific T cell clones to exert HLA restricted cytotoxicity, argues that the R9V peptide is naturally presented on certain cancer cells. This supports the view that p53 derived wild type peptides might serve as candidate......Mutations in the tumour suppressor gene p53 are among the most frequent genetic alterations in human malignancies, often associated with an accumulation of the p53 protein in the cytoplasm. We have generated a number of cytotoxic T lymphocyte (CTL) clones that specifically recognize the HLA-A*0201...

Genetic effects of combined chemical-X-ray treatments in male mouse germ cells

International Nuclear Information System (INIS)

Cattanach, B.M.; Rasberry, C.

1987-01-01

Several studies have shown that the yield of genetic damage induced by radiation in male mouse germ cells can be modified by chemical treatments. Pre-treatments with radio-protecting agents have given contradictory results but this appears to be largely attributable to the different germ cell stages tested and dependent upon the level of radiation damage induced. Pre-treatments which enhance the yield of genetic damage have been reported although, as yet, no tests have been conducted with radio-sensitizers. Another form of interaction between chemicals and radiation is specifically found with spermatogonial stem cells. Chemicals that kill cells can, by population depletion, substantially and predictably modify the genetic response to subsequent radiation exposure over a period of several days, or even weeks. Enhancement and reduction in the genetic yield can be attained, dependent upon the interval between treatments, with the modification also varying with the type of genetic damage scored. Post-treatment with one chemical (TEM) has been shown to reduce the genetic response to radiation exposure. (author)

Cell type specific DNA methylation in cord blood: A 450K-reference data set and cell count-based validation of estimated cell type composition

NARCIS (Netherlands)

Gervin, K. (Kristina); Page, C.M. (Christian Magnus); H.C.D. Aass (Hans Christian Dalsbotten); M.A.E. Jansen (Michelle); Fjeldstad, H.E. (Heidi Elisabeth); B.K. Andreassen (Bettina Kulle); L. Duijts (Liesbeth); J.B.J. van Meurs (Joyce); M.C. van Zelm (Menno); V.W.V. Jaddoe (Vincent); Nordeng, H. (Hedvig); Knudsen, G.P. (Gunn Peggy); P. Magnus (Per); W. Nystad (Wenche); Staff, A.C. (Anne Cathrine); J.F. Felix (Janine); R. Lyle (Robert)

2016-01-01

textabstractEpigenome-wide association studies of prenatal exposure to different environmental factors are becoming increasingly common. These studies are usually performed in umbilical cord blood. Since blood comprises multiple cell types with specific DNA methylation patterns, confounding caused

Alveolar epithelial type II cells induce T cell tolerance to specific antigen

DEFF Research Database (Denmark)

Lo, Bernice; Hansen, Søren; Evans, Kathy

2008-01-01

The lungs face the immunologic challenge of rapidly eliminating inhaled pathogens while maintaining tolerance to innocuous Ags. A break in this immune homeostasis may result in pulmonary inflammatory diseases, such as allergies or asthma. The observation that alveolar epithelial type II cells (Type...... II) constitutively express the class II MHC led us to hypothesize that Type II cells play a role in the adaptive immune response. Because Type II cells do not express detectable levels of the costimulatory molecules CD80 and CD86, we propose that Type II cells suppress activation of naive T cells...

Comprehensive meta-analysis of Signal Transducers and Activators of Transcription (STAT genomic binding patterns discerns cell-specific cis-regulatory modules

Directory of Open Access Journals (Sweden)

Kang Keunsoo

2013-01-01

Full Text Available Abstract Background Cytokine-activated transcription factors from the STAT (Signal Transducers and Activators of Transcription family control common and context-specific genetic programs. It is not clear to what extent cell-specific features determine the binding capacity of seven STAT members and to what degree they share genetic targets. Molecular insight into the biology of STATs was gained from a meta-analysis of 29 available ChIP-seq data sets covering genome-wide occupancy of STATs 1, 3, 4, 5A, 5B and 6 in several cell types. Results We determined that the genomic binding capacity of STATs is primarily defined by the cell type and to a lesser extent by individual family members. For example, the overlap of shared binding sites between STATs 3 and 5 in T cells is greater than that between STAT5 in T cells and non-T cells. Even for the top 1,000 highly enriched STAT binding sites, ~15% of STAT5 binding sites in mouse female liver are shared by other STATs in different cell types while in T cells ~90% of STAT5 binding sites are co-occupied by STAT3, STAT4 and STAT6. In addition, we identified 116 cis-regulatory modules (CRM, which are recognized by all STAT members across cell types defining a common JAK-STAT signature. Lastly, in liver STAT5 binding significantly coincides with binding of the cell-specific transcription factors HNF4A, FOXA1 and FOXA2 and is associated with cell-type specific gene transcription. Conclusions Our results suggest that genomic binding of STATs is primarily determined by the cell type and further specificity is achieved in part by juxtaposed binding of cell-specific transcription factors.

Genetic relationships among linear type traits, milk yield, body weight, fertility and somatic cell count in primiparous dairy cows

NARCIS (Netherlands)

Berry, D.P.; Buckley, F.; Dillon, P.P.; Evans, R.D.; Veerkamp, R.F.

2004-01-01

Phenotypic and genetic (co)variances among type traits, milk yield, body weight, fertility and somatic cell count were estimated. The data analysed included 3,058 primiparous spring-calving Holstein-Friesian cows from 80 farms throughout the south of Ireland. Heritability estimates for the type

Mammalian transcriptional hotspots are enriched for tissue specific enhancers near cell type specific highly expressed genes and are predicted to act as transcriptional activator hubs.

Science.gov (United States)

Joshi, Anagha

2014-12-30

Transcriptional hotspots are defined as genomic regions bound by multiple factors. They have been identified recently as cell type specific enhancers regulating developmentally essential genes in many species such as worm, fly and humans. The in-depth analysis of hotspots across multiple cell types in same species still remains to be explored and can bring new biological insights. We therefore collected 108 transcription-related factor (TF) ChIP sequencing data sets in ten murine cell types and classified the peaks in each cell type in three groups according to binding occupancy as singletons (low-occupancy), combinatorials (mid-occupancy) and hotspots (high-occupancy). The peaks in the three groups clustered largely according to the occupancy, suggesting priming of genomic loci for mid occupancy irrespective of cell type. We then characterized hotspots for diverse structural functional properties. The genes neighbouring hotspots had a small overlap with hotspot genes in other cell types and were highly enriched for cell type specific function. Hotspots were enriched for sequence motifs of key TFs in that cell type and more than 90% of hotspots were occupied by pioneering factors. Though we did not find any sequence signature in the three groups, the H3K4me1 binding profile had bimodal peaks at hotspots, distinguishing hotspots from mono-modal H3K4me1 singletons. In ES cells, differentially expressed genes after perturbation of activators were enriched for hotspot genes suggesting hotspots primarily act as transcriptional activator hubs. Finally, we proposed that ES hotspots might be under control of SetDB1 and not DNMT for silencing. Transcriptional hotspots are enriched for tissue specific enhancers near cell type specific highly expressed genes. In ES cells, they are predicted to act as transcriptional activator hubs and might be under SetDB1 control for silencing.

Genetic Modifiers of Sickle Cell Disease

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Steinberg, Martin H.; Sebastiani, Paola

2015-01-01

Sickle cell anemia is associated with unusual clinical heterogeneity for a Mendelian disorder. Fetal hemoglobin concentration and coincident ∝ thalassemia, both which directly affect the sickle erythrocyte, are the major modulators of the phenotype of disease. Understanding the genetics underlying the heritable subphenotypes of sickle cell anemia would be prognostically useful, could inform personalized therapeutics, and might help the discovery of new “druggable” pathophysiologic targets. Genotype-phenotype association studies have been used to identify novel genetic modifiers. In the future, whole genome sequencing with its promise of discovering hitherto unsuspected variants could add to our understanding of the genetic modifiers of this disease. PMID:22641398

Global Transcriptome Analysis of Primary Cerebrocortical Cells: Identification of Genes Regulated by Triiodothyronine in Specific Cell Types.

Science.gov (United States)

Gil-Ibañez, Pilar; García-García, Francisco; Dopazo, Joaquín; Bernal, Juan; Morte, Beatriz

2017-01-01

Thyroid hormones, thyroxine, and triiodothyronine (T3) are crucial for cerebral cortex development acting through regulation of gene expression. To define the transcriptional program under T3 regulation, we have performed RNA-Seq of T3-treated and untreated primary mouse cerebrocortical cells. The expression of 1145 genes or 7.7% of expressed genes was changed upon T3 addition, of which 371 responded to T3 in the presence of cycloheximide indicating direct transcriptional regulation. The results were compared with available transcriptomic datasets of defined cellular types. In this way, we could identify targets of T3 within genes enriched in astrocytes and neurons, in specific layers including the subplate, and in specific neurons such as prepronociceptin, cholecystokinin, or cortistatin neurons. The subplate and the prepronociceptin neurons appear as potentially major targets of T3 action. T3 upregulates mostly genes related to cell membrane events, such as G-protein signaling, neurotransmission, and ion transport and downregulates genes involved in nuclear events associated with the M phase of cell cycle, such as chromosome organization and segregation. Remarkably, the transcriptomic changes induced by T3 sustain the transition from fetal to adult patterns of gene expression. The results allow defining in molecular terms the elusive role of thyroid hormones on neocortical development. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cell type-specific suppression of mechanosensitive genes by audible sound stimulation.

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Kumeta, Masahiro; Takahashi, Daiji; Takeyasu, Kunio; Yoshimura, Shige H

2018-01-01

Audible sound is a ubiquitous environmental factor in nature that transmits oscillatory compressional pressure through the substances. To investigate the property of the sound as a mechanical stimulus for cells, an experimental system was set up using 94.0 dB sound which transmits approximately 10 mPa pressure to the cultured cells. Based on research on mechanotransduction and ultrasound effects on cells, gene responses to the audible sound stimulation were analyzed by varying several sound parameters: frequency, wave form, composition, and exposure time. Real-time quantitative PCR analyses revealed a distinct suppressive effect for several mechanosensitive and ultrasound-sensitive genes that were triggered by sounds. The effect was clearly observed in a wave form- and pressure level-specific manner, rather than the frequency, and persisted for several hours. At least two mechanisms are likely to be involved in this sound response: transcriptional control and RNA degradation. ST2 stromal cells and C2C12 myoblasts exhibited a robust response, whereas NIH3T3 cells were partially and NB2a neuroblastoma cells were completely insensitive, suggesting a cell type-specific response to sound. These findings reveal a cell-level systematic response to audible sound and uncover novel relationships between life and sound.

Cell-type specificity of ChIP-predicted transcription factor binding sites

Directory of Open Access Journals (Sweden)

Håndstad Tony

2012-08-01

Full Text Available Abstract Background Context-dependent transcription factor (TF binding is one reason for differences in gene expression patterns between different cellular states. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq identifies genome-wide TF binding sites for one particular context—the cells used in the experiment. But can such ChIP-seq data predict TF binding in other cellular contexts and is it possible to distinguish context-dependent from ubiquitous TF binding? Results We compared ChIP-seq data on TF binding for multiple TFs in two different cell types and found that on average only a third of ChIP-seq peak regions are common to both cell types. Expectedly, common peaks occur more frequently in certain genomic contexts, such as CpG-rich promoters, whereas chromatin differences characterize cell-type specific TF binding. We also find, however, that genotype differences between the cell types can explain differences in binding. Moreover, ChIP-seq signal intensity and peak clustering are the strongest predictors of common peaks. Compared with strong peaks located in regions containing peaks for multiple transcription factors, weak and isolated peaks are less common between the cell types and are less associated with data that indicate regulatory activity. Conclusions Together, the results suggest that experimental noise is prevalent among weak peaks, whereas strong and clustered peaks represent high-confidence binding events that often occur in other cellular contexts. Nevertheless, 30-40% of the strongest and most clustered peaks show context-dependent regulation. We show that by combining signal intensity with additional data—ranging from context independent information such as binding site conservation and position weight matrix scores to context dependent chromatin structure—we can predict whether a ChIP-seq peak is likely to be present in other cellular contexts.

Cell-type specific roles for PTEN in establishing a functional retinal architecture.

Directory of Open Access Journals (Sweden)

Robert Cantrup

Full Text Available The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture.In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam.We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and

WONOEP appraisal: new genetic approaches to study epilepsy

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Rossignol, Elsa; Kobow, Katja; Simonato, Michele; Loeb, Jeffrey A.; Grisar, Thierry; Gilby, Krista L.; Vinet, Jonathan; Kadam, Shilpa D.; Becker, Albert J.

2014-01-01

Objective New genetic investigation techniques, including next-generation sequencing, epigenetic profiling, cell lineage mapping, targeted genetic manipulation of specific neuronal cell types, stem cell reprogramming and optogenetic manipulations within epileptic networks are progressively unravelling the mysteries of epileptogenesis and ictogenesis. These techniques have opened new avenues to discover the molecular basis of epileptogenesis and to study the physiological impacts of mutations in epilepsy-associated genes on a multilayer level, from cells to circuits. Methods This manuscript reviews recently published applications of these new genetic technologies in the study of epilepsy, as well as work presented by the authors at the genetic session of the XII Workshop on the Neurobiology of Epilepsy in Quebec, Canada. Results Next-generation sequencing is providing investigators with an unbiased means to assess the molecular causes of sporadic forms of epilepsy and have revealed the complexity and genetic heterogeneity of sporadic epilepsy disorders. To assess the functional impact of mutations in these newly identified genes on specific neuronal cell-types during brain development, new modeling strategies in animals, including conditional genetics in mice and in utero knockdown approaches, are enabling functional validation with exquisite cell-type and temporal specificity. In addition, optogenetics, using cell-type specific Cre recombinase driver lines, is enabling investigators to dissect networks involved in epilepsy. Genetically-encoded cell-type labeling is also providing new means to assess the role of the non-neuronal components of epileptic networks such as glial cells. Furthermore, beyond its role in revealing coding variants involved in epileptogenesis, next-generation sequencing can be used to assess the epigenetic modifications that lead to sustained network hyperexcitability in epilepsy, including methylation changes in gene promoters and non

Engineering the human pluripotent stem cell microenvironment to direct cell fate.

Science.gov (United States)

Hazeltine, Laurie B; Selekman, Joshua A; Palecek, Sean P

2013-11-15

Human pluripotent stem cells (hPSCs), including both embryonic stem cells and induced pluripotent stem cells, offer a potential cell source for research, drug screening, and regenerative medicine applications due to their unique ability to self-renew or differentiate to any somatic cell type. Before the full potential of hPSCs can be realized, robust protocols must be developed to direct their fate. Cell fate decisions are based on components of the surrounding microenvironment, including soluble factors, substrate or extracellular matrix, cell-cell interactions, mechanical forces, and 2D or 3D architecture. Depending on their spatio-temporal context, these components can signal hPSCs to either self-renew or differentiate to cell types of the ectoderm, mesoderm, or endoderm. Researchers working at the interface of engineering and biology have identified various factors which can affect hPSC fate, often based on lessons from embryonic development, and they have utilized this information to design in vitro niches which can reproducibly direct hPSC fate. This review highlights culture systems that have been engineered to promote self-renewal or differentiation of hPSCs, with a focus on studies that have elucidated the contributions of specific microenvironmental cues in the context of those culture systems. We propose the use of microsystem technologies for high-throughput screening of spatial-temporal presentation of cues, as this has been demonstrated to be a powerful approach for differentiating hPSCs to desired cell types. Copyright © 2013 Elsevier Inc. All rights reserved.

Mapping Mammalian Cell-type-specific Transcriptional Regulatory Networks Using KD-CAGE and ChIP-seq Data in the TC-YIK Cell Line

Science.gov (United States)

Lizio, Marina; Ishizu, Yuri; Itoh, Masayoshi; Lassmann, Timo; Hasegawa, Akira; Kubosaki, Atsutaka; Severin, Jessica; Kawaji, Hideya; Nakamura, Yukio; Suzuki, Harukazu; Hayashizaki, Yoshihide; Carninci, Piero; Forrest, Alistair R. R.

2015-01-01

Mammals are composed of hundreds of different cell types with specialized functions. Each of these cellular phenotypes are controlled by different combinations of transcription factors. Using a human non islet cell insulinoma cell line (TC-YIK) which expresses insulin and the majority of known pancreatic beta cell specific genes as an example, we describe a general approach to identify key cell-type-specific transcription factors (TFs) and their direct and indirect targets. By ranking all human TFs by their level of enriched expression in TC-YIK relative to a broad collection of samples (FANTOM5), we confirmed known key regulators of pancreatic function and development. Systematic siRNA mediated perturbation of these TFs followed by qRT-PCR revealed their interconnections with NEUROD1 at the top of the regulation hierarchy and its depletion drastically reducing insulin levels. For 15 of the TF knock-downs (KD), we then used Cap Analysis of Gene Expression (CAGE) to identify thousands of their targets genome-wide (KD-CAGE). The data confirm NEUROD1 as a key positive regulator in the transcriptional regulatory network (TRN), and ISL1, and PROX1 as antagonists. As a complimentary approach we used ChIP-seq on four of these factors to identify NEUROD1, LMX1A, PAX6, and RFX6 binding sites in the human genome. Examining the overlap between genes perturbed in the KD-CAGE experiments and genes with a ChIP-seq peak within 50 kb of their promoter, we identified direct transcriptional targets of these TFs. Integration of KD-CAGE and ChIP-seq data shows that both NEUROD1 and LMX1A work as the main transcriptional activators. In the core TRN (i.e., TF-TF only), NEUROD1 directly transcriptionally activates the pancreatic TFs HSF4, INSM1, MLXIPL, MYT1, NKX6-3, ONECUT2, PAX4, PROX1, RFX6, ST18, DACH1, and SHOX2, while LMX1A directly transcriptionally activates DACH1, SHOX2, PAX6, and PDX1. Analysis of these complementary datasets suggests the need for caution in interpreting Ch

The female gametophyte: an emerging model for cell type-specific systems biology in plant development

Directory of Open Access Journals (Sweden)

Marc William Schmid

2015-11-01

Full Text Available Systems biology, a holistic approach describing a system emerging from the interactions of its molecular components, critically depends on accurate qualitative determination and quantitative measurements of these components. Development and improvement of large-scale profiling methods (omics now facilitates comprehensive measurements of many relevant molecules. For multicellular organisms, such as animals, fungi, algae, and plants, the complexity of the system is augmented by the presence of specialized cell types and organs, and a complex interplay within and between them. Cell type-specific analyses are therefore crucial for the understanding of developmental processes and environmental responses. This review first gives an overview of current methods used for large-scale profiling of specific cell types exemplified by recent advances in plant biology. The focus then lies on suitable model systems to study plant development and cell type specification. We introduce the female gametophyte of flowering plants as an ideal model to study fundamental developmental processes. Moreover, the female reproductive lineage is of importance for the emergence of evolutionary novelties such as an unequal parental contribution to the tissue nurturing the embryo or the clonal production of seeds by asexual reproduction (apomixis. Understanding these processes is not only interesting from a developmental or evolutionary perspective, but bears great potential for further crop improvement and the simplification of breeding efforts. We finally highlight novel methods, which are already available or which will likely soon facilitate large-scale profiling of the specific cell types of the female gametophyte in both model and non-model species. We conclude that it may take only few years until an evolutionary systems biology approach toward female gametogenesis may decipher some of its biologically most interesting and economically most valuable processes.

What Is Direct-to-Consumer Genetic Testing?

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... consumer genetic testing? What kinds of direct-to-consumer genetic tests are available? What is genetic ancestry testing? What are the benefits and risks of direct-to-consumer genetic testing? ...

Histochemical studies on genetical control of hormonal enzyme inducibility in the mouse. I. Non-specific esterase activity and regional histology of the epididymis

DEFF Research Database (Denmark)

Blecher, S R; Kirkeby, S

1978-01-01

As a base line for future cell genetical studies the authors record the distribution of non-specific esterase reaction in the various histologically distinguishable cell types of the mouse epididymis. The findings are correlated with previous descriptions of the lobar structure of the organ...

Non-genetic engineering of cells for drug delivery and cell-based therapy.

Science.gov (United States)

Wang, Qun; Cheng, Hao; Peng, Haisheng; Zhou, Hao; Li, Peter Y; Langer, Robert

2015-08-30

Cell-based therapy is a promising modality to address many unmet medical needs. In addition to genetic engineering, material-based, biochemical, and physical science-based approaches have emerged as novel approaches to modify cells. Non-genetic engineering of cells has been applied in delivering therapeutics to tissues, homing of cells to the bone marrow or inflammatory tissues, cancer imaging, immunotherapy, and remotely controlling cellular functions. This new strategy has unique advantages in disease therapy and is complementary to existing gene-based cell engineering approaches. A better understanding of cellular systems and different engineering methods will allow us to better exploit engineered cells in biomedicine. Here, we review non-genetic cell engineering techniques and applications of engineered cells, discuss the pros and cons of different methods, and provide our perspectives on future research directions. Copyright © 2014 Elsevier B.V. All rights reserved.

Differential 3’ processing of specific transcripts expands regulatory and protein diversity across neuronal cell types

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Jereb, Saša; Hwang, Hun-Way; Van Otterloo, Eric; Govek, Eve-Ellen; Fak, John J; Yuan, Yuan; Hatten, Mary E

2018-01-01

Alternative polyadenylation (APA) regulates mRNA translation, stability, and protein localization. However, it is unclear to what extent APA regulates these processes uniquely in specific cell types. Using a new technique, cTag-PAPERCLIP, we discovered significant differences in APA between the principal types of mouse cerebellar neurons, the Purkinje and granule cells, as well as between proliferating and differentiated granule cells. Transcripts that differed in APA in these comparisons were enriched in key neuronal functions and many differed in coding sequence in addition to 3’UTR length. We characterize Memo1, a transcript that shifted from expressing a short 3’UTR isoform to a longer one during granule cell differentiation. We show that Memo1 regulates granule cell precursor proliferation and that its long 3’UTR isoform is targeted by miR-124, contributing to its downregulation during development. Our findings provide insight into roles for APA in specific cell types and establish a platform for further functional studies. PMID:29578408

Spermatogenic Cell-Specific Gene Mutation in Mice via CRISPR-Cas9.

Science.gov (United States)

Bai, Meizhu; Liang, Dan; Wang, Yinghua; Li, Qing; Wu, Yuxuan; Li, Jinsong

2016-05-20

Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals. However, conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process, restricting rapid study of the gene function in vivo. CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique, which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes. Here, we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step. We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells. We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene. Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally. Consistently, male progeny from female founders were infertile and females could transmit the transgenes to the next generation. Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Genetically modified T cells in cancer therapy: opportunities and challenges

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Michaela Sharpe

2015-04-01

Full Text Available Tumours use many strategies to evade the host immune response, including downregulation or weak immunogenicity of target antigens and creation of an immune-suppressive tumour environment. T cells play a key role in cell-mediated immunity and, recently, strategies to genetically modify T cells either through altering the specificity of the T cell receptor (TCR or through introducing antibody-like recognition in chimeric antigen receptors (CARs have made substantial advances. The potential of these approaches has been demonstrated in particular by the successful use of genetically modified T cells to treat B cell haematological malignancies in clinical trials. This clinical success is reflected in the growing number of strategic partnerships in this area that have attracted a high level of investment and involve large pharmaceutical organisations. Although our understanding of the factors that influence the safety and efficacy of these therapies has increased, challenges for bringing genetically modified T-cell immunotherapy to many patients with different tumour types remain. These challenges range from the selection of antigen targets and dealing with regulatory and safety issues to successfully navigating the routes to commercial development. However, the encouraging clinical data, the progress in the scientific understanding of tumour immunology and the improvements in the manufacture of cell products are all advancing the clinical translation of these important cellular immunotherapies.

Birthdating studies reshape models for pituitary gland cell specification.

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Davis, Shannon W; Mortensen, Amanda H; Camper, Sally A

2011-04-15

The intermediate and anterior lobes of the pituitary gland are derived from an invagination of oral ectoderm that forms Rathke's pouch. During gestation proliferating cells are enriched around the pouch lumen, and they appear to delaminate as they exit the cell cycle and differentiate. During late mouse gestation and the postnatal period, anterior lobe progenitors re-enter the cell cycle and expand the populations of specialized, hormone-producing cells. At birth, all cell types are present, and their localization appears stratified based on cell type. We conducted a birth dating study of Rathke's pouch derivatives to determine whether the location of specialized cells at birth is correlated with the timing of cell cycle exit. We find that all of the anterior lobe cell types initiate differentiation concurrently with a peak between e11.5 and e13.5. Differentiation of intermediate lobe melanotropes is delayed relative to anterior lobe cell types. We discovered that specialized cell types are not grouped together based on birth date and are dispersed throughout the anterior lobe. Thus, the apparent stratification of specialized cells at birth is not correlated with cell cycle exit. Thus, the currently popular model of cell specification, dependent upon timing of extrinsic, directional gradients of signaling molecules, needs revision. We propose that signals intrinsic to Rathke's pouch are necessary for cell specification between e11.5 and e13.5 and that cell-cell communication likely plays an important role in regulating this process. Copyright © 2011 Elsevier Inc. All rights reserved.

Protein conservation and variation suggest mechanisms of cell type-specific modulation of signaling pathways.

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Martin H Schaefer

2014-06-01

Full Text Available Many proteins and signaling pathways are present in most cell types and tissues and yet perform specialized functions. To elucidate mechanisms by which these ubiquitous pathways are modulated, we overlaid information about cross-cell line protein abundance and variability, and evolutionary conservation onto functional pathway components and topological layers in the pathway hierarchy. We found that the input (receptors and the output (transcription factors layers evolve more rapidly than proteins in the intermediary transmission layer. In contrast, protein expression variability decreases from the input to the output layer. We observed that the differences in protein variability between the input and transmission layer can be attributed to both the network position and the tendency of variable proteins to physically interact with constitutively expressed proteins. Differences in protein expression variability and conservation are also accompanied by the tendency of conserved and constitutively expressed proteins to acquire somatic mutations, while germline mutations tend to occur in cell type-specific proteins. Thus, conserved core proteins in the transmission layer could perform a fundamental role in most cell types and are therefore less tolerant to germline mutations. In summary, we propose that the core signal transmission machinery is largely modulated by a variable input layer through physical protein interactions. We hypothesize that the bow-tie organization of cellular signaling on the level of protein abundance variability contributes to the specificity of the signal response in different cell types.

Persistence of collagen type II-specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis

International Nuclear Information System (INIS)

Londei, M.; Savill, C.M.; Verhoef, A.; Brennan, F.; Leech, Z.A.; Feldmann, M.; Duance, V.; Maini, R.N.

1989-01-01

Rheumatoid arthritis is an autoimmune disease characterized by T-cell infiltration of the synovium of joints. Analysis of the phenotype and antigen specificity of the infiltrating cells may thus provide insight into the pathogenesis of rheumatoid arthritis. T cells were cloned with interleukin 2, a procedure that selects for in vivo-activated cells. All clones had the CD4 CDW29 phenotype. Their antigen specificity was tested by using a panel of candidate joint autoantigens. Four of 17 reacted against autologous blood mononuclear cells. Two clones proliferated in response to collagen type II. After 21 months, another set of clones was derived from synovial tissue of the same joint. One of eight clones tested showed a strong proliferative response against collagen type II. The uncloned synovial T cells of a third operation from another joint also responded to collagen type II. The persistence of collagen type II-specific T cells in active rheumatoid joints over a period of 3 years suggests that collagen type II could be one of the autoantigens involved in perpetuating the inflammatory process in rheumatoid arthritis

Direct detection of diverse metabolic changes in virally transformed and tax-expressing cells by mass spectrometry.

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Prabhakar Sripadi

Full Text Available BACKGROUND: Viral transformation of a cell starts at the genetic level, followed by changes in the proteome and the metabolome of the host. There is limited information on the broad metabolic changes in HTLV transformed cells. METHODS AND PRINCIPAL FINDINGS: Here, we report the detection of key changes in metabolites and lipids directly from human T-lymphotropic virus type 1 and type 3 (HTLV1 and HTLV3 transformed, as well as Tax1 and Tax3 expressing cell lines by laser ablation electrospray ionization (LAESI mass spectrometry (MS. Comparing LAESI-MS spectra of non-HTLV1 transformed and HTLV1 transformed cells revealed that glycerophosphocholine (PC lipid components were dominant in the non-HTLV1 transformed cells, and PC(O-32:1 and PC(O-34:1 plasmalogens were displaced by PC(30:0 and PC(32:0 species in the HTLV1 transformed cells. In HTLV1 transformed cells, choline, phosphocholine, spermine and glutathione, among others, were downregulated, whereas creatine, dopamine, arginine and AMP were present at higher levels. When comparing metabolite levels between HTLV3 and Tax3 transfected 293T cells, there were a number of common changes observed, including decreased choline, phosphocholine, spermine, homovanillic acid, and glycerophosphocholine and increased spermidine and N-acetyl aspartic acid. These results indicate that the lipid metabolism pathway as well as the creatine and polyamine biosynthesis pathways are commonly deregulated after expression of HTLV3 and Tax3, indicating that the noted changes are likely due to Tax3 expression. N-acetyl aspartic acid is a novel metabolite that is upregulated in all cell types and all conditions tested. CONCLUSIONS AND SIGNIFICANCE: We demonstrate the high throughput in situ metabolite profiling of HTLV transformed and Tax expressing cells, which facilitates the identification of virus-induced perturbations in the biochemical processes of the host cells. We found virus type-specific (HTLV1 vs. HTLV3

Direct detection of diverse metabolic changes in virally transformed and tax-expressing cells by mass spectrometry.

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Sripadi, Prabhakar; Shrestha, Bindesh; Easley, Rebecca L; Carpio, Lawrence; Kehn-Hall, Kylene; Chevalier, Sebastien; Mahieux, Renaud; Kashanchi, Fatah; Vertes, Akos

2010-09-07

Viral transformation of a cell starts at the genetic level, followed by changes in the proteome and the metabolome of the host. There is limited information on the broad metabolic changes in HTLV transformed cells. Here, we report the detection of key changes in metabolites and lipids directly from human T-lymphotropic virus type 1 and type 3 (HTLV1 and HTLV3) transformed, as well as Tax1 and Tax3 expressing cell lines by laser ablation electrospray ionization (LAESI) mass spectrometry (MS). Comparing LAESI-MS spectra of non-HTLV1 transformed and HTLV1 transformed cells revealed that glycerophosphocholine (PC) lipid components were dominant in the non-HTLV1 transformed cells, and PC(O-32:1) and PC(O-34:1) plasmalogens were displaced by PC(30:0) and PC(32:0) species in the HTLV1 transformed cells. In HTLV1 transformed cells, choline, phosphocholine, spermine and glutathione, among others, were downregulated, whereas creatine, dopamine, arginine and AMP were present at higher levels. When comparing metabolite levels between HTLV3 and Tax3 transfected 293T cells, there were a number of common changes observed, including decreased choline, phosphocholine, spermine, homovanillic acid, and glycerophosphocholine and increased spermidine and N-acetyl aspartic acid. These results indicate that the lipid metabolism pathway as well as the creatine and polyamine biosynthesis pathways are commonly deregulated after expression of HTLV3 and Tax3, indicating that the noted changes are likely due to Tax3 expression. N-acetyl aspartic acid is a novel metabolite that is upregulated in all cell types and all conditions tested. We demonstrate the high throughput in situ metabolite profiling of HTLV transformed and Tax expressing cells, which facilitates the identification of virus-induced perturbations in the biochemical processes of the host cells. We found virus type-specific (HTLV1 vs. HTLV3), expression-specific (Tax1 vs. Tax3) and cell-type-specific (T lymphocytes vs. kidney

Molecular and Genetic Determinants of Glioma Cell Invasion

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Kenta Masui

2017-12-01

Full Text Available A diffusely invasive nature is a major obstacle in treating a malignant brain tumor, “diffuse glioma”, which prevents neurooncologists from surgically removing the tumor cells even in combination with chemotherapy and radiation. Recently updated classification of diffuse gliomas based on distinct genetic and epigenetic features has culminated in a multilayered diagnostic approach to combine histologic phenotypes and molecular genotypes in an integrated diagnosis. However, it is still a work in progress to decipher how the genetic aberrations contribute to the aggressive nature of gliomas including their highly invasive capacity. Here we depict a set of recent discoveries involving molecular genetic determinants of the infiltrating nature of glioma cells, especially focusing on genetic mutations in receptor tyrosine kinase pathways and metabolic reprogramming downstream of common cancer mutations. The specific biology of glioma cell invasion provides an opportunity to explore the genotype-phenotype correlation in cancer and develop novel glioma-specific therapeutic strategies for this devastating disease.

Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells.

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Kaitsuka, Taku; Tomizawa, Kazuhito

2015-11-06

Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.

Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells

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Taku Kaitsuka

2015-11-01

Full Text Available Protein transduction using cell-penetrating peptides (CPPs is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.

Genetic Correction and Hepatic Differentiation of Hemophilia B-specific Human Induced Pluripotent Stem Cells.

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He, Qiong; Wang, Hui-Hui; Cheng, Tao; Yuan, Wei-Ping; Ma, Yu-Po; Jiang, Yong-Ping; Ren, Zhi-Hua

2017-09-27

Objective To genetically correct a disease-causing point mutation in human induced pluripotent stem cells (iPSCs) derived from a hemophilia B patient. Methods First, the disease-causing mutation was detected by sequencing the encoding area of human coagulation factor IX (F IX) gene. Genomic DNA was extracted from the iPSCs, and the primers were designed to amplify the eight exons of F IX. Next, the point mutation in those iPSCs was genetically corrected using CRISPR/Cas9 technology in the presence of a 129-nucleotide homologous repair template that contained two synonymous mutations. Then, top 8 potential off-target sites were subsequently analyzed using Sanger sequencing. Finally, the corrected clones were differentiated into hepatocyte-like cells, and the secretion of F IX was validated by immunocytochemistry and ELISA assay. Results The cell line bore a missense mutation in the 6 th coding exon (c.676 C>T) of F IX gene. Correction of the point mutation was achieved via CRISPR/Cas9 technology in situ with a high efficacy at about 22% (10/45) and no off-target effects detected in the corrected iPSC clones. F IX secretion, which was further visualized by immunocytochemistry and quantified by ELISA in vitro, reached about 6 ng/ml on day 21 of differentiation procedure. Conclusions Mutations in human disease-specific iPSCs could be precisely corrected by CRISPR/Cas9 technology, and corrected cells still maintained hepatic differentiation capability. Our findings might throw a light on iPSC-based personalized therapies in the clinical application, especially for hemophilia B.

Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots

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Yoichiro Fukao

2016-01-01

Full Text Available The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex, respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.

Cell-type-specific role for nucleus accumbens neuroligin-2 in depression and stress susceptibility.

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Heshmati, Mitra; Aleyasin, Hossein; Menard, Caroline; Christoffel, Daniel J; Flanigan, Meghan E; Pfau, Madeline L; Hodes, Georgia E; Lepack, Ashley E; Bicks, Lucy K; Takahashi, Aki; Chandra, Ramesh; Turecki, Gustavo; Lobo, Mary Kay; Maze, Ian; Golden, Sam A; Russo, Scott J

2018-01-30

Behavioral coping strategies are critical for active resilience to stress and depression; here we describe a role for neuroligin-2 (NLGN-2) in the nucleus accumbens (NAc). Neuroligins (NLGN) are a family of neuronal postsynaptic cell adhesion proteins that are constituents of the excitatory and inhibitory synapse. Importantly, NLGN-3 and NLGN-4 mutations are strongly implicated as candidates underlying the development of neuropsychiatric disorders with social disturbances such as autism, but the role of NLGN-2 in neuropsychiatric disease states is unclear. Here we show a reduction in NLGN-2 gene expression in the NAc of patients with major depressive disorder. Chronic social defeat stress in mice also decreases NLGN-2 selectively in dopamine D1-positive cells, but not dopamine D2-positive cells, within the NAc of stress-susceptible mice. Functional NLGN-2 knockdown produces bidirectional, cell-type-specific effects: knockdown in dopamine D1-positive cells promotes subordination and stress susceptibility, whereas knockdown in dopamine D2-positive cells mediates active defensive behavior. These findings establish a behavioral role for NAc NLGN-2 in stress and depression; provide a basis for targeted, cell-type specific therapy; and highlight the role of active behavioral coping mechanisms in stress susceptibility.

Qualitative analysis of mouse specific-locus mutations: information on genetic organization, gene expression, and the chromosomal nature of induced lesions

International Nuclear Information System (INIS)

Russell, L.B.

1982-01-01

Analysis of mouse specific-locus (SL) mutations at three loci has identified over 33 distinct complementation groups - most of which are probably overlapping deficiencies - and 13 to 14 new functional units. The complementation maps that have been generated for the d-se and c regions include numerous vital functions; however, some of the genes in these regions are non-vital. At such loci, hypomorphic mutants must represent intragenic alterations, and some viable nulls could conceivably be intragenic lesions also. Analysis of SL mutations has provided information on genetic expression. Homozygous deficiencies can be completely viable or can kill at any one of a range of developmental stages. Heterozygonus deficiencies of up to 6 cM or more in genetic length have been recovered and propagated. The time of death of homozygous and the degree of inviability of heterozygous deficiencies are related more to specific content of the missing segment than to its length. Combinations of deficiencies with x-autosome translocations that inactivate the homologous region in a mosaic fashion have shown that organismic lethals are not necessarily cell lethal. The spectrum of mutations induced depends on the nature of the mutagen and the type of germ cell exposed. Radiation of spermatogonia produces intragenic as well as null mutations. Spontaneous mutations have an admixture of types not present in populations of mutations induced in germ cells, and this raises doubts concerning the accuracy of doubling-dose calculations in genetic risk estimation. The analysis of SL mutations has yielded genetic tools for the construction of detailed gene-dosage series, cis-trans comparisons, the mapping of known genes and identification of new genes, genetic rescue of various types, and the identification and isolation of DNA sequences

Cell-Type Specific DNA Methylation Patterns Define Human Breast Cellular Identity

Czech Academy of Sciences Publication Activity Database

Novák, Petr; Stampfer, M.R.; Munoz-Rodriguez, J.L.; Garbe, J.C.; Ehrich, M.; Futscher, B. W.; Jensen, T.J.

2012-01-01

Roč. 7, č. 12 (2012), e52299 E-ISSN 1932-6203 Institutional research plan: CEZ:AV0Z50510513 Institutional support: RVO:60077344 Keywords : MAMMARY EPITHELIAL-CELLS * PLURIPOTENT STEM-CELLS * CPG ISLAND SHORES Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.730, year: 2012

Cell-type-specific roles for COX-2 in UVB-induced skin cancer

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Herschman, Harvey

2014-01-01

In human tumors, and in mouse models, cyclooxygenase-2 (COX-2) levels are frequently correlated with tumor development/burden. In addition to intrinsic tumor cell expression, COX-2 is often present in fibroblasts, myofibroblasts and endothelial cells of the tumor microenvironment, and in infiltrating immune cells. Intrinsic cancer cell COX-2 expression is postulated as only one of many sources for prostanoids required for tumor promotion/progression. Although both COX-2 inhibition and global Cox-2 gene deletion ameliorate ultraviolet B (UVB)-induced SKH-1 mouse skin tumorigenesis, neither manipulation can elucidate the cell type(s) in which COX-2 expression is required for tumorigenesis; both eliminate COX-2 activity in all cells. To address this question, we created Cox-2 flox/flox mice, in which the Cox-2 gene can be eliminated in a cell-type-specific fashion by targeted Cre recombinase expression. Cox-2 deletion in skin epithelial cells of SKH-1 Cox-2 flox/flox;K14Cre + mice resulted, following UVB irradiation, in reduced skin hyperplasia and increased apoptosis. Targeted epithelial cell Cox-2 deletion also resulted in reduced tumor incidence, frequency, size and proliferation rate, altered tumor cell differentiation and reduced tumor vascularization. Moreover, Cox-2 flox/flox;K14Cre + papillomas did not progress to squamous cell carcinomas. In contrast, Cox-2 deletion in SKH-1 Cox-2 flox/flox; LysMCre + myeloid cells had no effect on UVB tumor induction. We conclude that (i) intrinsic epithelial COX-2 activity plays a major role in UVB-induced skin cancer, (ii) macrophage/myeloid COX-2 plays no role in UVB-induced skin cancer and (iii) either there may be another COX-2-dependent prostanoid source(s) that drives UVB skin tumor induction or there may exist a COX-2-independent pathway(s) to UVB-induced skin cancer. PMID:24469308

Patient-specific cardiovascular progenitor cells derived from integration-free induced pluripotent stem cells for vascular tissue regeneration.

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Hu, Jiang; Wang, Yongyu; Jiao, Jiao; Liu, Zhongning; Zhao, Chao; Zhou, Zhou; Zhang, Zhanpeng; Forde, Kaitlynn; Wang, Lunchang; Wang, Jiangang; Baylink, David J; Zhang, Xiao-Bing; Gao, Shaorong; Yang, Bo; Chen, Y Eugene; Ma, Peter X

2015-12-01

Tissue-engineered blood vessels (TEBVs) are promising in regenerating a live vascular replacement. However, the vascular cell source is limited, and it is crucial to develop a scaffold that accommodates new type of vascular progenitor cells and facilitates in vivo lineage specification of the cells into functional vascular smooth muscle cells (VSMCs) to regenerate vascular tissue. In the present study, integration-free human induced pluripotent stem cells (hiPSCs) were established from patient peripheral blood mononuclear cells through episomal vector nucleofection of reprogramming factors. The established hiPSCs were then induced into mesoderm-originated cardiovascular progenitor cells (CVPCs) with a highly efficient directed lineage specification method. The derived CVPCs were demonstrated to be able to differentiate into functional VSMCs. Subcutaneous implantation of CVPCs seeded on macroporous nanofibrous poly(l-lactide) scaffolds led to in vivo VSMC lineage specification and matrix deposition inside the scaffolds. In summary, we established integration-free patient-specific hiPSCs from peripheral blood mononuclear cells, derived CVPCs through directed lineage specification, and developed an advanced scaffold for these progenitor cells to further differentiate in vivo into VSMCs and regenerate vascular tissue in a subcutaneous implantation model. This study has established an efficient patient-specific approach towards in vivo regeneration of vascular tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Direct measurement of catalase activity in living cells and tissue biopsies

International Nuclear Information System (INIS)

Scaglione, Christine N.; Xu, Qijin; Ramanujan, V. Krishnan

2016-01-01

Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. - Highlights: • A novel, direct measurement of Catalase enzyme activity via, oxygen sensing method. • Steady-stateprofiles of Catalase activity follow the Michaelis-Menten Kinetics. • Catalase-specific activity demonstrated using genetic and pharmacological tools. • Overcomes limitations of spectroscopic methods and indirect calorimetric approaches. • Clear

Direct measurement of catalase activity in living cells and tissue biopsies

Energy Technology Data Exchange (ETDEWEB)

Scaglione, Christine N.; Xu, Qijin; Ramanujan, V. Krishnan, E-mail: Ramanujanv@csmc.edu

2016-01-29

Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. - Highlights: • A novel, direct measurement of Catalase enzyme activity via, oxygen sensing method. • Steady-stateprofiles of Catalase activity follow the Michaelis-Menten Kinetics. • Catalase-specific activity demonstrated using genetic and pharmacological tools. • Overcomes limitations of spectroscopic methods and indirect calorimetric approaches. • Clear

Histone modification profiles are predictive for tissue/cell-type specific expression of both protein-coding and microRNA genes

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Zhang Michael Q

2011-05-01

Full Text Available Abstract Background Gene expression is regulated at both the DNA sequence level and through modification of chromatin. However, the effect of chromatin on tissue/cell-type specific gene regulation (TCSR is largely unknown. In this paper, we present a method to elucidate the relationship between histone modification/variation (HMV and TCSR. Results A classifier for differentiating CD4+ T cell-specific genes from housekeeping genes using HMV data was built. We found HMV in both promoter and gene body regions to be predictive of genes which are targets of TCSR. For example, the histone modification types H3K4me3 and H3K27ac were identified as the most predictive for CpG-related promoters, whereas H3K4me3 and H3K79me3 were the most predictive for nonCpG-related promoters. However, genes targeted by TCSR can be predicted using other type of HMVs as well. Such redundancy implies that multiple type of underlying regulatory elements, such as enhancers or intragenic alternative promoters, which can regulate gene expression in a tissue/cell-type specific fashion, may be marked by the HMVs. Finally, we show that the predictive power of HMV for TCSR is not limited to protein-coding genes in CD4+ T cells, as we successfully predicted TCSR targeted genes in muscle cells, as well as microRNA genes with expression specific to CD4+ T cells, by the same classifier which was trained on HMV data of protein-coding genes in CD4+ T cells. Conclusion We have begun to understand the HMV patterns that guide gene expression in both tissue/cell-type specific and ubiquitous manner.

Genome-Wide Progesterone Receptor Binding: Cell Type-Specific and Shared Mechanisms in T47D Breast Cancer Cells and Primary Leiomyoma Cells

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Huang, Lei; Owen, Jonas K.; Xie, Anna; Navarro, Antonia; Monsivais, Diana; Coon V, John S.; Kim, J. Julie; Dai, Yang; Bulun, Serdar E.

2012-01-01

Background Progesterone, via its nuclear receptor (PR), exerts an overall tumorigenic effect on both uterine fibroid (leiomyoma) and breast cancer tissues, whereas the antiprogestin RU486 inhibits growth of these tissues through an unknown mechanism. Here, we determined the interaction between common or cell-specific genome-wide binding sites of PR and mRNA expression in RU486-treated uterine leiomyoma and breast cancer cells. Principal Findings ChIP-sequencing revealed 31,457 and 7,034 PR-binding sites in breast cancer and uterine leiomyoma cells, respectively; 1,035 sites overlapped in both cell types. Based on the chromatin-PR interaction in both cell types, we statistically refined the consensus progesterone response element to G•ACA• • •TGT•C. We identified two striking differences between uterine leiomyoma and breast cancer cells. First, the cis-regulatory elements for HSF, TEF-1, and C/EBPα and β were statistically enriched at genomic RU486/PR-targets in uterine leiomyoma, whereas E2F, FOXO1, FOXA1, and FOXF sites were preferentially enriched in breast cancer cells. Second, 51.5% of RU486-regulated genes in breast cancer cells but only 6.6% of RU486-regulated genes in uterine leiomyoma cells contained a PR-binding site within 5 kb from their transcription start sites (TSSs), whereas 75.4% of RU486-regulated genes contained a PR-binding site farther than 50 kb from their TSSs in uterine leiomyoma cells. RU486 regulated only seven mRNAs in both cell types. Among these, adipophilin (PLIN2), a pro-differentiation gene, was induced via RU486 and PR via the same regulatory region in both cell types. Conclusions Our studies have identified molecular components in a RU486/PR-controlled gene network involved in the regulation of cell growth, cell migration, and extracellular matrix function. Tissue-specific and common patterns of genome-wide PR binding and gene regulation may determine the therapeutic effects of antiprogestins in uterine fibroids and

Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA.

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Lu, Song; Chow, Christie C; Zhou, Junwei; Leung, Po Sing; Tsui, Stephen K; Lui, Kathy O

2016-01-01

In this chapter, we describe a highly efficient genetic modification strategy for human pancreatic progenitor cells using modified mRNA-encoding GFP and Neurogenin-3. The properties of modified mRNA offer an invaluable platform to drive protein expression, which has broad applicability in pathway regulation, directed differentiation, and lineage specification. This approach can also be used to regulate expression of other pivotal transcription factors during pancreas development and might have potential therapeutic values in regenerative medicine.

Direct and dynamic detection of HIV-1 in living cells.

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Jonas Helma

Full Text Available In basic and applied HIV research, reliable detection of viral components is crucial to monitor progression of infection. While it is routine to detect structural viral proteins in vitro for diagnostic purposes, it previously remained impossible to directly and dynamically visualize HIV in living cells without genetic modification of the virus. Here, we describe a novel fluorescent biosensor to dynamically trace HIV-1 morphogenesis in living cells. We generated a camelid single domain antibody that specifically binds the HIV-1 capsid protein (CA at subnanomolar affinity and fused it to fluorescent proteins. The resulting fluorescent chromobody specifically recognizes the CA-harbouring HIV-1 Gag precursor protein in living cells and is applicable in various advanced light microscopy systems. Confocal live cell microscopy and super-resolution microscopy allowed detection and dynamic tracing of individual virion assemblies at the plasma membrane. The analysis of subcellular binding kinetics showed cytoplasmic antigen recognition and incorporation into virion assembly sites. Finally, we demonstrate the use of this new reporter in automated image analysis, providing a robust tool for cell-based HIV research.

Type 2 Diabetes Mellitus and Cardiovascular Disease: Genetic and Epigenetic Links

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Salvatore De Rosa

2018-01-01

Full Text Available Type 2 diabetes mellitus (DM is a common metabolic disorder predisposing to diabetic cardiomyopathy and atherosclerotic cardiovascular disease (CVD, which could lead to heart failure through a variety of mechanisms, including myocardial infarction and chronic pressure overload. Pathogenetic mechanisms, mainly linked to hyperglycemia and chronic sustained hyperinsulinemia, include changes in metabolic profiles, intracellular signaling pathways, energy production, redox status, increased susceptibility to ischemia, and extracellular matrix remodeling. The close relationship between type 2 DM and CVD has led to the common soil hypothesis, postulating that both conditions share common genetic and environmental factors influencing this association. However, although the common risk factors of both CVD and type 2 DM, such as obesity, insulin resistance, dyslipidemia, inflammation, and thrombophilia, can be identified in the majority of affected patients, less is known about how these factors influence both conditions, so that efforts are still needed for a more comprehensive understanding of this relationship. The genetic, epigenetic, and environmental backgrounds of both type 2 DM and CVD have been more recently studied and updated. However, the underlying pathogenetic mechanisms have seldom been investigated within the broader shared background, but rather studied in the specific context of type 2 DM or CVD, separately. As the precise pathophysiological links between type 2 DM and CVD are not entirely understood and many aspects still require elucidation, an integrated description of the genetic, epigenetic, and environmental influences involved in the concomitant development of both diseases is of paramount importance to shed new light on the interlinks between type 2 DM and CVD. This review addresses the current knowledge of overlapping genetic and epigenetic aspects in type 2 DM and CVD, including microRNAs and long non-coding RNAs, whose

Laser-based direct-write techniques for cell printing

Energy Technology Data Exchange (ETDEWEB)

Schiele, Nathan R; Corr, David T [Biomedical Engineering Department, Rensselaer Polytechnic Institute, Troy, NY (United States); Huang Yong [Department of Mechanical Engineering, Clemson University, Clemson, SC (United States); Raof, Nurazhani Abdul; Xie Yubing [College of Nanoscale Science and Engineering, University at Albany, SUNY, Albany, NY (United States); Chrisey, Douglas B, E-mail: schien@rpi.ed, E-mail: chrisd@rpi.ed [Material Science and Engineering Department, Rensselaer Polytechnic Institute, Troy, NY (United States)

2010-09-15

Fabrication of cellular constructs with spatial control of cell location ({+-}5 {mu}m) is essential to the advancement of a wide range of applications including tissue engineering, stem cell and cancer research. Precise cell placement, especially of multiple cell types in co- or multi-cultures and in three dimensions, can enable research possibilities otherwise impossible, such as the cell-by-cell assembly of complex cellular constructs. Laser-based direct writing, a printing technique first utilized in electronics applications, has been adapted to transfer living cells and other biological materials (e.g., enzymes, proteins and bioceramics). Many different cell types have been printed using laser-based direct writing, and this technique offers significant improvements when compared to conventional cell patterning techniques. The predominance of work to date has not been in application of the technique, but rather focused on demonstrating the ability of direct writing to pattern living cells, in a spatially precise manner, while maintaining cellular viability. This paper reviews laser-based additive direct-write techniques for cell printing, and the various cell types successfully laser direct-written that have applications in tissue engineering, stem cell and cancer research are highlighted. A particular focus is paid to process dynamics modeling and process-induced cell injury during laser-based cell direct writing. (topical review)

Laser-based direct-write techniques for cell printing

International Nuclear Information System (INIS)

Schiele, Nathan R; Corr, David T; Huang Yong; Raof, Nurazhani Abdul; Xie Yubing; Chrisey, Douglas B

2010-01-01

Fabrication of cellular constructs with spatial control of cell location (±5 μm) is essential to the advancement of a wide range of applications including tissue engineering, stem cell and cancer research. Precise cell placement, especially of multiple cell types in co- or multi-cultures and in three dimensions, can enable research possibilities otherwise impossible, such as the cell-by-cell assembly of complex cellular constructs. Laser-based direct writing, a printing technique first utilized in electronics applications, has been adapted to transfer living cells and other biological materials (e.g., enzymes, proteins and bioceramics). Many different cell types have been printed using laser-based direct writing, and this technique offers significant improvements when compared to conventional cell patterning techniques. The predominance of work to date has not been in application of the technique, but rather focused on demonstrating the ability of direct writing to pattern living cells, in a spatially precise manner, while maintaining cellular viability. This paper reviews laser-based additive direct-write techniques for cell printing, and the various cell types successfully laser direct-written that have applications in tissue engineering, stem cell and cancer research are highlighted. A particular focus is paid to process dynamics modeling and process-induced cell injury during laser-based cell direct writing. (topical review)

Detection of GAD65 Autoreactive T-Cells by HLA Class I Tetramers in Type 1 Diabetic Patients

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Laura Giuliani

2009-01-01

Full Text Available Type 1 diabetes (T1D is an autoimmune disease, in which pancreatic β cells are destroyed in genetically predisposed individuals. While the direct contribution of autoantibodies to the disease pathogenesis is controversial, it is generally recognised that the mechanism of β cell destruction is mediated by autoreactive T cells that had escaped the thymic selection. We aimed to design a method to detect circulating CD8+ T cells autoreactive against an epitope of the glutamic acid decarboxylase autoantigen, isoform 65 (GAD65 ex vivo in T1D patients by using HLA class I tetramers. Low frequencies of GAD65 peptide-specific CD8+ cytotoxic T lymphocytes were detected in peripheral blood lymphocytes (PBMC of normal controls after GAD65 peptide-specific stimulation. Conversely, their frequencies were significantly higher than in controls in PBMC of T1D patients after GAD65 peptide stimulation. These preliminary data are encouraging in order to develop a reliable assay to be employed in large-scale screening studies.

A magic bullet to specifically eliminate mutated mitochondrial genomes from patients' cells

Science.gov (United States)

Moraes, Carlos T

2014-01-01

When mitochondrial diseases result from mutations found in the mitochondrial DNA, engineered mitochondrial-targeted nucleases such as mitochondrial-targeted zinc finger nucleases are shown to specifically eliminate the mutated molecules, leaving the wild-type mitochondrial DNA intact to replicate and restore normal copy number. In this issue, Gammage and colleagues successfully apply this improved technology on patients' cells with two types of genetic alterations responsible for neuropathy ataxia and retinitis pigmentosa (NARP) syndrome and Kearns Sayre syndrome and progressive external ophthalmoplegia (PEO). PMID:24623377

Development of a Direct Methanol Fuel Cell with Lightweight Disc Type Current Collectors

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Yean-Der Kuan

2014-05-01

Full Text Available The direct methanol fuel cell (DMFC adopts methanol solution as a fuel suitable for low power portable applications. A miniature, lightweight, passive air-breathing design is therefore desired. This paper presents a novel planar disc-type DMFC with multiple cells containing a novel developed lightweight current collector at both the anode and cathode sides. The present lightweight current collector adopts FR4 Glass/Epoxy as the substrate with the current collecting areas located at the corresponding membrane electrolyte assembly (MEA areas. The current collecting areas are fabricated by sequentially coating a corrosion resistant layer and electrical conduction layer via the thermal evaporation technique. The anode current collector has carved flow channels for fuel transport and production. The cathode current collector has drilled holes for passive air breathing. In order to ensure feasibility in the present concept a 3-cell prototype DMFC module with lightweight disc type current collectors is designed and constructed. Experiments were conducted to measure the cell performance. The results show that the highest cell power output is 54.88 mW·cm−2 and successfully demonstrate the feasibility of this novel design.

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

International Nuclear Information System (INIS)

Fujii, Hodaka

2007-01-01

Binding of interleukin-2 (IL-2) to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2 receptor (IL-2R)-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

Science.gov (United States)

Fujii, Hodaka

2007-01-01

Binding of IL-2 to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2R-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. However, Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling. PMID:17266928

C-type Lectin Receptors for Tumor Eradication: Future Directions

Energy Technology Data Exchange (ETDEWEB)

Streng-Ouwehand, Ingeborg; Unger, Wendy W. J.; Kooyk, Yvette van, E-mail: y.vankooyk@vumc.nl [Department of Molecular Cell Biology and Immunology, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam (Netherlands)

2011-08-08

Dendritic cells are key regulators in directing immune responses and therefore are under extensive research for the induction of anti-tumor responses. DCs express a large array of receptors by which they scan their surroundings for recognition and uptake of pathogens. One of the receptor-families is the C-type lectins (CLR), which bind carbohydrate structures and internalize antigens upon recognition. Intracellular routing of antigen through CLR enhances loading and presentation of antigen through MHC class I and II, inducing antigen-specific CD4{sup +} and CD8{sup +} T-cell proliferation and skewing T-helper cells. These characteristics make CLRs very interesting targets for DC-based immunotherapy. Profound research has been done on targeting specific tumor antigens to CLR using either antibodies or the natural ligands such as glycan structures. In this review we will focus on the current data showing the potency of CLR-targeting and discuss improvements that can be achieved to enhance anti-tumor activity in the near future.

First Evidence for the Disease-Stage, Cell-Type, and Virus Specificity of microRNAs during Human Immunodeficiency Virus Type-1 Infection

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Lauren Fowler

2016-05-01

Full Text Available The potential involvement of host microRNAs (miRNAs in HIV infection is well documented, and evidence suggests that HIV modulates and also dysregulates host miRNAs involved in maintaining the host innate immune system. Moreover, the dysregulation of host miRNAs by HIV also effectively interferes directly with the host gene expression. In this study, we have simultaneously evaluated the expression of host miRNAs in both CD4+ and CD8+ T-cells derived from HIV-positive (HIV+ individuals (viremic and aviremic individuals while receiving highly active antiretroviral therapy (HAART, therapy-naïve long-term non-progressors (LTNP, and HIV-negative (HIV– healthy controls. miRNAs were run on Affymetrix V2 chips, and the differential expression between HIV+ and HIV− samples, along with intergroup comparisons, was derived using PARTEK software, using an FDR of 5% and an adjusted p-value < 0.05. The miR-199a-5p was found to be HIV-specific and expressed in all HIV+ groups as opposed to HIV– controls. Moreover, these are the first studies to reveal clearly the highly discriminatory miRNAs at the level of the disease state, cell type, and HIV-specific miRNAs.

Dual gene activation and knockout screen reveals directional dependencies in genetic networks. | Office of Cancer Genomics

Science.gov (United States)

Understanding the direction of information flow is essential for characterizing how genetic networks affect phenotypes. However, methods to find genetic interactions largely fail to reveal directional dependencies. We combine two orthogonal Cas9 proteins from Streptococcus pyogenes and Staphylococcus aureus to carry out a dual screen in which one gene is activated while a second gene is deleted in the same cell. We analyze the quantitative effects of activation and knockout to calculate genetic interaction and directionality scores for each gene pair.

Development of Augmented Leukemia/Lymphoma-Specific T-Cell Immunotherapy for Deployment with Haploidentical, Hematompoietic Progenitor-Cell Transplant

Science.gov (United States)

2011-05-01

cells exhibited redirected specific lysis of a genetically modified EL4 target expressing 92% human CD19 (Figure 6a). T cells not genetically modified...but propagated by crosslinking CD3 using g-irradiated aAPC loaded with OKT3 did not appreciably lyse CD19 EL4 cells (Figure 6b). DISCUSSION We and...by genetically modified T cells. Lysis by chromium release assay of CD19+ EL4 tumor cells compared with CD19 parental EL4 cells by (a) T cells

Cell-specific type I IFN signatures in autoimmunity and viral infection: what makes the difference?

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Chieko Kyogoku

Full Text Available Gene expression profiling of peripheral blood mononuclear cells (PBMCs has revealed a crucial role for type I interferon (IFN in the pathogenesis of systemic lupus erythematosus (SLE. However, it is unclear how particular leucocyte subsets contribute to the overall type I IFN signature of PBMCs and whole blood samples.Furthermore, a detailed analysis describing the differences in the IFN signature in autoimmune diseases from that observed after viral infection has not been performed to date. Therefore, in this study, the transcriptional responses in peripheral T helper cells (CD4(+ and monocyte subsets (CD16(- inflammatory and CD16(+ resident monocytes isolated from patients with SLE, healthy donors (ND immunised with the yellow fever vaccine YFV-17Dand untreated controls were compared by global gene expression profiling.It was striking that all of the transcripts that were regulated in response to viral exposure were also found to be differentially regulated in SLE, albeit with markedly lower fold-change values. In addition to this common IFN signature, a pathogenic IFN-associated gene signature was detected in the CD4(+ T cells and monocytes from the lupus patients. IL-10, IL-9 and IL-15-mediated JAK/STAT signalling was shown to be involved in the pathological amplification of IFN responses observed in SLE. Type I IFN signatures identified were successfully applied for the monitoring of interferon responses in PBMCs of an independent cohort of SLE patients and virus-infected individuals. Moreover, these cell-type specific gene signatures allowed a correct classification of PBMCs independent from their heterogenic cellular composition. In conclusion, our data show for the first time that monocytes and CD4 cells are sensitive biosensors to monitor type I interferon response signatures in autoimmunity and viral infection and how these transriptional responses are modulated in a cell- and disease-specific manner.

CD4+ T-Cell Reactivity to Orexin/Hypocretin in Patients With Narcolepsy Type 1.

Science.gov (United States)

Ramberger, Melanie; Högl, Birgit; Stefani, Ambra; Mitterling, Thomas; Reindl, Markus; Lutterotti, Andreas

2017-03-01

Narcolepsy type 1 is accompanied by a selective loss of orexin/hypocretin (hcrt) neurons in the lateral hypothalamus caused by yet unknown mechanisms. Epidemiologic and genetic associations strongly suggest an immune-mediated pathogenesis of the disease. We compared specific T-cell reactivity to orexin/hcrt peptides in peripheral blood mononuclear cells of narcolepsy type 1 patients to healthy controls by a carboxyfluorescein succinimidyl ester proliferation assay. Orexin/hcrt-specific T-cell reactivity was also determined by cytokine (interferon gamma and granulocyte-macrophage colony-stimulating factor) analysis. Individuals were considered as responders if the cell division index of CD3+CD4+ T cells and both stimulation indices of cytokine secretion exceeded the cutoff 3. Additionally, T-cell reactivity to orexin/hcrt had to be confirmed by showing reactivity to single peptides present in different peptide pools. Using these criteria, 3/15 patients (20%) and 0/13 controls (0%) showed orexin/hcrt-specific CD4+ T-cell proliferation (p = .2262). The heterogeneous reactivity pattern did not allow the identification of a preferential target epitope. A significant role of orexin/hcrt-specific T cells in narcolepsy type 1 patients could not be confirmed in this study. Further studies are needed to assess the exact role of CD4+ T cells and possible target antigens in narcolepsy type 1 patients. © Sleep Research Society 2016. Published by Oxford University Press [on behalf of the Sleep Research Society].

Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells

Science.gov (United States)

Brandt, Benjamin; Munemasa, Shintaro; Wang, Cun; Nguyen, Desiree; Yong, Taiming; Yang, Paul G; Poretsky, Elly; Belknap, Thomas F; Waadt, Rainer; Alemán, Fernando; Schroeder, Julian I

2015-01-01

A central question is how specificity in cellular responses to the eukaryotic second messenger Ca2+ is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca2+-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca2+-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca2+-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruple mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca2+-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca2+-dependent and Ca2+-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca2+-signaling on a cellular, genetic, and biochemical level. DOI: http://dx.doi.org/10.7554/eLife.03599.001 PMID:26192964

Genetic analysis of PAX3 for diagnosis of Waardenburg syndrome type I.

Science.gov (United States)

Matsunaga, Tatsuo; Mutai, Hideki; Namba, Kazunori; Morita, Noriko; Masuda, Sawako

2013-04-01

PAX3 genetic analysis increased the diagnostic accuracy for Waardenburg syndrome type I (WS1). Analysis of the three-dimensional (3D) structure of PAX3 helped verify the pathogenicity of a missense mutation, and multiple ligation-dependent probe amplification (MLPA) analysis of PAX3 increased the sensitivity of genetic diagnosis in patients with WS1. Clinical diagnosis of WS1 is often difficult in individual patients with isolated, mild, or non-specific symptoms. The objective of the present study was to facilitate the accurate diagnosis of WS1 through genetic analysis of PAX3 and to expand the spectrum of known PAX3 mutations. In two Japanese families with WS1, we conducted a clinical evaluation of symptoms and genetic analysis, which involved direct sequencing, MLPA analysis, quantitative PCR of PAX3, and analysis of the predicted 3D structure of PAX3. The normal-hearing control group comprised 92 subjects who had normal hearing according to pure tone audiometry. In one family, direct sequencing of PAX3 identified a heterozygous mutation, p.I59F. Analysis of PAX3 3D structures indicated that this mutation distorted the DNA-binding site of PAX3. In the other family, MLPA analysis and subsequent quantitative PCR detected a large, heterozygous deletion spanning 1759-2554 kb that eliminated 12-18 genes including a whole PAX3 gene.

Directed migration of cancer cells by the graded texture of the underlying matrix

Science.gov (United States)

Park, JinSeok; Kim, Deok-Ho; Kim, Hong-Nam; Wang, Chiaochun Joanne; Kwak, Moon Kyu; Hur, Eunmi; Suh, Kahp-Yang; An, Steven S.; Levchenko, Andre

2016-01-01

Living cells and the extracellular matrix (ECM) can display complex interactions that define key developmental, physiological and pathological processes. Here, we report a new type of directed migration — which we term ‘topotaxis’ — by which cell movement is guided by the gradient of the nanoscale topographic features in the cells’ ECM environment. We show that the direction of topotaxis is reflective of the effective cell stiffness, and that it depends on the balance of the ECM-triggered signalling pathways PI3K-Akt and ROCK-MLCK. In melanoma cancer cells, this balance can be altered by different ECM inputs, pharmacological perturbations or genetic alterations, particularly a loss of PTEN in aggressive melanoma cells. We conclude that topotaxis is a product of the material properties of cells and the surrounding ECM, and propose that the invasive capacity of many cancers may depend broadly on topotactic responses, providing a potentially attractive mechanism for controlling invasive and metastatic behaviour. PMID:26974411

Adoptive immunotherapy using PRAME-specific T cells in medulloblastoma.

Science.gov (United States)

Orlando, Domenico; Miele, Evelina; De Angelis, Biagio; Guercio, Marika; Boffa, Iolanda; Sinibaldi, Matilde; Po, Agnese; Caruana, Ignazio; Abballe, Luana; Carai, Andrea; Caruso, Simona; Camera, Antonio; Moseley, Annemarie; Hagedoorn, Renate S; Heemskerk, Mirjam H M; Giangaspero, Felice; Mastronuzzi, Angela; Ferretti, Elisabetta; Locatelli, Franco; Quintarelli, Concetta

2018-04-03

Medulloblastoma is the most frequent malignant childhood brain tumor with a high morbidity. Identification of new therapeutic targets would be instrumental in improving patient outcomes. We evaluated the expression of the tumor-associated antigen PRAME in biopsies from 60 medulloblastoma patients. PRAME expression was detectable in 82% of tissues independent of molecular and histopathologic subgroups. High PRAME expression also correlated with worse overall survival. We next investigated the relevance of PRAME as a target for immunotherapy. Medulloblastoma cells were targeted using genetically modified T cells with a PRAME-specific TCR (SLL TCR T cells). SLL TCR T cells efficiently killed medulloblastoma HLA-A*02+ DAOY cells as well as primary HLA-A*02+ medulloblastoma cells. Moreover, SLL TCR T cells controlled tumor growth in an orthotopic mouse model of medulloblastoma. To prevent unexpected T cell-related toxicity,an inducible caspase 9 (iC9) gene was introduced in frame with the SLL TCR; this safety switch triggered prompt elimination of genetically-modified T cells. Altogether, these data indicate that T cells genetically modified with a high-affinity, PRAME-specific TCR and iC9 may represent a promising innovative approach for treating HLA-A*02+ medulloblastoma patients. Copyright ©2018, American Association for Cancer Research.

Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP.

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Caleb C Roth

Full Text Available Nanosecond electrical pulse (nsEP exposure activates signaling pathways, produces oxidative stress, stimulates hormone secretion, causes cell swelling and induces apoptotic and necrotic death. The underlying biophysical connection(s between these diverse cellular reactions and nsEP has yet to be elucidated. Using global genetic analysis, we evaluated how two commonly studied cell types, U937 and Jurkat, respond to nsEP exposure. We hypothesized that by studying the genetic response of the cells following exposure, we would gain direct insight into the stresses experienced by the cell and in turn better understand the biophysical interaction taking place during the exposure. Using Ingenuity Systems software, we found genes associated with cell growth, movement and development to be significantly up-regulated in both cell types 4 h post exposure to nsEP. In agreement with our hypothesis, we also found that both cell lines exhibit significant biological changes consistent with mechanical stress induction. These results advance nsEP research by providing strong evidence that the interaction of nsEPs with cells involves mechanical stress.

[Scientific potential of phenomics - functional direction of genetics].

Science.gov (United States)

Marasanov, A V; Valtseva, E A

In this paper on the based on the integration of known theories, doctrines and concepts - principles of consistency and self-regulation of physiological functions (Pavlov I.P., 1950), the theory offunctional systems (Anokhin P.K., 1973), the theory of adaptive reactions (Selye H., 1960 ; Garkavi LKh et al, 1979), the doctrine of the dominant (Ukhtomsky A.A., 1966), doctrine on health (Baevsky R.M.), doctrine on the body type of the human by Merlin VS. conception on the “the interrelationship between the function and genetic apparatus” by Meyerson F.Z., Pshennikova M.G., Platonov V.N., and others, there is proposed to select phenomics - functional division of genetics considering the poolability of specific mechanisms of the body in an integral system of the adaptive act in favor of the development of a personalized approach to the diagnosis and prevention of non-communicable diseases, increasing life expectancy of working age into the particular scientific direction. The task of phenomics is the establishment of the phenotypic characteristics of the person, norms of the response of systems of his body, determination of the deviation of the level of the functioning of the each system from the norm of its response and the elaboration of the tactics for the correction of the functional state of the organism (the optimization of its life activity), with taking into account the directedness of the interaction of body systems. The description of the shaping of the mechanism of stereotyped response of the organism generated an important contribution to the development of phenomics. Stereotyped response being initiated by the non-specific response of the body is aimed at the shaping of the activity of its systems after a fashion of norms of the activity, promotes the recovery of the specificity of the body, plays an important role in the establishment of cause-effect relations of the disease.

In-Depth Analysis of Citrulline-Specific CD4 T-Cells in Rheumatoid Arthritis

Science.gov (United States)

2018-01-01

player in the activation of lymphoid , myeloid and mast cells , indicating MALT1’s crucial role in innate and adaptive signaling. Therefore, MALT1 is...for RA (IFRA) Program Session 7: Adaptive immunity vs. innate immunity and mesenchymal functions in RA Genetics, T cell specificity and T cell ...Program Session 7: Adaptive immunity vs. innate immunity and mesenchymal functions in RA Genetics, T cell specificity and T cell regulation in RA

In Depth Analysis of Citrulline Specific CD4 T Cells in Rheumatoid Arthritis

Science.gov (United States)

2018-01-01

activation of lymphoid , myeloid and mast cells , indicating MALT1’s crucial role in innate and adaptive signaling. Therefore, MALT1 is regarded a...Session 7: Adaptive immunity vs. innate immunity and mesenchymal functions in RA Genetics, T cell specificity and T cell regulation in RA Jane Buckner...IFRA) Program Session 7: Adaptive immunity vs. innate immunity and mesenchymal functions in RA Genetics, T cell specificity and T cell regulation in

Antigen-specific murine T cell clones produce soluble interleukin 2 receptor on stimulation with specific antigens

International Nuclear Information System (INIS)

Wagner, D.K.; York-Jolley, J.; Malek, T.R.; Berzofsky, J.A.; Nelson, D.L.

1986-01-01

In this study, monoclonal antibodies were used to the murine IL 2 receptor (IL 2R) termed 3C7 and 7D4, which bind to different epitopes on the murine IL 2R, to develop an ELISA to measure soluble murine IL 2R. Surprisingly, stimulated murine spleen cells not only expressed cell-associated IL 2R, but also produced a considerable level of cellfree IL 2R in the culture supernatant fluid. To assess the fine specificity of this response, myoglobin-immune murine T cell clones were stimulated with appropriate or inappropriate antigen and syngeneic or allogeneic presenting cells. Proliferation, measured by [ 3 H] thymidine incorporation, and levels of soluble IL 2R were determined at day 4. The production of soluble IL2R displayed the same epitope fine specificity, genetic restriction, and antigen dose-response as the proliferative response. Indeed, in some cases there was sharper discrimination of epitope specificity and genetic restriction with the soluble IL 2R levels. There was also reproducible clone-to-clone variation in the amount of soluble receptor produced in response to antigen among 12 T cell clones and lines tested. In time course experiments, proliferation was greatest at day 3, whereas soluble IL 2R levels continued to rise in subsequent days. To the authors' knowledge, this is the first demonstration of release of secretion of soluble IL 2R by murine T cells, and the first demonstration of the fine specificity and genetic restriction of the induction of soluble IL 2R by specific antigen

Phenotypic silencing of cytoplasmic genes using sequence-specific double-stranded short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA viruses

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Barik Sailen

2001-12-01

Full Text Available Abstract Background Post-transcriptional gene silencing (PTGS by short interfering RNA has opened up new directions in the phenotypic mutation of cellular genes. However, its efficacy on non-nuclear genes and its effect on the interferon pathway remain unexplored. Since directed mutation of RNA genomes is not possible through conventional mutagenesis, we have tested sequence-specific 21-nucleotide long double-stranded RNAs (dsRNAs for their ability to silence cytoplasmic RNA genomes. Results Short dsRNAs were generated against specific mRNAs of respiratory syncytial virus, a nonsegmented negative-stranded RNA virus with a cytoplasmic life cycle. At nanomolar concentrations, the dsRNAs specifically abrogated expression of the corresponding viral proteins, and produced the expected mutant phenotype ex vivo. The dsRNAs did not induce an interferon response, and did not inhibit cellular gene expression. The ablation of the viral proteins correlated with the loss of the specific mRNAs. In contrast, viral genomic and antigenomic RNA, which are encapsidated, were not directly affected. Conclusions Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases. RNA-directed RNA gene silencing does not require cloning, expression, and mutagenesis of viral cDNA, and thus, will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle. This will, for the first time, permit functional genomic studies, attenuated infections, reverse genetic analysis, and studies of host-virus signaling pathways using a wild type RNA virus, unencumbered by any superinfecting virus.

Genetic typing in a cluster of Legionella pneumophila infections

NARCIS (Netherlands)

van Ketel, R. J.; de Wever, B.

1989-01-01

Legionella pneumophila strains isolated from six patients, three air-conditioning- and cooling tower-derived strains, and three hot water supply-derived strains were analyzed by three genetic typing methods. The results of the whole-cell DNA restriction endonuclease analysis and the restriction

Cocaine Exposure Reorganizes Cell-Type and Input-Specific Connectivity in the Nucleus Accumbens

Science.gov (United States)

MacAskill, Andrew F.; Cassel, John M.; Carter, Adam G.

2014-01-01

Exposure to cocaine alters the structural and functional properties of medium spiny neurons (MSNs) in the Nucleus Accumbens (NAc). These changes suggest a rewiring of the NAc circuit, with an enhancement of excitatory synaptic connections onto MSNs. However, it is unknown how drug exposure alters the balance of long-range afferents onto different cell types in the NAc. Here we use whole-cell recordings, two-photon microscopy, optogenetics and pharmacogenetics to show how repeated cocaine alters connectivity in the mouse NAc medial shell. We first determine that cocaine selectively enhances amygdala innervation of D1-MSNs relative to D2-MSNs. We then show that amygdala activity is required for cocaine-induced changes to behavior and connectivity. Finally, we establish how heightened amygdala innervation can explain the structural and functional changes induced by cocaine. Our findings reveal how exposure to drugs of abuse fundamentally reorganizes cell-type and input-specific connectivity in the NAc. PMID:25108911

Transfer of mRNA Encoding Invariant NKT Cell Receptors Imparts Glycolipid Specific Responses to T Cells and γδT Cells.

Science.gov (United States)

Shimizu, Kanako; Shinga, Jun; Yamasaki, Satoru; Kawamura, Masami; Dörrie, Jan; Schaft, Niels; Sato, Yusuke; Iyoda, Tomonori; Fujii, Shin-Ichiro

2015-01-01

Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and β chain mRNA (the Vα24 and Vβ11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide (α-GalCer), they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and β chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer.

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

Science.gov (United States)

Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K

2015-02-01

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transferred genetic marker in mouse FM3A cells

International Nuclear Information System (INIS)

Ayusawa, D.; Koyama, H.; Shimizu, K.; Kaneda, S.; Takeishi, K.; Seno, T.

1986-01-01

Studies were made on the genetic consequences of methotrexate-directed thymidylate stress, focusing attention on a human thymidylate synthase gene that was introduced as a heterologous genetic marker into mouse thymidylate synthase-negative mutant cells. Thymidylate stress induced thymidylate synthase-negative segregants with concomitant loss of human thymidylate synthase activity with frequencies 1 to 2 orders of magnitude higher than the uninduced spontaneous level in some but not all transformant lines. Induction of the segregants was suppressed almost completely by cycloheximide and partially by caffeine. Thymidylate stress did not, however, induce mutations, as determined by measuring resistance to ouabain or 6-thioguanine. Thymidylate synthase-negative segregants were also induced by other means such as bromodeoxyuridine treatment and X-ray irradiation. In each of the synthase-negative segregants induced by thymidylate stress, a DNA segment including almost the whole coding region of the transferred human thymidylate synthase gene was deleted in a very specific manner, as shown by Southern blot analysis with a human Alu sequence and a human thymidylate synthase cDNA as probes. In the segregants that emerged spontaneously at low frequency, the entire transferred genetic marker was lost. In the segregants induced by X-ray irradiation, structural alterations of the genetic marker were random. These results show that thymidylate stress is a physiological factor that provokes the instability of this exogenously incorporated DNA in some specific manner and produces nonrandom genetic recombination in mammalian cells

Specific Cell (Re-)Programming: Approaches and Perspectives.

Science.gov (United States)

Hausburg, Frauke; Jung, Julia Jeannine; David, Robert

2018-01-01

Many disorders are manifested by dysfunction of key cell types or their disturbed integration in complex organs. Thereby, adult organ systems often bear restricted self-renewal potential and are incapable of achieving functional regeneration. This underlies the need for novel strategies in the field of cell (re-)programming-based regenerative medicine as well as for drug development in vitro. The regenerative field has been hampered by restricted availability of adult stem cells and the potentially hazardous features of pluripotent embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Moreover, ethical concerns and legal restrictions regarding the generation and use of ESCs still exist. The establishment of direct reprogramming protocols for various therapeutically valuable somatic cell types has overcome some of these limitations. Meanwhile, new perspectives for safe and efficient generation of different specified somatic cell types have emerged from numerous approaches relying on exogenous expression of lineage-specific transcription factors, coding and noncoding RNAs, and chemical compounds.It should be of highest priority to develop protocols for the production of mature and physiologically functional cells with properties ideally matching those of their endogenous counterparts. Their availability can bring together basic research, drug screening, safety testing, and ultimately clinical trials. Here, we highlight the remarkable successes in cellular (re-)programming, which have greatly advanced the field of regenerative medicine in recent years. In particular, we review recent progress on the generation of cardiomyocyte subtypes, with a focus on cardiac pacemaker cells. Graphical Abstract.

Genetically conditioned male sterility

International Nuclear Information System (INIS)

Gottschalk, W.

1976-01-01

A survey is given of two different types of genetically controlled male sterility in higher plants. 'Functional' male sterility is due to the action of mutated genes causing a misdifferentiation of the growing points in different specific ways. Under the influence of the genes of this group either the stamens or the archespore tissues are not differentiated. In other mutants functionable male germ cells are produced but cannot be used for fertilizing the egg cells because the anthers remain closed or anthers and stigma become spatially separated from each other. Other genes of the group are responsible for the transformation of stamens into carpels, i.e. for a change of the hermaphrodite flower into a unisexually female one. A second type of male sterility is due to the action of ms genes influencing the course of micro-sporogenesis directly. They cause the breakdown of this process in a specific meiotic stage characteristic for each gene of the group. This breakdown is introduced by the degeneration of PMCs, microspores, or pollen grains preventing the production of male germ cells. The female sex organs remain uninfluenced. (author)

Type II collagen in cartilage evokes peptide-specific tolerance and skews the immune response.

Science.gov (United States)

Malmström, V; Kjellén, P; Holmdahl, R

1998-06-01

T cell recognition of type II collagen (CII) is a crucial event in the induction of collagen-induced arthritis in the mouse. Several CII peptides have been shown to be of importance, dependent on which MHC haplotype the mouse carries. By sequencing the rat CII and comparing the sequence with mouse, human, bovine and chicken CII, we have found that the immunodominant peptides all differ at critical positions compared with the autologous mouse sequence. Transgenic expression of the immunodominant Aq-restricted heterologous CII 256-270 epitope inserted into type I collagen (TSC mice) or type II collagen (MMC-1 mice) led to epitope-specific tolerance. Immunization of TSC mice with chick CII led to arthritis and immune responses, dependent on the subdominant, Aq-restricted and chick-specific CII 190-200 epitope. Immunization of F1 mice, expressing both H-2q and H-2r as well as transgenic expression of the Aq-restricted CII 256-270 epitope in cartilage, with bovine CII, led to arthritis, dependent on the Ar-restricted, bovine-specific epitope CII 607-621. These data show that the immunodominance of CII recognition is directed towards heterologous determinants, and that T cells directed towards the corresponding autologous epitopes are tolerated without evidence of active suppression.

L1 Cell Adhesion Molecule-Specific Chimeric Antigen Receptor-Redirected Human T Cells Exhibit Specific and Efficient Antitumor Activity against Human Ovarian Cancer in Mice.

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Hao Hong

Full Text Available New therapeutic modalities are needed for ovarian cancer, the most lethal gynecologic malignancy. Recent clinical trials have demonstrated the impressive therapeutic potential of adoptive therapy using chimeric antigen receptor (CAR-redirected T cells to target hematological cancers, and emerging studies suggest a similar impact may be achieved for solid cancers. We sought determine whether genetically-modified T cells targeting the CE7-epitope of L1-CAM, a cell adhesion molecule aberrantly expressed in several cancers, have promise as an immunotherapy for ovarian cancer, first demonstrating that L1-CAM was highly over-expressed on a panel of ovarian cancer cell lines, primary ovarian tumor tissue specimens, and ascites-derived primary cancer cells. Human central memory derived T cells (TCM were then genetically modified to express an anti-L1-CAM CAR (CE7R, which directed effector function upon tumor antigen stimulation as assessed by in vitro cytokine secretion and cytotoxicity assays. We also found that CE7R+ T cells were able to target primary ovarian cancer cells. Intraperitoneal (i.p. administration of CE7R+ TCM induced a significant regression of i.p. established SK-OV-3 xenograft tumors in mice, inhibited ascites formation, and conferred a significant survival advantage compared with control-treated animals. Taken together, these studies indicate that adoptive transfer of L1-CAM-specific CE7R+ T cells may offer a novel and effective immunotherapy strategy for advanced ovarian cancer.

Genome-edited human stem cell-derived beta cells: a powerful tool for drilling down on type 2 diabetes GWAS biology.

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Beer, Nicola L; Gloyn, Anna L

2016-01-01

Type 2 diabetes (T2D) is a disease of pandemic proportions, one defined by a complex aetiological mix of genetic, epigenetic, environmental, and lifestyle risk factors. Whilst the last decade of T2D genetic research has identified more than 100 loci showing strong statistical association with disease susceptibility, our inability to capitalise upon these signals reflects, in part, a lack of appropriate human cell models for study. This review discusses the impact of two complementary, state-of-the-art technologies on T2D genetic research: the generation of stem cell-derived, endocrine pancreas-lineage cells and the editing of their genomes. Such models facilitate investigation of diabetes-associated genomic perturbations in a physiologically representative cell context and allow the role of both developmental and adult islet dysfunction in T2D pathogenesis to be investigated. Accordingly, we interrogate the role that patient-derived induced pluripotent stem cell models are playing in understanding cellular dysfunction in monogenic diabetes, and how site-specific nucleases such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system are helping to confirm genes crucial to human endocrine pancreas development. We also highlight the novel biology gleaned in the absence of patient lines, including an ability to model the whole phenotypic spectrum of diabetes phenotypes occurring both in utero and in adult cells, interrogating the non-coding 'islet regulome' for disease-causing perturbations, and understanding the role of other islet cell types in aberrant glycaemia. This article aims to reinforce the importance of investigating T2D signals in cell models reflecting appropriate species, genomic context, developmental time point, and tissue type.

Cell-type-specific predictive network yields novel insights into mouse embryonic stem cell self-renewal and cell fate.

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Karen G Dowell

Full Text Available Self-renewal, the ability of a stem cell to divide repeatedly while maintaining an undifferentiated state, is a defining characteristic of all stem cells. Here, we clarify the molecular foundations of mouse embryonic stem cell (mESC self-renewal by applying a proven Bayesian network machine learning approach to integrate high-throughput data for protein function discovery. By focusing on a single stem-cell system, at a specific developmental stage, within the context of well-defined biological processes known to be active in that cell type, we produce a consensus predictive network that reflects biological reality more closely than those made by prior efforts using more generalized, context-independent methods. In addition, we show how machine learning efforts may be misled if the tissue specific role of mammalian proteins is not defined in the training set and circumscribed in the evidential data. For this study, we assembled an extensive compendium of mESC data: ∼2.2 million data points, collected from 60 different studies, under 992 conditions. We then integrated these data into a consensus mESC functional relationship network focused on biological processes associated with embryonic stem cell self-renewal and cell fate determination. Computational evaluations, literature validation, and analyses of predicted functional linkages show that our results are highly accurate and biologically relevant. Our mESC network predicts many novel players involved in self-renewal and serves as the foundation for future pluripotent stem cell studies. This network can be used by stem cell researchers (at http://StemSight.org to explore hypotheses about gene function in the context of self-renewal and to prioritize genes of interest for experimental validation.

Presynaptic (Type III) cells in mouse taste buds sense sour (acid) taste.

Science.gov (United States)

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.

Perforin expression directly ex vivo by HIV-specific CD8 T-cells is a correlate of HIV elite control.

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Adam R Hersperger

2010-05-01

Full Text Available Many immune correlates of CD8(+ T-cell-mediated control of HIV replication, including polyfunctionality, proliferative ability, and inhibitory receptor expression, have been discovered. However, no functional correlates using ex vivo cells have been identified with the known ability to cause the direct elimination of HIV-infected cells. We have recently discovered the ability of human CD8(+ T-cells to rapidly upregulate perforin--an essential molecule for cell-mediated cytotoxicity--following antigen-specific stimulation. Here, we examined perforin expression capability in a large cross-sectional cohort of chronically HIV-infected individuals with varying levels of viral load: elite controllers (n = 35, viremic controllers (n = 29, chronic progressors (n = 27, and viremic nonprogressors (n = 6. Using polychromatic flow cytometry and standard intracellular cytokine staining assays, we measured perforin upregulation, cytokine production, and degranulation following stimulation with overlapping peptide pools encompassing all proteins of HIV. We observed that HIV-specific CD8(+ T-cells from elite controllers consistently display an enhanced ability to express perforin directly ex vivo compared to all other groups. This ability is not restricted to protective HLA-B haplotypes, does not require proliferation or the addition of exogenous factors, is not restored by HAART, and primarily originates from effector CD8(+ T-cells with otherwise limited functional capability. Notably, we found an inverse relationship between HIV-specific perforin expression and viral load. Thus, the capability of HIV-specific CD8(+ T-cells to rapidly express perforin defines a novel correlate of control in HIV infection.

Tissue-Specific Gain of RTK Signalling Uncovers Selective Cell Vulnerability during Embryogenesis.

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Yannan Fan

Full Text Available The successive events that cells experience throughout development shape their intrinsic capacity to respond and integrate RTK inputs. Cellular responses to RTKs rely on different mechanisms of regulation that establish proper levels of RTK activation, define duration of RTK action, and exert quantitative/qualitative signalling outcomes. The extent to which cells are competent to deal with fluctuations in RTK signalling is incompletely understood. Here, we employ a genetic system to enhance RTK signalling in a tissue-specific manner. The chosen RTK is the hepatocyte growth factor (HGF receptor Met, an appropriate model due to its pleiotropic requirement in distinct developmental events. Ubiquitously enhanced Met in Cre/loxP-based Rosa26(stopMet knock-in context (Del-R26(Met reveals that most tissues are capable of buffering enhanced Met-RTK signalling thus avoiding perturbation of developmental programs. Nevertheless, this ubiquitous increase of Met does compromise selected programs such as myoblast migration. Using cell-type specific Cre drivers, we genetically showed that altered myoblast migration results from ectopic Met expression in limb mesenchyme rather than in migrating myoblasts themselves. qRT-PCR analyses show that ectopic Met in limbs causes molecular changes such as downregulation in the expression levels of Notum and Syndecan4, two known regulators of morphogen gradients. Molecular and functional studies revealed that ectopic Met expression in limb mesenchyme does not alter HGF expression patterns and levels, but impairs HGF bioavailability. Together, our findings show that myoblasts, in which Met is endogenously expressed, are capable of buffering increased RTK levels, and identify mesenchymal cells as a cell type vulnerable to ectopic Met-RTK signalling. These results illustrate that embryonic cells are sensitive to alterations in the spatial distribution of RTK action, yet resilient to fluctuations in signalling levels of an

Genetic characterization of human T-cell lymphotropic virus type 1 in Mozambique: transcontinental lineages drive the HTLV-1 endemic.

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Ana Carolina P Vicente

2011-04-01

Full Text Available Human T-Cell Lymphotropic Virus Type 1 (HTLV-1 is the etiological agent of adult T-cell leukemia (ATL and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP. It has been estimated that 10-20 million people are infected worldwide, but no successful treatment is available. Recently, the epidemiology of this virus was addressed in blood donors from Maputo, showing rates from 0.9 to 1.2%. However, the origin and impact of HTLV endemic in this population is unknown.To assess the HTLV-1 molecular epidemiology in Mozambique and to investigate their relationship with HTLV-1 lineages circulating worldwide.Blood donors and HIV patients were screened for HTLV antibodies by using enzyme immunoassay, followed by Western Blot. PCR and sequencing of HTLV-1 LTR region were applied and genetic HTLV-1 subtypes were assigned by the neighbor-joining method. The mean genetic distance of Mozambican HTLV-1 lineages among the genetic clusters were determined. Human mitochondrial (mt DNA analysis was performed and individuals classified in mtDNA haplogroups.LTR HTLV-1 analysis demonstrated that all isolates belong to the Transcontinental subgroup of the Cosmopolitan subtype. Mozambican HTLV-1 sequences had a high inter-strain genetic distance, reflecting in three major clusters. One cluster is associated with the South Africa sequences, one is related with Middle East and India strains and the third is a specific Mozambican cluster. Interestingly, 83.3% of HIV/HTLV-1 co-infection was observed in the Mozambican cluster. The human mtDNA haplotypes revealed that all belong to the African macrohaplogroup L with frequencies representatives of the country.The Mozambican HTLV-1 genetic diversity detected in this study reveals that although the strains belong to the most prevalent and worldwide distributed Transcontinental subgroup of the Cosmopolitan subtype, there is a high HTLV diversity that could be correlated with at least 3 different HTLV-1 introductions

Direct Differentiation of Human Pluripotent Stem Cells into Haploid Spermatogenic Cells

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Charles A. Easley, IV

2012-09-01

Full Text Available Human embryonic stem cells (hESCs and induced pluripotent stem cells (hiPSCs have been shown to differentiate into primordial germ cells (PGCs but not into spermatogonia, haploid spermatocytes, or spermatids. Here, we show that hESCs and hiPSCs differentiate directly into advanced male germ cell lineages, including postmeiotic, spermatid-like cells, in vitro without genetic manipulation. Furthermore, our procedure mirrors spermatogenesis in vivo by differentiating PSCs into UTF1-, PLZF-, and CDH1-positive spermatogonia-like cells; HIWI- and HILI-positive spermatocyte-like cells; and haploid cells expressing acrosin, transition protein 1, and protamine 1 (proteins that are uniquely found in spermatids and/or sperm. These spermatids show uniparental genomic imprints similar to those of human sperm on two loci: H19 and IGF2. These results demonstrate that male PSCs have the ability to differentiate directly into advanced germ cell lineages and may represent a novel strategy for studying spermatogenesis in vitro.

Site-Specific Genome Engineering in Human Pluripotent Stem Cells.

Science.gov (United States)

Merkert, Sylvia; Martin, Ulrich

2016-06-24

The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies.

Engineered artificial antigen presenting cells facilitate direct and efficient expansion of tumor infiltrating lymphocytes

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Coukos George

2011-08-01

Full Text Available Abstract Background Development of a standardized platform for the rapid expansion of tumor-infiltrating lymphocytes (TILs with anti-tumor function from patients with limited TIL numbers or tumor tissues challenges their clinical application. Methods To facilitate adoptive immunotherapy, we applied genetically-engineered K562 cell-based artificial antigen presenting cells (aAPCs for the direct and rapid expansion of TILs isolated from primary cancer specimens. Results TILs outgrown in IL-2 undergo rapid, CD28-independent expansion in response to aAPC stimulation that requires provision of exogenous IL-2 cytokine support. aAPCs induce numerical expansion of TILs that is statistically similar to an established rapid expansion method at a 100-fold lower feeder cell to TIL ratio, and greater than those achievable using anti-CD3/CD28 activation beads or extended IL-2 culture. aAPC-expanded TILs undergo numerical expansion of tumor antigen-specific cells, remain amenable to secondary aAPC-based expansion, and have low CD4/CD8 ratios and FOXP3+ CD4+ cell frequencies. TILs can also be expanded directly from fresh enzyme-digested tumor specimens when pulsed with aAPCs. These "young" TILs are tumor-reactive, positively skewed in CD8+ lymphocyte composition, CD28 and CD27 expression, and contain fewer FOXP3+ T cells compared to parallel IL-2 cultures. Conclusion Genetically-enhanced aAPCs represent a standardized, "off-the-shelf" platform for the direct ex vivo expansion of TILs of suitable number, phenotype and function for use in adoptive immunotherapy.

Generation and genetic modification of induced pluripotent stem cells.

Science.gov (United States)

Schambach, Axel; Cantz, Tobias; Baum, Christopher; Cathomen, Toni

2010-07-01

The generation of induced pluripotent stem cells (iPSCs) enabled by exogenous expression of the canonical Oct4, Sox2, Klf4 and c-Myc reprogramming factors has opened new ways to create patient- or disease-specific pluripotent cells. iPSCs represent an almost inexhaustible source of cells for targeted differentiation into somatic effector cells and hence are likely to be invaluable for therapeutic applications and disease-related research. After an introduction on the biology of reprogramming we cover emerging technological advances, including new reprogramming approaches, small-molecule compounds and tailored genetic modification, and give an outlook towards potential clinical applications of iPSCs. Although this field is progressing rapidly, reprogramming is still an inefficient process. The reader will learn about innovative tools to generate patient-specific iPSCs and how to modify these established lines in a safe way. Ideally, the disease-causing mutation is edited directly in the genome using novel technologies based on artificial nucleases, such as zinc-finger nucleases. Human iPSCs create fascinating options with regard to disease modeling, drug testing, developmental studies and therapeutic applications. However, important hurdles have to be taken and more efficient protocols to be established to achieve the ambitious goal of bringing iPSCs into clinical use.

GUCY2C-directed CAR-T cells oppose colorectal cancer metastases without autoimmunity.

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Magee, Michael S; Kraft, Crystal L; Abraham, Tara S; Baybutt, Trevor R; Marszalowicz, Glen P; Li, Peng; Waldman, Scott A; Snook, Adam E

2016-01-01

Adoptive T-cell therapy (ACT) is an emerging paradigm in which T cells are genetically modified to target cancer-associated antigens and eradicate tumors. However, challenges treating epithelial cancers with ACT reflect antigen targets that are not tumor-specific, permitting immune damage to normal tissues, and preclinical testing in artificial xenogeneic models, preventing prediction of toxicities in patients. In that context, mucosa-restricted antigens expressed by cancers exploit anatomical compartmentalization which shields mucosae from systemic antitumor immunity. This shielding may be amplified with ACT platforms employing antibody-based chimeric antigen receptors (CARs), which mediate MHC-independent recog-nition of antigens. GUCY2C is a cancer mucosa antigen expressed on the luminal surfaces of the intestinal mucosa in mice and humans, and universally overexpressed by colorectal tumors, suggesting its unique utility as an ACT target. T cells expressing CARs directed by a GUCY2C-specific antibody fragment recognized GUCY2C, quantified by expression of activation markers and cytokines. Further, GUCY2C CAR-T cells lysed GUCY2C-expressing, but not GUCY2C-deficient, mouse colorectal cancer cells. Moreover, GUCY2C CAR-T cells reduced tumor number and morbidity and improved survival in mice harboring GUCY2C-expressing colorectal cancer metastases. GUCY2C-directed T cell efficacy reflected CAR affinity and surface expression and was achieved without immune-mediated damage to normal tissues in syngeneic mice. These observations highlight the potential for therapeutic translation of GUCY2C-directed CAR-T cells to treat metastatic tumors, without collateral autoimmunity, in patients with metastatic colorectal cancer.

Determining T-cell specificity to understand and treat disease

DEFF Research Database (Denmark)

Hadrup, Sine Reker; Newell, Evan W.

2017-01-01

Adaptive immune responses and immunopathogeneses are based on the ability of T cells to respond to specific antigens. Consequently, understanding T-cell recognition patterns in health and disease involves studying the complexity and genetic heterogeneity of the antigen recognition pathway, which...

Specific Intensity Direct Current (DC) Electric Field Improves Neural Stem Cell Migration and Enhances Differentiation towards βIII-Tubulin+ Neurons

Science.gov (United States)

Zhao, Huiping; Steiger, Amanda; Nohner, Mitch; Ye, Hui

2015-01-01

Control of stem cell migration and differentiation is vital for efficient stem cell therapy. Literature reporting electric field–guided migration and differentiation is emerging. However, it is unknown if a field that causes cell migration is also capable of guiding cell differentiation—and the mechanisms for these processes remain unclear. Here, we report that a 115 V/m direct current (DC) electric field can induce directional migration of neural precursor cells (NPCs). Whole cell patching revealed that the cell membrane depolarized in the electric field, and buffering of extracellular calcium via EGTA prevented cell migration under these conditions. Immunocytochemical staining indicated that the same electric intensity could also be used to enhance differentiation and increase the percentage of cell differentiation into neurons, but not astrocytes and oligodendrocytes. The results indicate that DC electric field of this specific intensity is capable of promoting cell directional migration and orchestrating functional differentiation, suggestively mediated by calcium influx during DC field exposure. PMID:26068466

Combinatorial Modulation of Signaling Pathways Reveals Cell-Type-Specific Requirements for Highly Efficient and Synchronous iPSC Reprogramming

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Simon E. Vidal

2014-10-01

Full Text Available The differentiated state of somatic cells provides barriers for the derivation of induced pluripotent stem cells (iPSCs. To address why some cell types reprogram more readily than others, we studied the effect of combined modulation of cellular signaling pathways. Surprisingly, inhibition of transforming growth factor β (TGF-β together with activation of Wnt signaling in the presence of ascorbic acid allows >80% of murine fibroblasts to acquire pluripotency after 1 week of reprogramming factor expression. In contrast, hepatic and blood progenitors predominantly required only TGF-β inhibition or canonical Wnt activation, respectively, to reprogram at efficiencies approaching 100%. Strikingly, blood progenitors reactivated endogenous pluripotency loci in a highly synchronous manner, and we demonstrate that expression of specific chromatin-modifying enzymes and reduced TGF-β/mitogen-activated protein (MAP kinase activity are intrinsic properties associated with the unique reprogramming response of these cells. Our observations define cell-type-specific requirements for the rapid and synchronous reprogramming of somatic cells.

Rapid generation of sub-type, region-specific neurons and neural networks from human pluripotent stem cell-derived neurospheres

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Aynun N. Begum

2015-11-01

Full Text Available Stem cell-based neuronal differentiation has provided a unique opportunity for disease modeling and regenerative medicine. Neurospheres are the most commonly used neuroprogenitors for neuronal differentiation, but they often clump in culture, which has always represented a challenge for neurodifferentiation. In this study, we report a novel method and defined culture conditions for generating sub-type or region-specific neurons from human embryonic and induced pluripotent stem cells derived neurosphere without any genetic manipulation. Round and bright-edged neurospheres were generated in a supplemented knockout serum replacement medium (SKSRM with 10% CO2, which doubled the expression of the NESTIN, PAX6 and FOXG1 genes compared with those cultured with 5% CO2. Furthermore, an additional step (AdSTEP was introduced to fragment the neurospheres and facilitate the formation of a neuroepithelial-type monolayer that we termed the “neurosphederm”. The large neural tube-type rosette (NTTR structure formed from the neurosphederm, and the NTTR expressed higher levels of the PAX6, SOX2 and NESTIN genes compared with the neuroectoderm-derived neuroprogenitors. Different layers of cortical, pyramidal, GABAergic, glutamatergic, cholinergic neurons appeared within 27 days using the neurosphederm, which is a shorter period than in traditional neurodifferentiation-protocols (42–60 days. With additional supplements and timeline dopaminergic and Purkinje neurons were also generated in culture too. Furthermore, our in vivo results indicated that the fragmented neurospheres facilitated significantly better neurogenesis in severe combined immunodeficiency (SCID mouse brains compared with the non-fragmented neurospheres. Therefore, this neurosphere-based neurodifferentiation protocol is a valuable tool for studies of neurodifferentiation, neuronal transplantation and high throughput screening assays.

Re-analysis of public genetic data reveals a rare X-chromosomal variant associated with type 2 diabetes.

Science.gov (United States)

Bonàs-Guarch, Sílvia; Guindo-Martínez, Marta; Miguel-Escalada, Irene; Grarup, Niels; Sebastian, David; Rodriguez-Fos, Elias; Sánchez, Friman; Planas-Fèlix, Mercè; Cortes-Sánchez, Paula; González, Santi; Timshel, Pascal; Pers, Tune H; Morgan, Claire C; Moran, Ignasi; Atla, Goutham; González, Juan R; Puiggros, Montserrat; Martí, Jonathan; Andersson, Ehm A; Díaz, Carlos; Badia, Rosa M; Udler, Miriam; Leong, Aaron; Kaur, Varindepal; Flannick, Jason; Jørgensen, Torben; Linneberg, Allan; Jørgensen, Marit E; Witte, Daniel R; Christensen, Cramer; Brandslund, Ivan; Appel, Emil V; Scott, Robert A; Luan, Jian'an; Langenberg, Claudia; Wareham, Nicholas J; Pedersen, Oluf; Zorzano, Antonio; Florez, Jose C; Hansen, Torben; Ferrer, Jorge; Mercader, Josep Maria; Torrents, David

2018-01-22

The reanalysis of existing GWAS data represents a powerful and cost-effective opportunity to gain insights into the genetics of complex diseases. By reanalyzing publicly available type 2 diabetes (T2D) genome-wide association studies (GWAS) data for 70,127 subjects, we identify seven novel associated regions, five driven by common variants (LYPLAL1, NEUROG3, CAMKK2, ABO, and GIP genes), one by a low-frequency (EHMT2), and one driven by a rare variant in chromosome Xq23, rs146662057, associated with a twofold increased risk for T2D in males. rs146662057 is located within an active enhancer associated with the expression of Angiotensin II Receptor type 2 gene (AGTR2), a modulator of insulin sensitivity, and exhibits allelic specific activity in muscle cells. Beyond providing insights into the genetics and pathophysiology of T2D, these results also underscore the value of reanalyzing publicly available data using novel genetic resources and analytical approaches.

Direct ex vivo detection of HLA-DR3-restricted cytomegalovirus- and Mycobacterium tuberculosis-specific CD4+ T cells.

Science.gov (United States)

Bronke, Corine; Palmer, Nanette M; Westerlaken, Geertje H A; Toebes, Mireille; van Schijndel, Gijs M W; Purwaha, Veenu; van Meijgaarden, Krista E; Schumacher, Ton N M; van Baarle, Debbie; Tesselaar, Kiki; Geluk, Annemieke

2005-09-01

In order to detect epitope-specific CD4+ T cells in mycobacterial or viral infections in the context of human class II major histocompatibility complex protein human leukocyte antigen (HLA)-DR3, two HLA-DR3 tetrameric molecules were successfully produced. One contained an immunodominant HLA-DR3-restricted T-cell epitope derived from the 65-kDa heat-shock protein of Mycobacterium tuberculosis, peptide 1-13. For the other tetramer, we used an HLA-DR3-restricted T-cell epitope derived from cytomegalovirus (CMV) pp65 lower matrix protein, peptide 510-522, which induced high levels of interferon (IFN)-gamma-producing CD4+ T cells in three of four HLA-DR3-positive CMV-seropositive individuals up to 0.84% of CD4+ T cells by intracellular cytokine staining. In peripheral blood mononuclear cells from M. tuberculosis-exposed, Mycobacterium bovis bacille Calmette-Guérin (BCG)-vaccinated, or CMV-seropositive individuals, we were able to directly detect with both tetramers epitope-specific T cells up to 0.62% and 0.45% of the CD4+ T-cell population reactive to M. tuberculosis and CMV, respectively. After a 6-day culture with peptide p510-522, the frequency of CMV-specific tetramer-binding T cells was expanded up to 9.90% tetramer+ CFSElow (5,6-carboxyfluorescein diacetate succinimidyl ester) cells within the CD4+ T-cell population, further confirming the specificity of the tetrameric molecules. Thus, HLA-DR3/peptide tetrameric molecules can be used to investigate HLA-DR3-restricted antigen-specific CD4+ T cells in clinical disease or after vaccination.

Mosaic Expression of Thyroid Hormone Regulatory Genes Defines Cell Type-Specific Dependency in the Developing Chicken Cerebellum.

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Delbaere, Joke; Van Herck, Stijn L J; Bourgeois, Nele M A; Vancamp, Pieter; Yang, Shuo; Wingate, Richard J T; Darras, Veerle M

2016-12-01

The cerebellum is a morphologically unique brain structure that requires thyroid hormones (THs) for the correct coordination of key cellular events driving its development. Unravelling the interplay between the multiple factors that can regulate intracellular TH levels is a key step to understanding their role in the regulation of these cellular processes. We therefore investigated the regional/cell-specific expression pattern of TH transporters and deiodinases in the cerebellum using the chicken embryo as a model. In situ hybridisation revealed expression of the TH transporters monocarboxylate transporter 8 (MCT8) and 10 (MCT10), L-type amino acid transporter 1 (LAT1) and organic anion transporting polypeptide 1C1 (OATP1C1) as well as the inactivating type 3 deiodinase (D3) in the fourth ventricle choroid plexus, suggesting a possible contribution of the resulting proteins to TH exchange and subsequent inactivation of excess hormone at the blood-cerebrospinal fluid barrier. Exclusive expression of LAT1 and the activating type 2 deiodinase (D2) mRNA was found at the level of the blood-brain barrier, suggesting a concerted function for LAT1 and D2 in the direct access of active T 3 to the developing cerebellum via the capillary endothelial cells. The presence of MCT8 mRNA in Purkinje cells and cerebellar nuclei during the first 2 weeks of embryonic development points to a potential role of this transporter in the uptake of T 3 in central neurons. At later stages, together with MCT10, detection of MCT8 signal in close association with the Purkinje cell dendritic tree suggests a role of both transporters in TH signalling during Purkinje cell synaptogenesis. MCT10 was also expressed in late-born cells in the rhombic lip lineage with a clear hybridisation signal in the outer external granular layer, indicating a potential role for MCT10 in the proliferation of granule cell precursors. By contrast, expression of D3 in the first-born rhombic lip-derived population may

Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery of genetic material via living cells.

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Ward, Eliot; Chan, Emma; Gustafsson, Kenth; Jayasinghe, Suwan N

2010-05-01

The investigations reported in this article demonstrate the ability of bio-electrosprays and cell electrospinning to deliver a genetic construct in association with living cells. Previous studies on both bio-electrosprays and cell electrospinning demonstrated great promise for tissue engineering and regenerative biology/medicine. The investigations described herein widen the applicability of these biotechniques by combining gene therapy protocols, resulting in a novel drug delivery methodology previously unexplored. In these studies a human cell line was transduced with recombinant self-inactivating lentiviral particles. These particles incorporated a green fluorescent protein fused to an endosomal targeting construct. This construct encodes a peptide, which can subsequently be detected on the surface of cells by specific T-cells. The transduced cell line was subsequently manipulated in association with either bio-electrospraying or cell electrospinning. Hence this demonstrates (i) the ability to safely handle genetically modified living cells and (ii) the ability to directly form pre-determined architectures bearing living therapeutic cells. This merged technology demonstrates a unique approach for directly forming living therapeutic architectures for controlled and targeted release of experimental cells/genes, as well as medical cell/gene therapeutics for a plethora of biological and medical applications. Hence, such developments could be applied to personalised medicine.

Region-specificity of GABAA receptor mediated effects on orientation and direction selectivity in cat visual cortical area 18.

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Jirmann, Kay-Uwe; Pernberg, Joachim; Eysel, Ulf T

2009-01-01

The role of GABAergic inhibition in orientation and direction selectivity has been investigated with the GABA(A)-Blocker bicuculline in the cat visual cortex, and results indicated a region specific difference of functional contributions of GABAergic inhibition in areas 17 and 18. In area 17 inhibition appeared mainly involved in sculpturing orientation and direction tuning, while in area 18 inhibition seemed more closely associated with temporal receptive field properties. However, different types of stimuli were used to test areas 17 and 18 and further studies performed in area 17 suggested an important influence of the stimulus type (single light bars vs. moving gratings) on the evoked responses (transient vs. sustained) and inhibitory mechanisms (GABA(A) vs. GABA(B)) which in turn might be more decisive for the specific results than the cortical region. To insert the missing link in this chain of arguments it was necessary to study GABAergic inhibition in area 18 with moving light bars, which has not been done so far. Therefore, in the present study we investigated area 18 cells responding to oriented moving light bars with extracellular recordings and reversible microiontophoretic blockade of GABAergig inhibition with bicuculline methiodide. The majority of neurons was characterized by a pronounced orientation specificity and variable degrees of direction selectivity. GABA(A)ergic inhibition significantly influenced preferred orientation and preferred direction in area 18. During the action of bicuculline orientation tuning width increased and orientation and direction selectivity indices decreased. Our results obtained in area 18 with moving bar stimuli, although in the proportion of affected cells similar to those described in area 17, quantitatively matched the findings for direction and orientation specificity obtained with moving gratings in area 18. Accordingly, stimulus type is not decisive in area 18 and the GABA(A) dependent, inhibitory intracortical

Genetic Approaches to Study Meiosis and Meiosis-Specific Gene Expression in Saccharomyces cerevisiae.

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Kassir, Yona; Stuart, David T

2017-01-01

The budding yeast Saccharomyces cerevisiae has a long history as a model organism for studies of meiosis and the cell cycle. The popularity of this yeast as a model is in large part due to the variety of genetic and cytological approaches that can be effectively performed with the cells. Cultures of the cells can be induced to synchronously progress through meiosis and sporulation allowing large-scale gene expression and biochemical studies to be performed. Additionally, the spore tetrads resulting from meiosis make it possible to characterize the haploid products of meiosis allowing investigation of meiotic recombination and chromosome segregation. Here we describe genetic methods for analysis progression of S. cerevisiae through meiosis and sporulation with an emphasis on strategies for the genetic analysis of regulators of meiosis-specific genes.

Isolation and genetic analysis of pure cells from forensic biological mixtures: The precision of a digital approach.

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Fontana, F; Rapone, C; Bregola, G; Aversa, R; de Meo, A; Signorini, G; Sergio, M; Ferrarini, A; Lanzellotto, R; Medoro, G; Giorgini, G; Manaresi, N; Berti, A

2017-07-01

Latest genotyping technologies allow to achieve a reliable genetic profile for the offender identification even from extremely minute biological evidence. The ultimate challenge occurs when genetic profiles need to be retrieved from a mixture, which is composed of biological material from two or more individuals. In this case, DNA profiling will often result in a complex genetic profile, which is then subject matter for statistical analysis. In principle, when more individuals contribute to a mixture with different biological fluids, their single genetic profiles can be obtained by separating the distinct cell types (e.g. epithelial cells, blood cells, sperm), prior to genotyping. Different approaches have been investigated for this purpose, such as fluorescent-activated cell sorting (FACS) or laser capture microdissection (LCM), but currently none of these methods can guarantee the complete separation of different type of cells present in a mixture. In other fields of application, such as oncology, DEPArray™ technology, an image-based, microfluidic digital sorter, has been widely proven to enable the separation of pure cells, with single-cell precision. This study investigates the applicability of DEPArray™ technology to forensic samples analysis, focusing on the resolution of the forensic mixture problem. For the first time, we report here the development of an application-specific DEPArray™ workflow enabling the detection and recovery of pure homogeneous cell pools from simulated blood/saliva and semen/saliva mixtures, providing full genetic match with genetic profiles of corresponding donors. In addition, we assess the performance of standard forensic methods for DNA quantitation and genotyping on low-count, DEPArray™-isolated cells, showing that pure, almost complete profiles can be obtained from as few as ten haploid cells. Finally, we explore the applicability in real casework samples, demonstrating that the described approach provides complete

Genetic dissection of histone deacetylase requirement in tumor cells

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Haberland, Michael; Johnson, Aaron; Mokalled, Mayssa H.; Montgomery, Rusty L.; Olson, Eric N.

2009-01-01

Histone deacetylase inhibitors (HDACi) represent a new group of drugs currently being tested in a wide variety of clinical applications. They are especially effective in preclinical models of cancer where they show antiproliferative action in many different types of cancer cells. Recently, the first HDACi was approved for the treatment of cutaneous T cell lymphomas. Most HDACi currently in clinical development act by unspecifically interfering with the enzymatic activity of all class I HDACs (HDAC1, 2, 3, and 8), and it is widely believed that the development of isoform-specific HDACi could lead to better therapeutic efficacy. The contribution of the individual class I HDACs to different disease states, however, has so far not been fully elucidated. Here, we use a genetic approach to dissect the involvement of the different class I HDACs in tumor cells. We show that deletion of a single HDAC is not sufficient to induce cell death, but that HDAC1 and 2 play redundant and essential roles in tumor cell survival. Their deletion leads to nuclear bridging, nuclear fragmentation, and mitotic catastrophe, mirroring the effects of HDACi on cancer cells. These findings suggest that pharmacological inhibition of HDAC1 and 2 may be sufficient for anticancer activity, providing an experimental framework for the development of isoform-specific HDAC inhibitors. PMID:19416910

Lim homeobox genes in the Ctenophore Mnemiopsis leidyi: the evolution of neural cell type specification

Directory of Open Access Journals (Sweden)

Simmons David K

2012-01-01

Full Text Available Abstract Background Nervous systems are thought to be important to the evolutionary success and diversification of metazoans, yet little is known about the origin of simple nervous systems at the base of the animal tree. Recent data suggest that ctenophores, a group of macroscopic pelagic marine invertebrates, are the most ancient group of animals that possess a definitive nervous system consisting of a distributed nerve net and an apical statocyst. This study reports on details of the evolution of the neural cell type specifying transcription factor family of LIM homeobox containing genes (Lhx, which have highly conserved functions in neural specification in bilaterian animals. Results Using next generation sequencing, the first draft of the genome of the ctenophore Mnemiopsis leidyi has been generated. The Lhx genes in all animals are represented by seven subfamilies (Lhx1/5, Lhx3/4, Lmx, Islet, Lhx2/9, Lhx6/8, and LMO of which four were found to be represented in the ctenophore lineage (Lhx1/5, Lhx3/4, Lmx, and Islet. Interestingly, the ctenophore Lhx gene complement is more similar to the sponge complement (sponges do not possess neurons than to either the cnidarian-bilaterian or placozoan Lhx complements. Using whole mount in situ hybridization, the Lhx gene expression patterns were examined and found to be expressed around the blastopore and in cells that give rise to the apical organ and putative neural sensory cells. Conclusion This research gives us a first look at neural cell type specification in the ctenophore M. leidyi. Within M. leidyi, Lhx genes are expressed in overlapping domains within proposed neural cellular and sensory cell territories. These data suggest that Lhx genes likely played a conserved role in the patterning of sensory cells in the ancestor of sponges and ctenophores, and may provide a link to the expression of Lhx orthologs in sponge larval photoreceptive cells. Lhx genes were later co-opted into patterning more

Legislation on direct-to-consumer genetic testing in seven European countries.

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Borry, Pascal; van Hellemondt, Rachel E; Sprumont, Dominique; Jales, Camilla Fittipaldi Duarte; Rial-Sebbag, Emmanuelle; Spranger, Tade Matthias; Curren, Liam; Kaye, Jane; Nys, Herman; Howard, Heidi

2012-07-01

An increasing number of private companies are now offering direct-to-consumer (DTC) genetic testing services. Although a lot of attention has been devoted to the regulatory framework of DTC genetic testing services in the USA, only limited information about the regulatory framework in Europe is available. We will report on the situation with regard to the national legislation on DTC genetic testing in seven European countries (Belgium, the Netherlands, Switzerland, Portugal, France, Germany, the United Kingdom). The paper will address whether these countries have legislation that specifically address the issue of DTC genetic testing or have relevant laws that is pertinent to the regulatory control of these services in their countries. The findings show that France, Germany, Portugal and Switzerland have specific legislation that defines that genetic tests can only be carried out by a medical doctor after the provision of sufficient information concerning the nature, meaning and consequences of the genetic test and after the consent of the person concerned. In the Netherlands, some DTC genetic tests could fall under legislation that provides the Minister the right to refuse to provide a license to operate if a test is scientifically unsound, not in accordance with the professional medical practice standards or if the expected benefit is not in balance with the (potential) health risks. Belgium and the United Kingdom allow the provision of DTC genetic tests.

Cell type-specific anti-cancer properties of valproic acid: independent effects on HDAC activity and Erk1/2 phosphorylation

DEFF Research Database (Denmark)

Gotfryd, Kamil; Skladchikova, Galina; Lepekhin, Eugene E

2010-01-01

lines (BT4C, BT4Cn, U87MG, N2a, PC12-E2, CSML0, CSML100, HeLa, L929, Swiss 3T3). Results: VPA induced significant histone deacetylase (HDAC) inhibition in most of the cell lines, but the degree of inhibition was highly cell type-specific. Moreover, cell growth, motility and the degree of Erk1......ABSTRACT: BACKGROUND: The anti-epileptic drug valproic acid (VPA) has attracted attention as an anti-cancer agent. Methods: The present study investigated effects of VPA exposure on histone deacetylase (HDAC) inhibition, cell growth, cell speed, and the degree of Erk1/2 phosphorylation in 10 cell....../2 phosphorylation were inhibited, activated, or unaffected by VPA in a cell type-specific manner. Importantly, no relationship was found between the effects of VPA on HDAC inhibition and changes in the degree of Erk1/2 phosphorylation, cell growth, or motility. In contrast, VPA-induced modulation of the MAPK...

Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.

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Warren, Luigi; Manos, Philip D; Ahfeldt, Tim; Loh, Yuin-Han; Li, Hu; Lau, Frank; Ebina, Wataru; Mandal, Pankaj K; Smith, Zachary D; Meissner, Alexander; Daley, George Q; Brack, Andrew S; Collins, James J; Cowan, Chad; Schlaeger, Thorsten M; Rossi, Derrick J

2010-11-05

Clinical application of induced pluripotent stem cells (iPSCs) is limited by the low efficiency of iPSC derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPSCs toward clinically useful cell types are lacking. Here we describe a simple, nonintegrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate antiviral responses. We show that this approach can reprogram multiple human cell types to pluripotency with efficiencies that greatly surpass established protocols. We further show that the same technology can be used to efficiently direct the differentiation of RNA-induced pluripotent stem cells (RiPSCs) into terminally differentiated myogenic cells. This technology represents a safe, efficient strategy for somatic cell reprogramming and directing cell fate that has broad applicability for basic research, disease modeling, and regenerative medicine. Copyright © 2010 Elsevier Inc. All rights reserved.

Direct chromosome-length haplotyping by single-cell sequencing

NARCIS (Netherlands)

Porubský, David; Sanders, Ashley D; van Wietmarschen, Niek; Falconer, Ester; Hills, Mark; Spierings, Diana C J; Bevova, Marianna R; Guryev, Victor; Lansdorp, Peter Michael

Haplotypes are fundamental to fully characterize the diploid genome of an individual, yet methods to directly chart the unique genetic makeup of each parental chromosome are lacking. Here we introduce single-cell DNA template strand sequencing (Strand-seq) as a novel approach to phasing diploid

A quantitative comparison of cell-type-specific microarray gene expression profiling methods in the mouse brain.

Directory of Open Access Journals (Sweden)

Benjamin W Okaty

Full Text Available Expression profiling of restricted neural populations using microarrays can facilitate neuronal classification and provide insight into the molecular bases of cellular phenotypes. Due to the formidable heterogeneity of intermixed cell types that make up the brain, isolating cell types prior to microarray processing poses steep technical challenges that have been met in various ways. These methodological differences have the potential to distort cell-type-specific gene expression profiles insofar as they may insufficiently filter out contaminating mRNAs or induce aberrant cellular responses not normally present in vivo. Thus we have compared the repeatability, susceptibility to contamination from off-target cell-types, and evidence for stress-responsive gene expression of five different purification methods--Laser Capture Microdissection (LCM, Translating Ribosome Affinity Purification (TRAP, Immunopanning (PAN, Fluorescence Activated Cell Sorting (FACS, and manual sorting of fluorescently labeled cells (Manual. We found that all methods obtained comparably high levels of repeatability, however, data from LCM and TRAP showed significantly higher levels of contamination than the other methods. While PAN samples showed higher activation of apoptosis-related, stress-related and immediate early genes, samples from FACS and Manual studies, which also require dissociated cells, did not. Given that TRAP targets actively translated mRNAs, whereas other methods target all transcribed mRNAs, observed differences may also reflect translational regulation.

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

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

2012-01-01

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

Modulation of intracellular Ca2+ via L-type calcium channels in heart cells by the autoantibody directed against the second extracellular loop of the alpha1-adrenoceptors.

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Bkaily, Ghassan; El-Bizri, Nesrine; Bui, Michel; Sukarieh, Rami; Jacques, Danielle; Fu, Michael L X

2003-03-01

The effects of methoxamine, a selective alpha1-adrenergic receptor agonist, and the autoantibody directed against the second extracellular loop of alpha1-adrenoceptors were studied on intracellular free Ca2+ levels using confocal microscopy and ionic currents using the whole-cell patch clamp technique in single cells of 10-day-old embryonic chick and 20-week-old fetal human hearts. We observed that like methoxamine, the autoantibody directed against the second extracellular loop of alpha1-adrenoreceptors significantly increased the L-type calcium current (I(Ca(L))) but had no effect on the T-type calcium current (I(Ca(T))), the delayed outward potassium current, or the fast sodium current. This effect of the autoantibody was prevented by a prestimulation of the receptors with methoxamine and vice versa. Moreover, treating the cells with prazosin, a selective alpha1-adrenergic receptor antagonist blocked the methoxamine and the autoantibody-induced increase in I(Ca(L)), respectively. In absence of prazosin, both methoxamine and the autoantibody showed a substantial enhancement in the frequency of cell contraction and that of the concomitant cytosolic and nuclear free Ca2+ variations. The subsequent addition of nifedipine, a specific L-type Ca2+ channel blocker, reversed not only the methoxamine or the autoantibody-induced effect but also completely abolished cell contraction. These results demonstrated that functional alpha1-adrenoceptors exist in both 10-day-old embryonic chick and 20-week-old human fetal hearts and that the autoantibody directed against the second extracellular loop of this type of receptors plays an important role in stimulating their activity via activation of L-type calcium channels. This loop seems to have a functional significance by being the target of alpha1-receptor agonists like methoxamine.

1 kb of the lactase-phlorizin hydrolase promoter directs post-weaning decline and small intestinal-specific expression in transgenic mice

DEFF Research Database (Denmark)

Troelsen, J T; Mehlum, A; Spodsberg, N

1994-01-01

Adult-type hypolactasia is a genetic condition making approximately one half of the human population intolerant to milk because of abdominal symptoms. The cause is a post-weaning down-regulation of the intestinal-specific enzyme lactase-phlorizin hydrolase (LPH) reducing the intestinal capacity...... to hydrolyze lactose. We here demonstrate that the stretch -17 to -994 in the pig LPH-promoter carries cis-elements which direct a small intestinal-specific expression and a post-weaning decline of a linked rabbit beta-globin gene. These data demonstrate that the post-weaning decline of LPH is mainly due...

Linking transcriptional and genetic tumor heterogeneity through allele analysis of single-cell RNA-seq data.

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Fan, Jean; Lee, Hae-Ock; Lee, Soohyun; Ryu, Da-Eun; Lee, Semin; Xue, Catherine; Kim, Seok Jin; Kim, Kihyun; Barkas, Nikolas; Park, Peter J; Park, Woong-Yang; Kharchenko, Peter V

2018-06-13

Characterization of intratumoral heterogeneity is critical to cancer therapy, as presence of phenotypically diverse cell populations commonly fuels relapse and resistance to treatment. Although genetic variation is a well-studied source of intratumoral heterogeneity, the functional impact of most genetic alterations remains unclear. Even less understood is the relative importance of other factors influencing heterogeneity, such as epigenetic state or tumor microenvironment. To investigate the relationship between genetic and transcriptional heterogeneity in a context of cancer progression, we devised a computational approach called HoneyBADGER to identify copy number variation and loss-of-heterozygosity in individual cells from single-cell RNA-sequencing data. By integrating allele and normalized expression information, HoneyBADGER is able to identify and infer the presence of subclone-specific alterations in individual cells and reconstruct underlying subclonal architecture. Examining several tumor types, we show that HoneyBADGER is effective at identifying deletion, amplifications, and copy-neutral loss-of-heterozygosity events, and is capable of robustly identifying subclonal focal alterations as small as 10 megabases. We further apply HoneyBADGER to analyze single cells from a progressive multiple myeloma patient to identify major genetic subclones that exhibit distinct transcriptional signatures relevant to cancer progression. Surprisingly, other prominent transcriptional subpopulations within these tumors did not line up with the genetic subclonal structure, and were likely driven by alternative, non-clonal mechanisms. These results highlight the need for integrative analysis to understand the molecular and phenotypic heterogeneity in cancer. Published by Cold Spring Harbor Laboratory Press.

Homeostasis or channelopathy? Acquired cell type-specific ion channel changes in temporal lobe epilepsy and their antiepileptic potential

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Wolfart, Jakob; Laker, Debora

2015-01-01

Neurons continuously adapt the expression and functionality of their ion channels. For example, exposed to chronic excitotoxicity, neurons homeostatically downscale their intrinsic excitability. In contrast, the “acquired channelopathy” hypothesis suggests that proepileptic channel characteristics develop during epilepsy. We review cell type-specific channel alterations under different epileptic conditions and discuss the potential of channels that undergo homeostatic adaptations, as targets for antiepileptic drugs (AEDs). Most of the relevant studies have been performed on temporal lobe epilepsy (TLE), a widespread AED-refractory, focal epilepsy. The TLE patients, who undergo epilepsy surgery, frequently display hippocampal sclerosis (HS), which is associated with degeneration of cornu ammonis subfield 1 pyramidal cells (CA1 PCs). Although the resected human tissue offers insights, controlled data largely stem from animal models simulating different aspects of TLE and other epilepsies. Most of the cell type-specific information is available for CA1 PCs and dentate gyrus granule cells (DG GCs). Between these two cell types, a dichotomy can be observed: while DG GCs acquire properties decreasing the intrinsic excitability (in TLE models and patients with HS), CA1 PCs develop channel characteristics increasing intrinsic excitability (in TLE models without HS only). However, thorough examination of data on these and other cell types reveals the coexistence of protective and permissive intrinsic plasticity within neurons. These mechanisms appear differentially regulated, depending on the cell type and seizure condition. Interestingly, the same channel molecules that are upregulated in DG GCs during HS-related TLE, appear as promising targets for future AEDs and gene therapies. Hence, GCs provide an example of homeostatic ion channel adaptation which can serve as a primer when designing novel anti-epileptic strategies. PMID:26124723

Insight into Genotype-Phenotype Associations through eQTL Mapping in Multiple Cell Types in Health and Immune-Mediated Disease.

Directory of Open Access Journals (Sweden)

James E Peters

2016-03-01

Full Text Available Genome-wide association studies (GWAS have transformed our understanding of the genetics of complex traits such as autoimmune diseases, but how risk variants contribute to pathogenesis remains largely unknown. Identifying genetic variants that affect gene expression (expression quantitative trait loci, or eQTLs is crucial to addressing this. eQTLs vary between tissues and following in vitro cellular activation, but have not been examined in the context of human inflammatory diseases. We performed eQTL mapping in five primary immune cell types from patients with active inflammatory bowel disease (n = 91, anti-neutrophil cytoplasmic antibody-associated vasculitis (n = 46 and healthy controls (n = 43, revealing eQTLs present only in the context of active inflammatory disease. Moreover, we show that following treatment a proportion of these eQTLs disappear. Through joint analysis of expression data from multiple cell types, we reveal that previous estimates of eQTL immune cell-type specificity are likely to have been exaggerated. Finally, by analysing gene expression data from multiple cell types, we find eQTLs not previously identified by database mining at 34 inflammatory bowel disease-associated loci. In summary, this parallel eQTL analysis in multiple leucocyte subsets from patients with active disease provides new insights into the genetic basis of immune-mediated diseases.

Non-genetic heterogeneity, criticality and cell differentiation.

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Pal, Mainak; Ghosh, Sayantari; Bose, Indrani

2014-11-27

The different cell types in a living organism acquire their identity through the process of cell differentiation in which multipotent progenitor cells differentiate into distinct cell types. Experimental evidence and analysis of large-scale microarray data establish the key role played by a two-gene motif in cell differentiation in a number of cell systems. The two genes express transcription factors which repress each other's expression and autoactivate their own production. A number of theoretical models have recently been proposed based on the two-gene motif to provide a physical understanding of how cell differentiation occurs. In this paper, we study a simple model of cell differentiation which assumes no cooperativity in the regulation of gene expression by the transcription factors. The latter repress each other's activity directly through DNA binding and indirectly through the formation of heterodimers. We specifically investigate how deterministic processes combined with stochasticity contribute in bringing about cell differentiation. The deterministic dynamics of our model give rise to a supercritical pitchfork bifurcation from an undifferentiated stable steady state to two differentiated stable steady states. The stochastic dynamics of our model are studied using the approaches based on the Langevin equations and the linear noise approximation. The simulation results provide a new physical understanding of recent experimental observations. We further propose experimental measurements of quantities like the variance and the lag-1 autocorrelation function in protein fluctuations as the early signatures of an approaching bifurcation point in the cell differentiation process.

Estrogen deficiency heterogeneously affects tissue specific stem cells in mice

Science.gov (United States)

Kitajima, Yuriko; Doi, Hanako; Ono, Yusuke; Urata, Yoshishige; Goto, Shinji; Kitajima, Michio; Miura, Kiyonori; Li, Tao-Sheng; Masuzaki, Hideaki

2015-01-01

Postmenopausal disorders are frequently observed in various organs, but their relationship with estrogen deficiency and mechanisms remain unclear. As tissue-specific stem cells have been found to express estrogen receptors, we examined the hypothesis that estrogen deficiency impairs stem cells, which consequently contributes to postmenopausal disorders. Six-week-old C57BL/6 female mice were ovariectomized, following which they received 17β-estradiol replacement or vehicle (control). Sham-operated mice were used as healthy controls. All mice were killed for evaluation 2 months after treatments. Compared with the healthy control, ovariectomy significantly decreased uterine weight, which was partially recovered by 17β-estradiol replacement. Ovariectomy significantly increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, but impaired their capacity to grow mixed cell-type colonies in vitro. Estrogen replacement further increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, without significantly affecting colony growth in vitro. The number of CD105-positive mesenchymal stem cells in bone marrow also significantly decreased after ovariectomy, but completely recovered following estrogen replacement. Otherwise, neither ovariectomy nor estrogen replacement changed the number of Pax7-positive satellite cells, which are a skeletal muscle-type stem cell. Estrogen deficiency heterogeneously affected tissue-specific stem cells, suggesting a likely and direct relationship with postmenopausal disorders. PMID:26245252

Do post-translational beta cell protein modifications trigger type 1 diabetes?

DEFF Research Database (Denmark)

Størling, Joachim; Overgaard, Anne Julie; Brorsson, Caroline Anna

2013-01-01

beta cell-specific neo-epitopes. We suggest that the current paradigm of type 1 diabetes as a classical autoimmune disease should be reconsidered since the immune response may not be directed against native beta cell proteins. A modified model for the pathogenetic events taking place in islets leading...... diabetes exists in the published literature. Furthermore, we report that cytokines change the expression levels of several genes encoding proteins involved in PTM processes in human islets, and that there are type 1 diabetes-associated polymorphisms in a number of these. In conclusion, data from...... the literature and presented experimental data support the notion that PTM of beta cell proteins may be involved in triggering beta cell destruction in type 1 diabetes. If the beta cell antigens recognised by the immune system foremost come from modified proteins rather than native ones, the concept of type 1...

Analyses of genetic relationships between linear type traits, fat-to-protein ratio, milk production traits, and somatic cell count in first-parity Czech Holstein cows

DEFF Research Database (Denmark)

Zink, V; Zavadilová, L; Lassen, Jan

2014-01-01

. The number of animals for each linear type trait was 59 454, except for locomotion, for which 53 424 animals were recorded. The numbers of animals with records of milk production data were 43 992 for milk yield, fat percentage, protein percentage, and fat-to-protein percentage ratio and 43 978 for fat yield...... and protein yield. In total, 27 098 somatic cell score records were available. The strongest positive genetic correlation between production traits and linear type traits was estimated between udder width and fat yield (0.51 ± 0.04), while the strongest negative correlation estimated was between body......Genetic and phenotypic correlations between production traits, selected linear type traits, and somatic cell score were estimated. The results could be useful for breeding programs involving Czech Holstein dairy cows or other populations. A series of bivariate analyses was applied whereby (co...

Olfactory map formation in the Drosophila brain: genetic specificity and neuronal variability.

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Brochtrup, Anna; Hummel, Thomas

2011-02-01

The development of the Drosophila olfactory system is a striking example of how genetic programs specify a large number of different neuron types and assemble them into functional circuits. To ensure precise odorant perception, each sensory neuron has to not only select a single olfactory receptor (OR) type out of a large genomic repertoire but also segregate its synaptic connections in the brain according to the OR class identity. Specification and patterning of second-order interneurons in the olfactory brain center occur largely independent of sensory input, followed by a precise point-to-point matching of sensory and relay neurons. Here we describe recent progress in the understanding of how cell-intrinsic differentiation programs and context-dependent cellular interactions generate a stereotyped sensory map in the Drosophila brain. Recent findings revealed an astonishing morphological diversity among members of the same interneuron class, suggesting an unexpected variability in local microcircuits involved in insect sensory processing. Copyright © 2010 Elsevier Ltd. All rights reserved.

New frontier in regenerative medicine: site-specific gene correction in patient-specific induced pluripotent stem cells.

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Garate, Zita; Davis, Brian R; Quintana-Bustamante, Oscar; Segovia, Jose C

2013-06-01

Advances in cell and gene therapy are opening up new avenues for regenerative medicine. Because of their acquired pluripotency, human induced pluripotent stem cells (hiPSCs) are a promising source of autologous cells for regenerative medicine. They show unlimited self-renewal while retaining the ability, in principle, to differentiate into any cell type of the human body. Since Yamanaka and colleagues first reported the generation of hiPSCs in 2007, significant efforts have been made to understand the reprogramming process and to generate hiPSCs with potential for clinical use. On the other hand, the development of gene-editing platforms to increase homologous recombination efficiency, namely DNA nucleases (zinc finger nucleases, TAL effector nucleases, and meganucleases), is making the application of locus-specific gene therapy in human cells an achievable goal. The generation of patient-specific hiPSC, together with gene correction by homologous recombination, will potentially allow for their clinical application in the near future. In fact, reports have shown targeted gene correction through DNA-Nucleases in patient-specific hiPSCs. Various technologies have been described to reprogram patient cells and to correct these patient hiPSCs. However, no approach has been clearly more efficient and safer than the others. In addition, there are still significant challenges for the clinical application of these technologies, such as inefficient differentiation protocols, genetic instability resulting from the reprogramming process and hiPSC culture itself, the efficacy and specificity of the engineered DNA nucleases, and the overall homologous recombination efficiency. To summarize advances in the generation of gene corrected patient-specific hiPSCs, this review focuses on the available technological platforms, including their strengths and limitations regarding future therapeutic use of gene-corrected hiPSCs.

Nanoparticles for Site Specific Genome Editing

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McNeer, Nicole Ali

Triplex-forming peptide nucleic acids (PNAs) can be used to coordinate the recombination of short 50-60 by "donor DNA" fragments into genomic DNA, resulting in site-specific correction of genetic mutations or the introduction of advantageous genetic modifications. Site-specific gene editing in hematopoietic stem and progenitor cells (HSPCs) could result in treatment or cure of inherited disorders of the blood such as beta-thalassemia. Gene editing in HSPCs and differentiated T cells could help combat HIV/AIDs by modifying receptors, such as CCR5, necessary for R5-tropic HIV entry. However, translation of genome modification technologies to clinical practice is limited by challenges in intracellular delivery, especially in difficult-to-transfect hematolymphoid cells. In vivo gene editing could also provide novel treatment for systemic monogenic disorders such as cystic fibrosis, an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane receptor. Here, we have engineered biodegradable nanoparticles to deliver oligonucleotides for site-specific genome editing of disease-relevant genes in human cells, with high efficiency, low toxicity, and editing of clinically relevant cell types. We designed nanoparticles to edit the human beta-globin and CCR5 genes in hematopoietic cells. We show that poly(lactic-co-glycolic acid) (PLGA) nanoparticles can delivery PNA and donor DNA for site-specific gene modification in human hematopoietic cells in vitro and in vivo in NOD-scid IL2rgammanull mice. Nanoparticles delivered by tail vein localized to hematopoietic compartments in the spleen and bone marrow of humanized mice, resulting in modification of the beta-globin and CCR5 genes. Modification frequencies ranged from 0.005 to 20% of cells depending on the organ and cell type, without detectable toxicity. This project developed highly versatile methods for delivery of therapeutics to hematolymphoid cells and hematopoietic stem cells, and will help to

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

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Martell, Jeffrey D; Deerinck, Thomas J; Lam, Stephanie S; Ellisman, Mark H; Ting, Alice Y

2018-01-01

Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3′-diaminobenzidine (DAB) and hydrogen peroxide (H2O2). PMID:28796234

Ethical and clinical practice considerations for genetic counselors related to direct-to-consumer marketing of genetic tests.

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Wade, Christopher H; Wilfond, Benjamin S

2006-11-15

Several companies utilize direct-to-consumer (DTC) advertising for genetic tests and some, but not all, bypass clinician involvement by offering DTC purchase of the tests. This article examines how DTC marketing strategies may affect genetic counselors, using available cardiovascular disease susceptibility tests as an illustration. The interpretation of these tests is complex and includes consideration of clinical validity and utility, and the further complications of gene-environment interactions and pleiotropy. Although it is unclear to what extent genetic counselors will encounter clients who have been exposed to DTC marketing strategies, these strategies may influence genetic counseling interactions if they produce directed interest in specific tests and unrealistic expectations for the tests' capacity to predict disease. Often, a client's concern about risk for cardiovascular diseases is best addressed by established clinical tests and a family history assessment. Ethical dilemmas may arise for genetic counselors who consider whether to accept clients who request test interpretation or to order DTC-advertised tests that require a clinician's authorization. Genetic counselors' obligations to care for clients extend to interpreting DTC tests, although this obligation may be fulfilled by referral or consultation with specialists. Genetic counselors do not have an obligation to order DTC-advertised tests that have minimal clinical validity and utility at a client's request. This can be a justified restriction on autonomy based on consideration of risks to the client, the costs, and the implications for society. Published 2006 Wiley-Liss, Inc.

Estimating the actual subject-specific genetic correlations in behavior genetics.

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Molenaar, Peter C M

2012-10-01

Generalization of the standard behavior longitudinal genetic factor model for the analysis of interindividual phenotypic variation to a genetic state space model for the analysis of intraindividual variation enables the possibility to estimate subject-specific heritabilities.

Will a mAb-Based Immunotherapy Directed against Cancer Stem Cells Be Feasible?

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Silvia Santamaria

2017-11-01

Full Text Available The cancer stem cell (CSC hypothesis suggests that within a tumor, there is a small subpopulation of cells with stem cell properties responsible for tumor maintenance and metastasis generation. This hypothesis also implies that new antitumor drugs, rather than targeting the bulk of the tumor mass, would be more effective if they directly targeted the CSC subpopulation. The CSCs from several types of tumors have been identified with mAbs recognizing surface antigens in these cells; however, antigens specifically or exclusively expressed in the CSC population have not yet been identified. Thus, questioning the possibility of using therapeutic antibodies directed against the CSCs. Here, we review the possibilities of using antibodies directly targeting the CSCs as therapeutic agents in the form of naked antibodies, antibodies conjugated to nanoparticles, or antibody cocktails.

Computational neuroanatomy: mapping cell-type densities in the mouse brain, simulations from the Allen Brain Atlas

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Grange, Pascal

2015-09-01

The Allen Brain Atlas of the adult mouse (ABA) consists of digitized expression profiles of thousands of genes in the mouse brain, co-registered to a common three-dimensional template (the Allen Reference Atlas).This brain-wide, genome-wide data set has triggered a renaissance in neuroanatomy. Its voxelized version (with cubic voxels of side 200 microns) is available for desktop computation in MATLAB. On the other hand, brain cells exhibit a great phenotypic diversity (in terms of size, shape and electrophysiological activity), which has inspired the names of some well-studied cell types, such as granule cells and medium spiny neurons. However, no exhaustive taxonomy of brain cell is available. A genetic classification of brain cells is being undertaken, and some cell types have been chraracterized by their transcriptome profiles. However, given a cell type characterized by its transcriptome, it is not clear where else in the brain similar cells can be found. The ABA can been used to solve this region-specificity problem in a data-driven way: rewriting the brain-wide expression profiles of all genes in the atlas as a sum of cell-type-specific transcriptome profiles is equivalent to solving a quadratic optimization problem at each voxel in the brain. However, the estimated brain-wide densities of 64 cell types published recently were based on one series of co-registered coronal in situ hybridization (ISH) images per gene, whereas the online ABA contains several image series per gene, including sagittal ones. In the presented work, we simulate the variability of cell-type densities in a Monte Carlo way by repeatedly drawing a random image series for each gene and solving the optimization problem. This yields error bars on the region-specificity of cell types.

The Genetic Architecture of Type 1 Diabetes

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Samuel T Jerram

2017-08-01

Full Text Available Type 1 diabetes (T1D is classically characterised by the clinical need for insulin, the presence of disease-associated serum autoantibodies, and an onset in childhood. The disease, as with other autoimmune diseases, is due to the interaction of genetic and non-genetic effects, which induce a destructive process damaging insulin-secreting cells. In this review, we focus on the nature of this interaction, and how our understanding of that gene–environment interaction has changed our understanding of the nature of the disease. We discuss the early onset of the disease, the development of distinct immunogenotypes, and the declining heritability with increasing age at diagnosis. Whilst Human Leukocyte Antigens (HLA have a major role in causing T1D, we note that some of these HLA genes have a protective role, especially in children, whilst other non-HLA genes are also important. In adult-onset T1D, the disease is often not insulin-dependent at diagnosis, and has a dissimilar immunogenotype with reduced genetic predisposition. Finally, we discuss the putative nature of the non-genetic factors and how they might interact with genetic susceptibility, including preliminary studies of the epigenome associated with T1D.

Introduction of Exogenous HSV-TK Suicide Gene Increases Safety of Keratinocyte-Derived Induced Pluripotent Stem Cells by Providing Genetic "Emergency Exit" Switch.

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Sułkowski, Maciej; Konieczny, Paweł; Chlebanowska, Paula; Majka, Marcin

2018-01-09

Since their invention in 2006, induced Pluripotent Stem (iPS) cells remain a great promise for regenerative medicine circumventing the ethical issues linked to Embryonic Stem (ES) cell research. iPS cells can be generated in a patient-specific manner as an unlimited source of various cell types for in vitro drug screening, developmental biology studies and regenerative use. Having the capacity of differentiating into the cells of all three primary germ layers, iPS cells have high potential to form teratoma tumors. This remains their main disadvantage and hazard which, until resolved, prevents utilization of iPS cells in clinic. Here, we present an approach for increasing iPS cells safety by introducing genetic modification-exogenous suicide gene Herpes Simplex Virus Thymidine Kinase ( HSV-TK ). Its expression results in specific vulnerability of genetically modified cells to prodrug-ganciclovir (GCV). We show that HSV-TK expressing cells can be eradicated both in vitro and in vivo with high specificity and efficiency with low doses of GCV. Described strategy increases iPS cells safety for future clinical applications by generating "emergency exit" switch allowing eradication of transplanted cells in case of their malfunction.

Comparative radiation genetics. What we learnt from our studies on Medaka germ cell mutagenesis

International Nuclear Information System (INIS)

Shima, Akihiro

2004-01-01

Having been interested in studying germ cell mutagenesis from the biodiversity viewpoint, in 1985 we started developing a nonmammalian specific-locus test (SLT) system using the Medaka, Oryzias latipes. The tester strain with five marker loci, which is a prerequisite for SLT, was established by consecutive crossings of five spontaneous single mutants followed by selection based on the phenotype of each mutant. The genetic endpoints available were dominant lethal mutations (DLM), total specific-locus mutations (TSLM) and viable specific-locus mutations. Using γ-rays, ethylnitrosourea and Fe-ion beam as mutagens to which wild type males or females were exposed, we screened approx. 1.6 million F1 embryos that correspond to approx. 4.7 million loci. In an attempt to best express the comparative sensitivity of Medaka germ cells to the genetic effects of γ-rays, the gametic doubling doses for acute and high-dose γ-rays were estimated. Extensive sex differences within the wild type (HNI) strain as well as strain differences in male germ cells between the two wild type strains (HNI and Sakura) were notably found in doubling doses for DLM and TSLM. Interestingly, among these values, the doubling dose for TSLM in spermatogonia of the HNI strain (0.33 Gy) nearly coincided with that estimated from the Russell 7-locus system of mice (0.44 Gy). Our data also suggested that the initial genomic changes induced in male germ cells would not straightforwardly manifest themselves as phenotypic effects in F1 progeny, but that twofold checks, one a prefertilization check in the gonads against genomic alterations using DNA repair machinery as well as apoptotic response, and the other postfertilization check in developing embryos through dominant lethal effects. should operate to restore or ameliorate those genomic changes. More mechanistically, AP/PCR-RAPD DNA fingerprinting was employed in order to scan as wider regions of the zygotic genome as possible. These anonymous DNA markers

Usefulness of microsatellite typing in population genetic studies of Trypanosoma cruzi

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Macedo Andrea M

2001-01-01

Full Text Available Through microsatellite analysis of 53 monoclonal populations of Trypanosoma cruzi, we found a remarkable degree of genetic polymorphism with no single multilocus genotype being observed more than once. The microsatellite profile proved to be stable during 70 generations of the CL Brener clone in culture. The microsatellite profiling presented also high diagnostic sensitivity since DNA amplifications could be achieved with less than 100 fg DNA, corresponding to half parasite total DNA content. Based on these technical attributes the microsatellite assay turns out to be an important tool for direct typing T. cruzi in biological samples. By using this approach we were able to type T. cruzi in feces of artificially infected bugs and in single cells sorted by FACS. The microsatellites have shown to be excellent markers for T. cruzi phylogenetic reconstruction. We used maximum parsimony based on the minimum number of mutational steps to build an unrooted Wagner network, which confirms previous conclusions based on the analysis of the D7 domain of the LSU rDNA gene that T. cruzi is composed by two major groups. We also obtained evidence that strains belonging to rRNA group 2 are subdivided into two genetically distant clusters, and that one of these clusters is more related to rRNA group 1/2. These results suggest different origins for these strains.

Direct PCR amplification of the HVSI region in mitochondrial DNA from buccal cell swabs

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Kovačević-Grujičić Nataša

2012-01-01

Full Text Available Amplification of human mitochondrial DNA (mtDNA has been widely used in population genetics, human evolutionary and molecular anthropology studies. mtDNA hypervariable segments I and II (HVSI and HVSII were shown to be a suitable tool in genetic analyses due to the unique properties of mtDNA, such as the lack of recombination, maternal mode of inheritance, rapid evolutionary rate and high population-specific polymorphisms. Here we present a rapid and low-cost method for direct PCR amplification of a 330 bp fragment of HVSI from buccal cell samples. Avoiding the DNA isolation step makes this method appropriate for the analysis of a large number of samples in a short period of time. Since the transportation of samples and fieldwork conditions can affect the quality of samples and subsequent DNA analysis, we tested the effects of long-term storage of buccal cell swabs on the suitability of such samples for direct PCR amplification. We efficiently amplified a 330 bp fragment of HVSI even after the long-term storage of buccal cells at room temperature, +4°C or at -20°C, for up to eight months. All examined PCR products were successfully sequenced, regardless of sample storage time and conditions. Our results suggest that the direct PCR amplification of the HVSI region from buccal cells is a method well suited for large-scale mtDNA population studies.[Acknowledgments. This work was supported by the Ministry of Education and Science of the Republic of Serbia (Grant no. III 47025.

Genetic specificity of face recognition.

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Shakeshaft, Nicholas G; Plomin, Robert

2015-10-13

Specific cognitive abilities in diverse domains are typically found to be highly heritable and substantially correlated with general cognitive ability (g), both phenotypically and genetically. Recent twin studies have found the ability to memorize and recognize faces to be an exception, being similarly heritable but phenotypically substantially uncorrelated both with g and with general object recognition. However, the genetic relationships between face recognition and other abilities (the extent to which they share a common genetic etiology) cannot be determined from phenotypic associations. In this, to our knowledge, first study of the genetic associations between face recognition and other domains, 2,000 18- and 19-year-old United Kingdom twins completed tests assessing their face recognition, object recognition, and general cognitive abilities. Results confirmed the substantial heritability of face recognition (61%), and multivariate genetic analyses found that most of this genetic influence is unique and not shared with other cognitive abilities.

Cardiovascular Disease Modeling Using Patient-Specific Induced Pluripotent Stem Cells

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Atsushi Tanaka

2015-08-01

Full Text Available The generation of induced pluripotent stem cells (iPSCs has opened up a new scientific frontier in medicine. This technology has made it possible to obtain pluripotent stem cells from individuals with genetic disorders. Because iPSCs carry the identical genetic anomalies related to those disorders, iPSCs are an ideal platform for medical research. The pathophysiological cellular phenotypes of genetically heritable heart diseases such as arrhythmias and cardiomyopathies, have been modeled on cell culture dishes using disease-specific iPSC-derived cardiomyocytes. These model systems can potentially provide new insights into disease mechanisms and drug discoveries. This review focuses on recent progress in cardiovascular disease modeling using iPSCs, and discusses problems and future perspectives concerning their use.

What Is Direct-to-Consumer Genetic Testing?

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... consumer genetic testing. Additional information about direct-to-consumer marketing of genetic tests and related research questions are ... for Links Data Files & API Site Map Subscribe Customer Support USA.gov Copyright Privacy Accessibility FOIA Viewers & ...

Alpha1a-Adrenoceptor Genetic Variant Triggers Vascular Smooth Muscle Cell Hyperproliferation and Agonist Induced Hypertrophy via EGFR Transactivation Pathway.

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Irina Gradinaru

Full Text Available α1a Adrenergic receptors (α1aARs are the predominant AR subtype in human vascular smooth muscle cells (SMCs. α1aARs in resistance vessels are crucial in the control of blood pressure, yet the impact of naturally occurring human α1aAR genetic variants in cardiovascular disorders remains poorly understood. To this end, we present novel findings demonstrating that 3D cultures of vascular SMCs expressing human α1aAR-247R (247R genetic variant demonstrate significantly increased SMC contractility compared with cells expressing the α1aAR-WT (WT receptor. Stable expression of 247R genetic variant also triggers MMP/EGFR-transactivation dependent serum- and agonist-independent (constitutive hyperproliferation and agonist-dependent hypertrophy of SMCs. Agonist stimulation reduces contractility Using pathway-specific inhibitors we determined that the observed hyperproliferation of 247R-expressing cells is triggered via β-arrestin1/Src/MMP-2/EGFR/ERK-dependent mechanism. MMP-2-specific siRNA inhibited 247R-triggered hyperproliferation indicating MMP-2 involvement in 247R-triggered hyperproliferation in SMCs. β-arrestin1-specific shRNA also inhibited 247R-triggered hyperproliferation but did not affect hypertrophy in 247R-expressing SMCs, indicating that agonist-dependent hypertrophy is independent of β-arrestin1. Our data reveal that in different cardiovascular cells the same human receptor genetic variant can activate alternative modulators of the same signaling pathway. Thus, our findings in SMCs demonstrate that depending on the type of cells expressing the same receptor (or receptor variant, different target-specific inhibitors could be used to modulate aberrant hyperproliferative or hypertrophic pathways in order to restore normal phenotype.

Transsynaptic transport of wheat germ agglutinin expressed in a subset of type II taste cells of transgenic mice

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Mosinger Bedrich

2008-10-01

Full Text Available Abstract Background Anatomical tracing of neural circuits originating from specific subsets of taste receptor cells may shed light on interactions between taste cells within the taste bud and taste cell-to nerve interactions. It is unclear for example, if activation of type II cells leads to direct activation of the gustatory nerves, or whether the information is relayed through type III cells. To determine how WGA produced in T1r3-expressing taste cells is transported into gustatory neurons, transgenic mice expressing WGA-IRES-GFP driven by the T1r3 promoter were generated. Results Immunohistochemistry showed co-expression of WGA, GFP and endogenous T1r3 in the taste bud cells of transgenic mice: the only taste cells immunoreactive for WGA were the T1r3-expressing cells. The WGA antibody also stained intragemmal nerves. WGA, but not GFP immunoreactivity was found in the geniculate and petrosal ganglia of transgenic mice, indicating that WGA was transported across synapses. WGA immunoreactivity was also found in the trigeminal ganglion, suggesting that T1r3-expressing cells make synapses with trigeminal neurons. In the medulla, WGA was detected in the nucleus of the solitary tract but also in the nucleus ambiguus, the vestibular nucleus, the trigeminal nucleus and in the gigantocellular reticular nucleus. WGA was not detected in the parabrachial nucleus, or the gustatory cortex. Conclusion These results show the usefulness of genetically encoded WGA as a tracer for the first and second order neurons that innervate a subset of taste cells, but not for higher order neurons, and demonstrate that the main route of output from type II taste cells is the gustatory neuron, not the type III cells.

Different role of zinc transporter 8 between type 1 diabetes mellitus and type 2 diabetes mellitus.

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Yi, Bo; Huang, Gan; Zhou, Zhiguang

2016-07-01

Diabetes can be simply classified into type 1 diabetes mellitus and type 2 diabetes mellitus. Zinc transporter 8 (ZnT8), a novel islet autoantigen, is specifically expressed in insulin-containing secretory granules of β-cells. Genetic studies show that the genotypes of SLC30A8 can determine either protective or diabetogenic response depending on environmental and lifestyle factors. The ZnT8 protein expression, as well as zinc content in β-cells, was decreased in diabetic mice. Thus, ZnT8 might participate in insulin biosynthesis and release, and subsequently involved deteriorated β-cell function through direct or indirect mechanisms in type 1 diabetes mellitus and type 2 diabetes mellitus. From a clinical feature standpoint, the prevalence of ZnT8A is gradiently increased in type 2 diabetes mellitus, latent autoimmune diabetes in adults and type 1 diabetes mellitus. The frequency and epitopes of ZnT8-specific T cells and cytokine release by ZnT8-specific T cells are also different in diabetic patients and healthy controls. Additionally, the response to ZnT8 administration is also different in type 1 diabetes mellitus and type 2 diabetes mellitus. In the present review, we summarize the literature about clinical aspects of ZnT8 in the pathogenesis of diabetes, and suggest that ZnT8 might play a different role between type 1 diabetes mellitus and type 2 diabetes mellitus. © 2015 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Introduction of Exogenous HSV-TK Suicide Gene Increases Safety of Keratinocyte-Derived Induced Pluripotent Stem Cells by Providing Genetic “Emergency Exit” Switch

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Maciej Sułkowski

2018-01-01

Full Text Available Since their invention in 2006, induced Pluripotent Stem (iPS cells remain a great promise for regenerative medicine circumventing the ethical issues linked to Embryonic Stem (ES cell research. iPS cells can be generated in a patient-specific manner as an unlimited source of various cell types for in vitro drug screening, developmental biology studies and regenerative use. Having the capacity of differentiating into the cells of all three primary germ layers, iPS cells have high potential to form teratoma tumors. This remains their main disadvantage and hazard which, until resolved, prevents utilization of iPS cells in clinic. Here, we present an approach for increasing iPS cells safety by introducing genetic modification—exogenous suicide gene Herpes Simplex Virus Thymidine Kinase (HSV-TK. Its expression results in specific vulnerability of genetically modified cells to prodrug—ganciclovir (GCV. We show that HSV-TK expressing cells can be eradicated both in vitro and in vivo with high specificity and efficiency with low doses of GCV. Described strategy increases iPS cells safety for future clinical applications by generating “emergency exit” switch allowing eradication of transplanted cells in case of their malfunction.

Tracking by flow cytometry antigen-specific follicular helper T cells in wild-type animals after protein vaccination.

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Chakarov, Svetoslav; Fazilleau, Nicolas

2015-01-01

Flow cytometry is a valuable technology used in immunology to characterize and enumerate the different cell subpopulations specific for a nonself-antigen in the context of an ongoing immune response. Among them, follicular helper T cells are the cognate regulators of B cells in secondary lymphoid tissues. Thus, tracking them is of high interest especially in the context of protein vaccination. For this purpose, transgenic antigen-receptor mouse models have been largely used. It is now clear that transgenic models are not always the best means to study the dynamics of the immune response since they can modify the response. In this chapter, we describe how to track endogenous antigen-specific follicular helper T cells by flow cytometry after protein vaccination in nonmodified wild-type animals, which ultimately provides a comprehensive way to enumerate, characterize, and isolate these particular cells in vivo.

Supernatural T cells: genetic modification of T cells for cancer therapy.

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Kershaw, Michael H; Teng, Michele W L; Smyth, Mark J; Darcy, Phillip K

2005-12-01

Immunotherapy is receiving much attention as a means of treating cancer, but complete, durable responses remain rare for most malignancies. The natural immune system seems to have limitations and deficiencies that might affect its ability to control malignant disease. An alternative to relying on endogenous components in the immune repertoire is to generate lymphocytes with abilities that are greater than those of natural T cells, through genetic modification to produce 'supernatural' T cells. This Review describes how such T cells can circumvent many of the barriers that are inherent in the tumour microenvironment while optimizing T-cell specificity, activation, homing and antitumour function.

Genetic Risk Score Modelling for Disease Progression in New-Onset Type 1 Diabetes Patients

DEFF Research Database (Denmark)

Brorsson, Caroline A; Nielsen, Lotte B; Andersen, Marie-Louise

2016-01-01

Genome-wide association studies (GWAS) have identified over 40 type 1 diabetes risk loci. The clinical impact of these loci on β-cell function during disease progression is unknown. We aimed at testing whether a genetic risk score could predict glycemic control and residual β-cell function in type...... 1 diabetes (T1D). As gene expression may represent an intermediate phenotype between genetic variation and disease, we hypothesized that genes within T1D loci which are expressed in islets and transcriptionally regulated by proinflammatory cytokines would be the best predictors of disease...... constructed a genetic risk score based on the cumulative number of risk alleles carried in children with newly diagnosed T1D. With each additional risk allele carried, HbA1c levels increased significantly within first year after diagnosis. Network and gene ontology (GO) analyses revealed that several...

Genet-specific spawning patterns in Acropora palmata

Science.gov (United States)

Miller, M. W.; Williams, D. E.; Fisch, J.

2016-12-01

The broadcast spawning elkhorn coral, Acropora palmata, requires outcrossing among different genets for effective fertilization. Hence, a low density of genets in parts of its range emphasizes the need for precise synchrony among neighboring genets as sperm concentration dilutes rapidly in open-ocean conditions. We documented the genet-specific nightly occurrence of spawning of A. palmata over 8 yr in a depauperate population in the Florida Keys to better understand this potential reproductive hurdle. The observed population failed to spawn within the predicted monthly window (nights 2-6 after the full moon in August) in three of the 8 yr of observation; negligible spawning was observed in a fourth year. Moreover, genet-specific patterns are evident in that (1) certain genets have significantly greater odds of spawning overall and (2) certain genets predictably spawn on the earlier and others on the later lunar nights within the predicted window. Given the already low genet density in this population, this pattern implies a substantial degree of wasted reproductive effort and supports the hypothesis that depensatory factors are impairing recovery in this species.

Prospect of Induced Pluripotent Stem Cell Genetic Repair to Cure Genetic Diseases

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Jeanne Adiwinata Pawitan

2012-01-01

Full Text Available In genetic diseases, where the cells are already damaged, the damaged cells can be replaced by new normal cells, which can be differentiated from iPSC. To avoid immune rejection, iPSC from the patient’s own cell can be developed. However, iPSC from the patients’s cell harbors the same genetic aberration. Therefore, before differentiating the iPSCs into required cells, genetic repair should be done. This review discusses the various technologies to repair the genetic aberration in patient-derived iPSC, or to prevent the genetic aberration to cause further damage in the iPSC-derived cells, such as Zn finger and TALE nuclease genetic editing, RNA interference technology, exon skipping, and gene transfer method. In addition, the challenges in using the iPSC and the strategies to manage the hurdles are addressed.

Genetics and sport performance: current challenges and directions to the future

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João Paulo Limongi França GUILHERME

2014-03-01

Full Text Available In recent years there has been a great progress in molecular biology techniques, which has facilitated the researches on influence of genetics on human performance. There are specific regions of DNA that can vary between individuals. Such variations (i.e., polymorphisms may, in part, explain why some individuals have differentiated responses to certain stimuli, including the responses to sports training. In a particular sport, the presence of specific polymorphisms may contribute to high levels of performance. Since 1998, several polymorphisms have been associated with athletic phenotypes; however the accumulation of information generated over these 15 years shows that the influence of genetics to sport is extremely complex. In this review, we will summarise the current status of the field, discussing the implications of available knowledge for the practice of professionals involved with the sport and suggesting future directions for research. We also discuss topics related to the importance of polygenic profile characterization of athletes, methods for the identification of new polymorphisms associated with physical performance, the use of genetic testing for predicting competitive success, and how crucial is the genetic profile for the success athletes in competition.

Cell-type independent MYC target genes reveal a primordial signature involved in biomass accumulation.

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Hongkai Ji

Full Text Available The functions of key oncogenic transcription factors independent of context have not been fully delineated despite our richer understanding of the genetic alterations in human cancers. The MYC oncogene, which produces the Myc transcription factor, is frequently altered in human cancer and is a major regulatory hub for many cancers. In this regard, we sought to unravel the primordial signature of Myc function by using high-throughput genomic approaches to identify the cell-type independent core Myc target gene signature. Using a model of human B lymphoma cells bearing inducible MYC, we identified a stringent set of direct Myc target genes via chromatin immunoprecipitation (ChIP, global nuclear run-on assay, and changes in mRNA levels. We also identified direct Myc targets in human embryonic stem cells (ESCs. We further document that a Myc core signature (MCS set of target genes is shared in mouse and human ESCs as well as in four other human cancer cell types. Remarkably, the expression of the MCS correlates with MYC expression in a cell-type independent manner across 8,129 microarray samples, which include 312 cell and tissue types. Furthermore, the expression of the MCS is elevated in vivo in Eμ-Myc transgenic murine lymphoma cells as compared with premalignant or normal B lymphocytes. Expression of the MCS in human B cell lymphomas, acute leukemia, lung cancers or Ewing sarcomas has the highest correlation with MYC expression. Annotation of this gene signature reveals Myc's primordial function in RNA processing, ribosome biogenesis and biomass accumulation as its key roles in cancer and stem cells.

Adult-type myogenesis of the frog Xenopus laevis specifically suppressed by notochord cells but promoted by spinal cord cells in vitro.

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Yamane, Hitomi; Ihara, Setsunosuke; Kuroda, Masaaki; Nishikawa, Akio

2011-08-01

Larval-to-adult myogenic conversion occurs in the dorsal muscle but not in the tail muscle during Xenopus laevis metamorphosis. To know the mechanism for tail-specific suppression of adult myogenesis, response character was compared between adult myogenic cells (Ad-cells) and larval tail myogenic cells (La-cells) to a Sonic hedgehog (Shh) inhibitor, notochord (Nc) cells, and spinal cord (SC) cells in vitro. Cyclopamine, an Shh inhibitor, suppressed the differentiation of cultured Ad (but not La) cells, suggesting the significance of Shh signaling in promoting adult myogenesis. To test the possibility that Shh-producing axial elements (notochord and spinal cord) regulate adult myogenesis, Ad-cells or La-cells were co-cultured with Nc or SC cells. The results showed that differentiation of Ad-cells were strongly inhibited by Nc cells but promoted by SC cells. If Ad-cells were "separately" co-cultured with Nc cells without direct cell-cell interactions, adult differentiation was not inhibited but rather promoted, suggesting that Nc cells have two roles, one is a short-range suppression and another is a long-range promotion for adult myogenesis. Immunohistochemical analysis showed both notochord and spinal cord express the N-terminal Shh fragment throughout metamorphosis. The "spinal cord-promotion" and long-range effect by Nc cells on adult myogenesis is thus involved in Shh signaling, while the signaling concerning the short-range "Nc suppression" will be determined by future studies. Interestingly, these effects, "Nc suppression" and "SC promotion" were not observed for La-cells. Situation where the spinal cord/notochord cross-sectional ratio is quite larger in tadpole trunk than in the tail seems to contribute to trunk-specific promotion and tail-specific suppression of adult myogenesis during Xenopus metamorphosis.

iPSC-Derived Regulatory Dendritic Cells Inhibit Allograft Rejection by Generating Alloantigen-Specific Regulatory T Cells

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Songjie Cai

2017-05-01

Full Text Available Regulatory dendritic cell (DCregs-based immunotherapy is a potential therapeutic tool for transplant rejection. We generated DCregs from murine induced pluripotent stem cells (iPSCs, which could remain in a “stable immature stage” even under strong stimulation. Harnessing this characteristic, we hypothesized that iPS-DCregs worked as a negative vaccine to generate regulatory T cells (Tregs, and induced donor-specific allograft acceptance. We immunized naive CBA (H-2Kk mice with B6 (H-2Kb iPS-DCregs and found that Tregs (CD4+CD25+FOXP3+ significantly increased in CBA splenocytes. Moreover, immunized CBA recipients permanently accepted B6 cardiac grafts in a donor-specific pattern. We demonstrated mechanistically that donor-type iPS-DCregs triggered transforming growth factor β1 secretion, under which the donor-antigen peptides directed naive CD4+ T cells to differentiate into donor-specific FOXP3+ Tregs instead of into effector T cells in vivo. These findings highlight the potential of iPS-DCregs as a key cell therapy resource in clinical transplantation.

Genetic engineering with T cell receptors.

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Zhang, Ling; Morgan, Richard A

2012-06-01

In the past two decades, human gene transfer research has been translated from a laboratory technology to clinical evaluation. The success of adoptive transfer of tumor-reactive lymphocytes to treat the patients with metastatic melanoma has led to new strategies to redirect normal T cells to recognize tumor antigens by genetic engineering with tumor antigen-specific T cell receptor (TCR) genes. This new strategy can generate large numbers of defined antigen-specific cells for therapeutic application. Much progress has been made to TCR gene transfer systems by optimizing gene expression and gene transfer protocols. Vector and protein modifications have enabled excellent expression of introduced TCR chains in human lymphocytes with reduced mis-pairing between the introduced and endogenous TCR chains. Initial clinical studies have demonstrated that TCR gene-engineered T cells could mediate tumor regression in vivo. In this review, we discuss the progress and prospects of TCR gene-engineered T cells as a therapeutic strategy for treating patients with melanoma and other cancers. Published by Elsevier B.V.

Characterizing the genetic risk for Type 2 diabetes in a Malaysian multi-ethnic cohort.

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Abdullah, N; Abdul Murad, N A; Attia, J; Oldmeadow, C; Mohd Haniff, E A; Syafruddin, S E; Abd Jalal, N; Ismail, N; Ishak, M; Jamal, R; Scott, R J; Holliday, E G

2015-10-01

To characterize the association with Type 2 diabetes of known Type 2 diabetes risk variants in people in Malaysia of Malay, Chinese and Indian ancestry who participated in the Malaysian Cohort project. We genotyped 1604 people of Malay ancestry (722 cases, 882 controls), 1654 of Chinese ancestry (819 cases, 835 controls) and 1728 of Indian ancestry (851 cases, 877 controls). First, 62 candidate single-nucleotide polymorphisms previously associated with Type 2 diabetes were assessed for association via logistic regression within ancestral groups and then across ancestral groups using a meta-analysis. Second, estimated odds ratios were assessed for excess directional concordance with previously studied populations. Third, a genetic risk score aggregating allele dosage across the candidate single-nucleotide polymorphisms was tested for association within and across ancestral groups. After Bonferroni correction, seven individual single-nucleotide polymorphisms were associated with Type 2 diabetes in the combined Malaysian sample. We observed a highly significant excess in concordance of effect directions between Malaysian and previously studied populations. The genetic risk score was strongly associated with Type 2 diabetes in all Malaysian groups, explaining from 1.0 to 1.7% of total Type 2 diabetes risk variance. This study suggests there is substantial overlap of the genetic risk alleles underlying Type 2 diabetes in Malaysian and other populations. © 2015 The Authors. Diabetic Medicine © 2015 Diabetes UK.

Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells.

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Jaclyn C Scott

2010-10-01

Full Text Available The exogenous RNA interference (RNAi pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (siRNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2 cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

Tissue-specific expression of type IX collagen

International Nuclear Information System (INIS)

Nishimura, I.; Muragaki, Y.; Ninomiya, Y.; Olsen, B.R.; Hayashi, M.

1990-01-01

This paper reports on the tissue-specific expression of type IX collagen, a major component of cartilage fibrils. It contains molecules with three genetically distinct subunits. The subunits form three triple-helical (CO) domains separated by non-triple-helical (NC) sequences. One of the subunits in cartilage, α1(IX), contains a large amino-terminal globular domain, NC4, while a second subunit, α2(IX), contains a covalently attached chondroitin sulfate chain. The site of attachment for this chain is located within the non-triple-helical sequence NC3, which separates the amino-terminal and central triple-helical domains of the type IX molecules. The NC3 region is 5 amino acid residues longer in the α2(IX) chain than in the α1(IX) and α3(IX) chains. This may explain why type IX molecules tend to show a sharp angle in the NC3 region, and why monoclonal antibody molecules that are specific for the stub left after chondroitinase ABC digestion of the chondroitin sulfate side chain always are located on the outside of the angle

Direct-to-consumer sales of genetic services on the Internet.

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Gollust, Sarah E; Wilfond, Benjamin S; Hull, Sara Chandros

2003-01-01

PURPOSE The increasing use of the Internet to obtain genetics information and to order medical services without a prescription, combined with a rise in direct-to-consumer marketing for genetic testing, suggests the potential for the Internet to be used to sell genetic services. METHODS A systematic World Wide Web search was conducted in May 2002 to assess the availability of genetic services sold directly to consumers on the Internet. RESULTS Out of 105 sites that offered genetic services directly, most offered non-health-related services, including parentage confirmation testing (83%), identity testing (56%), and DNA banking (24%); however, health-related genetic tests were offered through 14 sites (13%). The health-related genetic tests available ranged from standard tests, such as hemochromatosis and cystic fibrosis, to more unconventional tests related to nutrition, behavior, and aging. Of these 14 sites, 5 described risks associated with the genetic services and 6 described the availability of counseling. CONCLUSIONS The availability of direct sales of health-related genetic tests creates the potential for inadequate pretest decision making, misunderstanding test results, and access to tests of questionable clinical value.

Genetic modification of stem cells for improved therapy of the infarcted myocardium.

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Haider, Husnain Kh; Mustafa, Anique; Feng, Yuliang; Ashraf, Muhammad

2011-10-03

The conventional treatment modalities for ischemic heart disease only provide symptomatic relief to the patient without repairing and regenerating the damaged myocardium. Stem cell transplantation has emerged as a promising alternative therapeutic approach for cardiovascular diseases. Stem cells possess the potential of differentiation to adopt morphofunctional cardiac and vasculogenic phenotypes to repopulate the scar tissue and restore regional blood flow in the ischemic myocardium. These beneficial therapeutic effects make stem cell transplantation the method of choice for the treatment of ischemic heart disease. The efficacy of stem cell transplantation may be augmented by genetic manipulation of the cells prior to transplantation. Not only will insertion of therapeutic transgene(s) into the stem cells support the survival and differentiation of cells in the unfavorable microenvironment of the ischemic myocardium, but also the genetically manipulated stem cells will serve as a source of the transgene expression product in the heart for therapeutic benefits. We provide an overview of the extensively studied stem cell types for cardiac regeneration, the various methods in which these cells have been genetically manipulated and rationale of genetic modification of stem cells for use in regenerative cardiovascular therapeutics.

Mesenchymal Stem Cells Sense Three Dimensional Type I Collagen through Discoidin Domain Receptor 1.

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Lund, A W; Stegemann, J P; Plopper, G E

2009-01-01

The extracellular matrix provides structural and organizational cues for tissue development and defines and maintains cellular phenotype during cell fate determination. Multipotent mesenchymal stem cells use this matrix to tightly regulate the balance between their differentiation potential and self-renewal in the native niche. When understood, the mechanisms that govern cell-matrix crosstalk during differentiation will allow for efficient engineering of natural and synthetic matrices to specifically direct and maintain stem cell phenotype. This work identifies the discoidin domain receptor 1 (DDR1), a collagen activated receptor tyrosine kinase, as a potential link through which stem cells sense and respond to the 3D organization of their extracellular matrix microenvironment. DDR1 is dependent upon both the structure and proteolytic state of its collagen ligand and is specifically expressed and localized in three dimensional type I collagen culture. Inhibition of DDR1 expression results in decreased osteogenic potential, increased cell spreading, stress fiber formation and ERK1/2 phosphorylation. Additionally, loss of DDR1 activity alters the cell-mediated organization of the naïve type I collagen matrix. Taken together, these results demonstrate a role for DDR1 in the stem cell response to and interaction with three dimensional type I collagen. Dynamic changes in cell shape in 3D culture and the tuning of the local ECM microstructure, directs crosstalk between DDR1 and two dimensional mechanisms of osteogenesis that can alter their traditional roles.

Stem cell monitoring with a direct or indirect labeling method

Energy Technology Data Exchange (ETDEWEB)

Kim, Min Hwan; Lee, Yong Jin [Molecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences (KIRAMS), Seoul (Korea, Republic of)

2016-12-15

The molecular imaging techniques allow monitoring of the transplanted cells in the same individuals over time, from early localization to the survival, migration, and differentiation. Generally, there are two methods of stem cell labeling: direct and indirect labeling methods. The direct labeling method introduces a labeling agent into the cell, which is stably incorporated or attached to the cells prior to transplantation. Direct labeling of cells with radionuclides is a simple method with relatively fewer adverse events related to genetic responses. However, it can only allow short-term distribution of transplanted cells because of the decreasing imaging signal with radiodecay, according to the physical half-lives, or the signal becomes more diffuse with cell division and dispersion. The indirect labeling method is based on the expression of a reporter gene transduced into the cell before transplantation, which is then visualized upon the injection of an appropriate probe or substrate. In this review, various imaging strategies to monitor the survival and behavior change of transplanted stem cells are covered. Taking these new approaches together, the direct and indirect labeling methods may provide new insights on the roles of in vivo stem cell monitoring, from bench to bedside.

Sensing of substratum rigidity and directional migration by fast-crawling cells

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Okimura, Chika; Sakumura, Yuichi; Shimabukuro, Katsuya; Iwadate, Yoshiaki

2018-05-01

Living cells sense the mechanical properties of their surrounding environment and respond accordingly. Crawling cells detect the rigidity of their substratum and migrate in certain directions. They can be classified into two categories: slow-moving and fast-moving cell types. Slow-moving cell types, such as fibroblasts, smooth muscle cells, mesenchymal stem cells, etc., move toward rigid areas on the substratum in response to a rigidity gradient. However, there is not much information on rigidity sensing in fast-moving cell types whose size is ˜10 μ m and migration velocity is ˜10 μ m /min . In this study, we used both isotropic substrata with different rigidities and an anisotropic substratum that is rigid on the x axis but soft on the y axis to demonstrate rigidity sensing by fast-moving Dictyostelium cells and neutrophil-like differentiated HL-60 cells. Dictyostelium cells exerted larger traction forces on a more rigid isotropic substratum. Dictyostelium cells and HL-60 cells migrated in the "soft" direction on the anisotropic substratum, although myosin II-null Dictyostelium cells migrated in random directions, indicating that rigidity sensing of fast-moving cell types differs from that of slow types and is induced by a myosin II-related process.

The long noncoding RNA RNCR2 directs mouse retinal cell specification

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Blackshaw Seth

2010-05-01

Full Text Available Abstract Background Recent work has identified that many long mRNA-like noncoding RNAs (lncRNAs are expressed in the developing nervous system. Despite their abundance, the function of these ncRNAs has remained largely unexplored. We have investigated the highly abundant lncRNA RNCR2 in regulation of mouse retinal cell differentiation. Results We find that the RNCR2 is selectively expressed in a subset of both mitotic progenitors and postmitotic retinal precursor cells. ShRNA-mediated knockdown of RNCR2 results in an increase of both amacrine cells and Müller glia, indicating a role for this lncRNA in regulating retinal cell fate specification. We further report that RNCR2 RNA, which is normally nuclear-retained, can be exported from the nucleus when fused to an IRES-GFP sequence. Overexpression of RNCR2-IRES-GFP phenocopies the effects of shRNA-mediated knockdown of RNCR2, implying that forced mislocalization of RNCR2 induces a dominant-negative phenotype. Finally, we use the IRES-GFP fusion approach to identify specific domains of RNCR2 that are required for repressing both amacrine and Müller glial differentiation. Conclusion These data demonstrate that the lncRNA RNCR2 plays a critical role in regulating mammalian retinal cell fate specification. Furthermore, we present a novel approach for generating dominant-negative constructs of lncRNAs, which may be generally useful in the functional analysis of this class of molecules.

Reversal of Human Papillomavirus-Specific T Cell Immune Suppression through TLR Agonist Treatment of Langerhans Cells Exposed to Human Papillomavirus Type 161

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Fahey, Laura M.; Raff, Adam B.; Da Silva, Diane M.; Kast, W. Martin

2009-01-01

Human papillomavirus (HPV) type 16 infects the epithelial layer of cervical mucosa and is causally associated with the generation of cervical cancer. Langerhans cells (LC) are the resident antigen-presenting cells at the site of infection and therefore are responsible for initiating an immune response against HPV16. On the contrary, LC exposed to HPV16 do not induce a specific T cell immune response, which leads to the immune evasion of HPV16. Demonstrating that Toll-like receptor 7 (TLR7) and TLR8 are expressed on human LC, we hypothesized that imidazoquinolines would activate LC exposed to HPV16, leading to the induction of an HPV16-specific cell-mediated immune response. Surprisingly both phenotypic and functional hallmarks of activation are not observed when LC are exposed to HPV16 virus-like particles (VLP) and treated with imiquimod (TLR7 agonist). However, we found that LC are activated by 3M-002 (TLR8 agonist) and resiquimod (TLR8/7 agonist). LC exposed to HPV16 VLP and subsequently treated with 3M-002 or resiquimod highly up-regulate surface activation markers, secrete pro-inflammatory cytokines and chemokines, induce CCL21-directed migration, and initiate an HPV16-specific CD8+ T cell response. These data strongly indicate that 3M-002 and resiquimod are promising therapeutics for treatment of HPV-infections and HPV-induced cervical lesions. This is an author-produced version of a manuscript accepted for publication in The Journal of Immunology (The JI). The American Association of Immunologists, Inc. (AAI), publisher of The JI, holds the copyright to this manuscript. This version of the manuscript has not yet been copyedited or subjected to editorial proofreading by The JI; hence, it may differ from the final version published in The JI (online and in print). AAI (The JI) is not liable for errors or omissions in this author-produced version of the manuscript or in any version derived from it by the U.S. National Institutes of Health or any other third

The Direct Methanol Liquid-Feed Fuel Cell

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Halpert, Gerald

1997-01-01

Until the early 1990's the idea of a practical direct methanol fuel cell from transportation and other applications was just that, an idea. Several types of fuel cells that operate under near ambient conditions were under development.

Direct trans-activation of the human cyclin D2 gene by the oncogene product Tax of human T-cell leukemia virus type I.

Science.gov (United States)

Huang, Y; Ohtani, K; Iwanaga, R; Matsumura, Y; Nakamura, M

2001-03-01

Cyclins are one of the pivotal determinants regulating cell cycle progression. We previously reported that the trans-activator Tax of human T-cell leukemia virus type I (HTLV-I) induces endogenous cyclin D2 expression along with cell cycle progression in a resting human T-cell line, Kit 225, suggesting a role of cyclin D2 in Tax-mediated cell cycle progression. The cyclin D2 gene has a typical E2F binding element, raising the possibility that induction of cyclin D2 expression is a consequence of cell cycle progression. In this study, we examined the role and molecular mechanism of induction of the endogenous human cyclin D2 gene by Tax. Introduction of p19(INK4d), a cyclin dependent kinase (CDK) inhibitor of the INK4 family specific for D-type CDK, inhibited Tax-mediated activation of E2F, indicating requirement of D-type CDK in Tax-mediated activation of E2F. Previously indicated E2F binding element and two NF-kappaB-like binding elements in the 1.6 kbp cyclin D2 promoter fragment had little, if any, effect on responsiveness to Tax. We found that trans-activation of the cyclin D2 promoter by Tax was mainly mediated by a newly identified NF-kappaB-like element with auxiliary contribution of a CRE-like element residing in sequences downstream of -444 which were by themselves sufficient for trans-activation by Tax. These results indicate that Tax directly trans-activates the cyclin D2 gene, resulting in growth promotion and perhaps leukemogenesis through activation of D-type CDK.

Genetic recombination of Herpes simplex virus, the role of the host cell and UV-irradiation of the virus

International Nuclear Information System (INIS)

Dasgupta, U.B.; Summers, W.C.; Yale Univ., New Haven, CT; Yale Univ., New Haven, CT

1980-01-01

Recombination frequencies for two sets of genetic markers of Herpes simplex virus were determined in various host cells with and without ultraviolet irradiation of the virus. UV irradiation increased the recombination frequency in all the cell types studied in direct proportion to the unrepaired lethal damage. In human skin fibroblasts derived from a patient with xeroderma pigmentosum (XP) of complementation group A, a given dose of UV stimulated recombination more than that in fibroblasts from normal individuals. On the other hand, UV stimulation of HSV recombination was slightly less than normal in fibroblasts derived from a patient with a variant form XP and from an ataxia telangiectasia patient. Caffeine, an agent known to inhibit repair of UV damage, reduced recombination in most of the cell types studied but did not suppress the UV-induced increase in recombination. These findings suggest that for virus DNA with the same number of unrepaired UV-lesions, each of the tested cell types promoted HSV-recombination to an equivalent extent. (orig.) [de

Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells

OpenAIRE

Kebriaei, Partow; Singh, Harjeet; Huls, M. Helen; Figliola, Matthew J.; Bassett, Roland; Olivares, Simon; Jena, Bipulendu; Dawson, Margaret J.; Kumaresan, Pappanaicken R.; Su, Shihuang; Maiti, Sourindra; Dai, Jianliang; Moriarity, Branden; Forget, Marie-Andrée; Senyukov, Vladimir

2016-01-01

BACKGROUND. T cells expressing antigen-specific chimeric antigen receptors (CARs) improve outcomes for CD19-expressing B cell malignancies. We evaluated a human application of T cells that were genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific CAR.

Cell-specific monitoring of protein synthesis in vivo.

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Nikos Kourtis

Full Text Available Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems.

Tests for genetic interactions in type 1 diabetes

DEFF Research Database (Denmark)

Morahan, Grant; Mehta, Munish; James, Ian

2011-01-01

Interactions between genetic and environmental factors lead to immune dysregulation causing type 1 diabetes and other autoimmune disorders. Recently, many common genetic variants have been associated with type 1 diabetes risk, but each has modest individual effects. Familial clustering of type 1...... diabetes has not been explained fully and could arise from many factors, including undetected genetic variation and gene interactions....

Cell type-specific localization of Ephs pairing with ephrin-B2 in the rat postnatal pituitary gland.

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Yoshida, Saishu; Kato, Takako; Kanno, Naoko; Nishimura, Naoto; Nishihara, Hiroto; Horiguchi, Kotaro; Kato, Yukio

2017-10-01

Sox2-expressing stem/progenitor cells in the anterior lobe of the pituitary gland form two types of micro-environments (niches): the marginal cell layer and dense cell clusters in the parenchyma. In relation to the mechanism of regulation of niches, juxtacrine signaling via ephrin and its receptor Eph is known to play important roles in various niches. The ephrin and Eph families are divided into two subclasses to create ephrin/Eph signaling in co-operation with confined partners. Recently, we reported that ephrin-B2 localizes specifically to both pituitary niches. However, the Ephs interacting with ephrin-B2 in these pituitary niches have not yet been identified. Therefore, the present study aims to identify the Ephs interacting with ephrin-B2 and the cells that produce them in the rat pituitary gland. In situ hybridization and immunohistochemistry demonstrated cell type-specific localization of candidate interacting partners for ephrin-B2, including EphA4 in cells located in the posterior lobe, EphB1 in gonadotropes, EphB2 in corticotropes, EphB3 in stem/progenitor cells and EphB4 in endothelial cells in the adult pituitary gland. In particular, double-immunohistochemistry showed cis-interactions between EphB3 and ephrin-B2 in the apical cell membranes of stem/progenitor cell niches throughout life and trans-interactions between EphB2 produced by corticotropes and ephrin-B2 located in the basolateral cell membranes of stem/progenitor cells in the early postnatal pituitary gland. These data indicate that ephrin-B2 plays a role in pituitary stem/progenitor cell niches by selective interaction with EphB3 in cis and EphB2 in trans.

B-Cell-Specific Diversion of Glucose Carbon Utilization Reveals a Unique Vulnerability in B Cell Malignancies.

Science.gov (United States)

Xiao, Gang; Chan, Lai N; Klemm, Lars; Braas, Daniel; Chen, Zhengshan; Geng, Huimin; Zhang, Qiuyi Chen; Aghajanirefah, Ali; Cosgun, Kadriye Nehir; Sadras, Teresa; Lee, Jaewoong; Mirzapoiazova, Tamara; Salgia, Ravi; Ernst, Thomas; Hochhaus, Andreas; Jumaa, Hassan; Jiang, Xiaoyan; Weinstock, David M; Graeber, Thomas G; Müschen, Markus

2018-04-05

B cell activation during normal immune responses and oncogenic transformation impose increased metabolic demands on B cells and their ability to retain redox homeostasis. While the serine/threonine-protein phosphatase 2A (PP2A) was identified as a tumor suppressor in multiple types of cancer, our genetic studies revealed an essential role of PP2A in B cell tumors. Thereby, PP2A redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway (PPP) to salvage oxidative stress. This unique vulnerability reflects constitutively low PPP activity in B cells and transcriptional repression of G6PD and other key PPP enzymes by the B cell transcription factors PAX5 and IKZF1. Reflecting B-cell-specific transcriptional PPP-repression, glucose carbon utilization in B cells is heavily skewed in favor of glycolysis resulting in lack of PPP-dependent antioxidant protection. These findings reveal a gatekeeper function of the PPP in a broad range of B cell malignancies that can be efficiently targeted by small molecule inhibition of PP2A and G6PD. Copyright © 2018 Elsevier Inc. All rights reserved.

Oriented collagen fibers direct tumor cell intravasation

KAUST Repository

Han, Weijing; Chen, Shaohua; Yuan, Wei; Fan, Qihui; Tian, Jianxiang; Wang, Xiaochen; Chen, Longqing; Zhang, Xixiang; Wei, Weili; Liu, Ruchuan; Qu, Junle; Jiao, Yang; Austin, Robert H.; Liu, Liyu

2016-01-01

that the local fiber alignment enhanced cell-ECM interactions. Specifically, metastatic MDA-MB-231 breast cancer cells followed the local fiber alignment direction during the intravasation into rigid Matrigel (∼10 mg/mL protein concentration).

Cell kinetics and genetic instabilities in differentiated type early gastric cancers with different mucin phenotype.

Science.gov (United States)

Shibata, Naomi; Watari, Jiro; Fujiya, Mikihiro; Tanno, Satoshi; Saitoh, Yusuke; Kohgo, Yutaka

2003-01-01

To clarify the biological impact and molecular pathogenesis of cellular phenotype in differentiated-type gastric cancers (DGCs), we investigated cell kinetics and genetic instabilities in early stage of DGCs. A total of 43 early gastric cancers (EGCs) were studied. EGCs were divided into 3 phenotypic categories: gastric (G type, n = 11), ordinary (O type, n = 20), and complete intestinal (CI type, n = 12) based on the combination of HGM, ConA, MUC2, and CD10. Proliferative index (PI), apoptotic index (AI), and p53 overexpression were investigated by immunohistochemical staining with anti-Ki-67, the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method, and p53 antibody, respectively. Using a high-resolution fluorescent microsatellite analysis system, microsatellite instability (MSI) and loss of heterozygosity (LOH) were examined. Frameshift mutation analysis of transforming growth factor-beta type II receptor (TGF-betaRII) and bcl-2-associated X (BAX) in cancers with MSI was also performed. The mean AI/PI ratio values were 0.04 for G-type, 0.10 for O-type, and 0.13 for CI-type cancers--significantly lower in G type than in O and CI types (P = 0.02 and P = 0.001, respectively). No difference in the incidence of MSI and LOH was seen among the 3 cellular phenotypes. However, the major pattern of MSI, which showed drastic and widely dispersed changes and is related to an increased risk for cancer, was significantly higher in G and O types than in CI type (P cancers. These results indicate that G-type cancers are likely to show more aggressive behaviors than CI-type cancers, and that O-type cancers show the intermediate characteristics of both types. However, the molecular pathogenesis of each phenotypic cancer is not associated with microsatellite alterations. Copyright 2003, Elsevier Science (USA). All rights reserved.

Identifying States along the Hematopoietic Stem Cell Differentiation Hierarchy with Single Cell Specificity via Raman Spectroscopy.

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Ilin, Yelena; Choi, Ji Sun; Harley, Brendan A C; Kraft, Mary L

2015-11-17

A major challenge for expanding specific types of hematopoietic cells ex vivo for the treatment of blood cell pathologies is identifying the combinations of cellular and matrix cues that direct hematopoietic stem cells (HSC) to self-renew or differentiate into cell populations ex vivo. Microscale screening platforms enable minimizing the number of rare HSCs required to screen the effects of numerous cues on HSC fate decisions. These platforms create a strong demand for label-free methods that accurately identify the fate decisions of individual hematopoietic cells at specific locations on the platform. We demonstrate the capacity to identify discrete cells along the HSC differentiation hierarchy via multivariate analysis of Raman spectra. Notably, cell state identification is accurate for individual cells and independent of the biophysical properties of the functionalized polyacrylamide gels upon which these cells are cultured. We report partial least-squares discriminant analysis (PLS-DA) models of single cell Raman spectra enable identifying four dissimilar hematopoietic cell populations across the HSC lineage specification. Successful discrimination was obtained for a population enriched for long-term repopulating HSCs (LT-HSCs) versus their more differentiated progeny, including closely related short-term repopulating HSCs (ST-HSCs) and fully differentiated lymphoid (B cells) and myeloid (granulocytes) cells. The lineage-specific differentiation states of cells from these four subpopulations were accurately identified independent of the stiffness of the underlying biomaterial substrate, indicating subtle spectral variations that discriminated these populations were not masked by features from the culture substrate. This approach enables identifying the lineage-specific differentiation stages of hematopoietic cells on biomaterial substrates of differing composition and may facilitate correlating hematopoietic cell fate decisions with the extrinsic cues that

A powerful method for transcriptional profiling of specific cell types in eukaryotes: laser-assisted microdissection and RNA sequencing.

Directory of Open Access Journals (Sweden)

Marc W Schmid

Full Text Available The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM, linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq. As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms.

Genetic recombination pathways and their application for genome modification of human embryonic stem cells.

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Nieminen, Mikko; Tuuri, Timo; Savilahti, Harri

2010-10-01

Human embryonic stem cells are pluripotent cells derived from early human embryo and retain a potential to differentiate into all adult cell types. They provide vast opportunities in cell replacement therapies and are expected to become significant tools in drug discovery as well as in the studies of cellular and developmental functions of human genes. The progress in applying different types of DNA recombination reactions for genome modification in a variety of eukaryotic cell types has provided means to utilize recombination-based strategies also in human embryonic stem cells. Homologous recombination-based methods, particularly those utilizing extended homologous regions and those employing zinc finger nucleases to boost genomic integration, have shown their usefulness in efficient genome modification. Site-specific recombination systems are potent genome modifiers, and they can be used to integrate DNA into loci that contain an appropriate recombination signal sequence, either naturally occurring or suitably pre-engineered. Non-homologous recombination can be used to generate random integrations in genomes relatively effortlessly, albeit with a moderate efficiency and precision. DNA transposition-based strategies offer substantially more efficient random strategies and provide means to generate single-copy insertions, thus potentiating the generation of genome-wide insertion libraries applicable in genetic screens. 2010 Elsevier Inc. All rights reserved.

Generation of multiple cell types in Bacillus subtilis.

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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.

Direct and inverted repeats elicit genetic instability by both exploiting and eluding DNA double-strand break repair systems in mycobacteria.

Directory of Open Access Journals (Sweden)

Ewelina A Wojcik

Full Text Available Repetitive DNA sequences with the potential to form alternative DNA conformations, such as slipped structures and cruciforms, can induce genetic instability by promoting replication errors and by serving as a substrate for DNA repair proteins, which may lead to DNA double-strand breaks (DSBs. However, the contribution of each of the DSB repair pathways, homologous recombination (HR, non-homologous end-joining (NHEJ and single-strand annealing (SSA, to this sort of genetic instability is not fully understood. Herein, we assessed the genome-wide distribution of repetitive DNA sequences in the Mycobacterium smegmatis, Mycobacterium tuberculosis and Escherichia coli genomes, and determined the types and frequencies of genetic instability induced by direct and inverted repeats, both in the presence and in the absence of HR, NHEJ, and SSA. All three genomes are strongly enriched in direct repeats and modestly enriched in inverted repeats. When using chromosomally integrated constructs in M. smegmatis, direct repeats induced the perfect deletion of their intervening sequences ~1,000-fold above background. Absence of HR further enhanced these perfect deletions, whereas absence of NHEJ or SSA had no influence, suggesting compromised replication fidelity. In contrast, inverted repeats induced perfect deletions only in the absence of SSA. Both direct and inverted repeats stimulated excision of the constructs from the attB integration sites independently of HR, NHEJ, or SSA. With episomal constructs, direct and inverted repeats triggered DNA instability by activating nucleolytic activity, and absence of the DSB repair pathways (in the order NHEJ>HR>SSA exacerbated this instability. Thus, direct and inverted repeats may elicit genetic instability in mycobacteria by 1 directly interfering with replication fidelity, 2 stimulating the three main DSB repair pathways, and 3 enticing L5 site-specific recombination.

Direct and inverted repeats elicit genetic instability by both exploiting and eluding DNA double-strand break repair systems in mycobacteria.

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Wojcik, Ewelina A; Brzostek, Anna; Bacolla, Albino; Mackiewicz, Pawel; Vasquez, Karen M; Korycka-Machala, Malgorzata; Jaworski, Adam; Dziadek, Jaroslaw

2012-01-01

Repetitive DNA sequences with the potential to form alternative DNA conformations, such as slipped structures and cruciforms, can induce genetic instability by promoting replication errors and by serving as a substrate for DNA repair proteins, which may lead to DNA double-strand breaks (DSBs). However, the contribution of each of the DSB repair pathways, homologous recombination (HR), non-homologous end-joining (NHEJ) and single-strand annealing (SSA), to this sort of genetic instability is not fully understood. Herein, we assessed the genome-wide distribution of repetitive DNA sequences in the Mycobacterium smegmatis, Mycobacterium tuberculosis and Escherichia coli genomes, and determined the types and frequencies of genetic instability induced by direct and inverted repeats, both in the presence and in the absence of HR, NHEJ, and SSA. All three genomes are strongly enriched in direct repeats and modestly enriched in inverted repeats. When using chromosomally integrated constructs in M. smegmatis, direct repeats induced the perfect deletion of their intervening sequences ~1,000-fold above background. Absence of HR further enhanced these perfect deletions, whereas absence of NHEJ or SSA had no influence, suggesting compromised replication fidelity. In contrast, inverted repeats induced perfect deletions only in the absence of SSA. Both direct and inverted repeats stimulated excision of the constructs from the attB integration sites independently of HR, NHEJ, or SSA. With episomal constructs, direct and inverted repeats triggered DNA instability by activating nucleolytic activity, and absence of the DSB repair pathways (in the order NHEJ>HR>SSA) exacerbated this instability. Thus, direct and inverted repeats may elicit genetic instability in mycobacteria by 1) directly interfering with replication fidelity, 2) stimulating the three main DSB repair pathways, and 3) enticing L5 site-specific recombination.

Synchronization and Arrest of the Budding Yeast Cell Cycle Using Chemical and Genetic Methods.

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Rosebrock, Adam P

2017-01-03

The cell cycle of budding yeast can be arrested at specific positions by different genetic and chemical methods. These arrests enable study of cell cycle phase-specific phenotypes that would be missed during examination of asynchronous cultures. Some methods for arrest are reversible, with kinetics that enable release of cells back into a synchronous cycling state. Benefits of chemical and genetic methods include scalability across a large range of culture sizes from a few milliliters to many liters, ease of execution, the absence of specific equipment requirements, and synchronization and release of the entire culture. Of note, cell growth and division are decoupled during arrest and block-release experiments. Cells will continue transcription, translation, and accumulation of protein while arrested. If allowed to reenter the cell cycle, cells will do so as a population of mixed, larger-than-normal cells. Despite this important caveat, many aspects of budding yeast physiology are accessible using these simple chemical and genetic tools. Described here are methods for the block and release of cells in G 1 phase and at the M/G 1 transition using α-factor mating pheromone and the temperature-sensitive cdc15-2 allele, respectively, in addition to methods for arresting the cell cycle in early S phase and at G 2 /M by using hydroxyurea and nocodazole, respectively. © 2017 Cold Spring Harbor Laboratory Press.

The use of genetic transformation in the study of ovarian-specific gene expression

International Nuclear Information System (INIS)

Manzi, A.; Andone, S.; Rotoli, D.; Capua, M.R.; Gargiulo, G.; Graziani, F.; Malva, C.

1998-01-01

We are using genetic and molecular approaches to understand the mechanisms controlling the establishment of the cellular specificity of expression during oogenesis. Female-sterile mutations have been isolated and the molecular analysis is revealing interesting cell-cell interaction systems that work not only during oogenesis but also at other developmental stages. We will review in this paper our most recent studies on genes involved in ovarian development. (author)

Specification of Drosophila corpora cardiaca neuroendocrine cells from mesoderm is regulated by Notch signaling.

Directory of Open Access Journals (Sweden)

Sangbin Park

2011-08-01

Full Text Available Drosophila neuroendocrine cells comprising the corpora cardiaca (CC are essential for systemic glucose regulation and represent functional orthologues of vertebrate pancreatic α-cells. Although Drosophila CC cells have been regarded as developmental orthologues of pituitary gland, the genetic regulation of CC development is poorly understood. From a genetic screen, we identified multiple novel regulators of CC development, including Notch signaling factors. Our studies demonstrate that the disruption of Notch signaling can lead to the expansion of CC cells. Live imaging demonstrates localized emergence of extra precursor cells as the basis of CC expansion in Notch mutants. Contrary to a recent report, we unexpectedly found that CC cells originate from head mesoderm. We show that Tinman expression in head mesoderm is regulated by Notch signaling and that the combination of Daughterless and Tinman is sufficient for ectopic CC specification in mesoderm. Understanding the cellular, genetic, signaling, and transcriptional basis of CC cell specification and expansion should accelerate discovery of molecular mechanisms regulating ontogeny of organs that control metabolism.

Genetics Home Reference: glutaric acidemia type I

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... Glutaric acidemia type 1 in patients of Lumbee heritage from North Carolina. Mol Genet Metab. 2006 May; ... 825-8. Review. Citation on PubMed More from Genetics Home Reference Bulletins Genetics Home Reference Celebrates Its ...

Direct sorbitol proton exchange membrane fuel cell using moderate catalyst loadings

International Nuclear Information System (INIS)

Oyarce, Alejandro; Gonzalez, Carlos; Lima, Raquel Bohn; Lindström, Rakel Wreland; Lagergren, Carina; Lindbergh, Göran

2014-01-01

Highlights: •The performance of a direct sorbitol fuel cell was evaluated at different temperatures. •The performance was compared to the performance of a direct glucose fuel cell. •The mass specific peak power density of the direct sorbitol fuel cell was 3.6 mW mg −1 totalcatalystloading at 80 °C. •Both sorbitol and glucose fuel cell suffer from deactivation. -- Abstract: Recent progress in biomass hydrolysis has made it interesting to study the use of sorbitol for electricity generation. In this study, sorbitol and glucose are used as fuels in proton exchange membrane fuel cells having 0.9 mg cm −2 PtRu/C at the anode and 0.3 mg cm −2 Pt/C at the cathode. The sorbitol oxidation was found to have slower kinetics than glucose oxidation. However, at low temperatures the direct sorbitol fuel cell shows higher performance than the direct glucose fuel cell, attributed to a lower degree of catalyst poisoning. The performance of both fuel cells is considerably improved at higher temperatures. High temperatures lower the poisoning, allowing the direct glucose fuel cell to reach a higher performance than the direct sorbitol fuel cell. The mass specific peak power densities of the direct sorbitol and direct glucose fuel cells at 65 °C was 3.2 mW mg −1 catalyst and 3.5 mW mg −1 catalyst , respectively. Both of these values are one order of magnitude larger than mass specific peak power densities of earlier reported direct glucose fuel cells using proton exchange membranes. Furthermore, both the fuel cells showed a considerably decrease in performance with time, which is partially attributed to sorbitol and glucose crossover poisoning the Pt/C cathode

Aberrant DNA Methylation in Human iPSCs Associates with MYC-Binding Motifs in a Clone-Specific Manner Independent of Genetics.

Science.gov (United States)

Panopoulos, Athanasia D; Smith, Erin N; Arias, Angelo D; Shepard, Peter J; Hishida, Yuriko; Modesto, Veronica; Diffenderfer, Kenneth E; Conner, Clay; Biggs, William; Sandoval, Efren; D'Antonio-Chronowska, Agnieszka; Berggren, W Travis; Izpisua Belmonte, Juan Carlos; Frazer, Kelly A

2017-04-06

Induced pluripotent stem cells (iPSCs) show variable methylation patterns between lines, some of which reflect aberrant differences relative to embryonic stem cells (ESCs). To examine whether this aberrant methylation results from genetic variation or non-genetic mechanisms, we generated human iPSCs from monozygotic twins to investigate how genetic background, clone, and passage number contribute. We found that aberrantly methylated CpGs are enriched in regulatory regions associated with MYC protein motifs and affect gene expression. We classified differentially methylated CpGs as being associated with genetic and/or non-genetic factors (clone and passage), and we found that aberrant methylation preferentially occurs at CpGs associated with clone-specific effects. We further found that clone-specific effects play a strong role in recurrent aberrant methylation at specific CpG sites across different studies. Our results argue that a non-genetic biological mechanism underlies aberrant methylation in iPSCs and that it is likely based on a probabilistic process involving MYC that takes place during or shortly after reprogramming. Published by Elsevier Inc.

Synthetically derived bat influenza A-like viruses reveal a cell type- but not species-specific tropism.

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Moreira, Étori Aguiar; Locher, Samira; Kolesnikova, Larissa; Bolte, Hardin; Aydillo, Teresa; García-Sastre, Adolfo; Schwemmle, Martin; Zimmer, Gert

2016-10-24

Two novel influenza A-like viral genome sequences have recently been identified in Central and South American fruit bats and provisionally designated "HL17NL10" and "HL18NL11." All efforts to isolate infectious virus from bats or to generate these viruses by reverse genetics have failed to date. Recombinant vesicular stomatitis virus (VSV) encoding the hemagglutinin-like envelope glycoproteins HL17 or HL18 in place of the VSV glycoprotein were generated to identify cell lines that are susceptible to bat influenza A-like virus entry. More than 30 cell lines derived from various species were screened but only a few cell lines were found to be susceptible, including Madin-Darby canine kidney type II (MDCK II) cells. The identification of cell lines susceptible to VSV chimeras allowed us to recover recombinant HL17NL10 and HL18NL11 viruses from synthetic DNA. Both influenza A-like viruses established a productive infection in MDCK II cells; however, HL18NL11 replicated more efficiently than HL17NL10 in this cell line. Unlike conventional influenza A viruses, bat influenza A-like viruses started the infection preferentially at the basolateral membrane of polarized MDCK II cells; however, similar to conventional influenza A viruses, bat influenza A-like viruses were released primarily from the apical site. The ability of HL18NL11 or HL17NL10 viruses to infect canine and human cells might reflect a zoonotic potential of these recently identified bat viruses.

Cellular Immunotherapy for Carcinoma Using Genetically Modified EGFR-Specific T Lymphocytes

Directory of Open Access Journals (Sweden)

Xikun Zhou

2013-05-01

Full Text Available Epidermal growth factor receptor (EGFR is overexpressed in a variety of human malignancies, including pancreatic cancer, breast cancer, colon cancer, and non-small cell lung cancer. Overexpression of EGFR is a predictive marker of therapeutic response and several lines of evidence suggest that EGFR is an excellent target for tumor therapy. However, the effective antitumor capacity of EGFR-specific T cells against EGFR-overexpressing tumor cells has not been fully elucidated. In our previous study, we identified an anti-EGFR single-chain variable fragment (scFv with specific and high affinity after screening by ribosome display. In this study, the anticancer potential of anti-EGFR scFv was investigated on the basis of cell-targeted therapy. A chimeric antigen receptor (CAR targeting EGFR was constructed and expressed on the cell membrane of T lymphocytes. These CAR-modified T cells demonstrated antitumor efficacy both in vitro and in vivo. In addition, the safety evaluation showed that CAR-modified lymphocytes have no or very minimal acute systemic toxicity. Taken together, our study provided the experimental basis for clinical application of genetically engineered lymphocytes; moreover, we also evaluate a new and interesting cell therapy protocol.

Studying the Genetics of Complex Disease With Ancestry-Specific Human Phenotype Networks: The Case of Type 2 Diabetes in East Asian Populations.

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Qiu, Jingya; Moore, Jason H; Darabos, Christian

2016-05-01

Genome-wide association studies (GWAS) have led to the discovery of over 200 single nucleotide polymorphisms (SNPs) associated with type 2 diabetes mellitus (T2DM). Additionally, East Asians develop T2DM at a higher rate, younger age, and lower body mass index than their European ancestry counterparts. The reason behind this occurrence remains elusive. With comprehensive searches through the National Human Genome Research Institute (NHGRI) GWAS catalog literature, we compiled a database of 2,800 ancestry-specific SNPs associated with T2DM and 70 other related traits. Manual data extraction was necessary because the GWAS catalog reports statistics such as odds ratio and P-value, but does not consistently include ancestry information. Currently, many statistics are derived by combining initial and replication samples from study populations of mixed ancestry. Analysis of all-inclusive data can be misleading, as not all SNPs are transferable across diverse populations. We used ancestry data to construct ancestry-specific human phenotype networks (HPN) centered on T2DM. Quantitative and visual analysis of network models reveal the genetic disparities between ancestry groups. Of the 27 phenotypes in the East Asian HPN, six phenotypes were unique to the network, revealing the underlying ancestry-specific nature of some SNPs associated with T2DM. We studied the relationship between T2DM and five phenotypes unique to the East Asian HPN to generate new interaction hypotheses in a clinical context. The genetic differences found in our ancestry-specific HPNs suggest different pathways are involved in the pathogenesis of T2DM among different populations. Our study underlines the importance of ancestry in the development of T2DM and its implications in pharmocogenetics and personalized medicine. © 2016 The Authors. *Genetic Epidemiology Published by Wiley Periodicals, Inc.

Genetic heterogeneity in type 1 Gaucher disease: Multiple genotypes in Ashkenazic and non-Ashkenazic individuals

International Nuclear Information System (INIS)

Tsuji, Shoji; Martin, B.M.; Stubblefield, B.K.; LaMarca, M.E.; Ginns, E.I.; Barranger, J.A.

1988-01-01

Nucleotide sequence analysis of a genomic clone from an Ashkenazic Jewish patient with type 1 Gaucher disease revealed a single-base mutation (adenosine to guanosine transition) in exon 9 of the glucocerebrosidase gene. This change results in the amino acid substitution of serine for asparagine. Transient expression studies following oligonucleotide-directed mutagenesis of the normal cDNA confirmed that the mutation results in loss of glucocerebrosidase activity. Allele-specific hybridization with oligonucleotide probes demonstrated that this mutation was found exclusively in type 1 phenotype. None of the 6 type 2 patients, 11 type 3 patients, or 12 normal controls had this allele. In contrast, 15 of 24 type 1 patients had one allele with this mutation, and 3 others were homozygous for the mutation. Furthermore, some of the Ashkenazic Jewish type 1 patients had only one allele with this mutation, suggesting that even in this population there is allelic heterozygosity. These findings indicate that there are multiple allelic mutations responsible for type 1 Gaucher disease in both the Jewish and non-Jewish populations. Allelic-specific hybridization demonstrating this mutation in exon 9, used in conjunction with the Nci I restriction fragment length polymorphism described as a marker for neuronopathic Gaucher disease, provides a tool for diagnosis and genetic counseling that is ∼80% informative in all Gaucher patients studied

Genetic heterogeneity in type 1 Gaucher disease: Multiple genotypes in Ashkenazic and non-Ashkenazic individuals

Energy Technology Data Exchange (ETDEWEB)

Tsuji, Shoji; Martin, B.M.; Stubblefield, B.K.; LaMarca, M.E.; Ginns, E.I. (National Institute of Mental Health, Bethesda, MD (USA)); Barranger, J.A. (Childrens Hospital of Los Angeles, CA (USA))

1988-04-01

Nucleotide sequence analysis of a genomic clone from an Ashkenazic Jewish patient with type 1 Gaucher disease revealed a single-base mutation (adenosine to guanosine transition) in exon 9 of the glucocerebrosidase gene. This change results in the amino acid substitution of serine for asparagine. Transient expression studies following oligonucleotide-directed mutagenesis of the normal cDNA confirmed that the mutation results in loss of glucocerebrosidase activity. Allele-specific hybridization with oligonucleotide probes demonstrated that this mutation was found exclusively in type 1 phenotype. None of the 6 type 2 patients, 11 type 3 patients, or 12 normal controls had this allele. In contrast, 15 of 24 type 1 patients had one allele with this mutation, and 3 others were homozygous for the mutation. Furthermore, some of the Ashkenazic Jewish type 1 patients had only one allele with this mutation, suggesting that even in this population there is allelic heterozygosity. These findings indicate that there are multiple allelic mutations responsible for type 1 Gaucher disease in both the Jewish and non-Jewish populations. Allelic-specific hybridization demonstrating this mutation in exon 9, used in conjunction with the Nci I restriction fragment length polymorphism described as a marker for neuronopathic Gaucher disease, provides a tool for diagnosis and genetic counseling that is {approx}80% informative in all Gaucher patients studied.

Chemical compound-based direct reprogramming for future clinical applications

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Takeda, Yukimasa; Harada, Yoshinori; Yoshikawa, Toshikazu; Dai, Ping

2018-01-01

Recent studies have revealed that a combination of chemical compounds enables direct reprogramming from one somatic cell type into another without the use of transgenes by regulating cellular signaling pathways and epigenetic modifications. The generation of induced pluripotent stem (iPS) cells generally requires virus vector-mediated expression of multiple transcription factors, which might disrupt genomic integrity and proper cell functions. The direct reprogramming is a promising alternative to rapidly prepare different cell types by bypassing the pluripotent state. Because the strategy also depends on forced expression of exogenous lineage-specific transcription factors, the direct reprogramming in a chemical compound-based manner is an ideal approach to further reduce the risk for tumorigenesis. So far, a number of reported research efforts have revealed that combinations of chemical compounds and cell-type specific medium transdifferentiate somatic cells into desired cell types including neuronal cells, glial cells, neural stem cells, brown adipocytes, cardiomyocytes, somatic progenitor cells, and pluripotent stem cells. These desired cells rapidly converted from patient-derived autologous fibroblasts can be applied for their own transplantation therapy to avoid immune rejection. However, complete chemical compound-induced conversions remain challenging particularly in adult human-derived fibroblasts compared with mouse embryonic fibroblasts (MEFs). This review summarizes up-to-date progress in each specific cell type and discusses prospects for future clinical application toward cell transplantation therapy. PMID:29739872

Clinical and genetic aspects of testicular germ cell tumours

NARCIS (Netherlands)

Holzik, Martijn F. Lutke; Sijmons, Rolf H.; Hoekstra-Weebers, Josette E. H. M.; Sleijfer, Dirk Th.; Hoekstra, Harald J.

2008-01-01

In this paper we review clinical and genetic aspects of testicular germ cell tumours (TGCTs). TGCT is the most common type of malignant disorder in men aged 15-40 years. Its incidence has increased sharply in recent years. Fortunately, survival of patients with TGCT has improved enormously, which

Preliminary investigation of genetic characterization of native and endemic fowl types of Sri Lanka

International Nuclear Information System (INIS)

Silva, L.P.; Rajapaksha, W.R.A.K.J.S.

2005-01-01

The Red Jungle Fowl (Gallus gallus) is generally considered to be main ancestor of the domestic fowl (Callus domesticus). However, it is also believed that other wild Callus species might have contributed to the modern genetic make-up of the domestic fowl, one wild species being the Ceylon Jungle Fowl (Gallus lafayetti), endemic to Sri Lanka, which could have contributed to the domestic stock of Sri Lankan native poultry. The present study was conducted in order to investigate the origin of native fowl in Sri Lanka and to establish genetic relationships among them and the Ceylon Jungle Fowl. Morphological characters of endemic, indigenous and exotic fowl types were recorded. These included Ceylon Jungle fowl; eleven types of native chicken from Sri Lanka; and two exotic chicken breeds (Cornish and Rhode Island Red). Blood samples were collected for DNA extraction. Randomly Amplified Polymorphic DNA (RAPD) analysis was carried out using sixteen non-specific primers. The results of morphological characterization revealed many variations in plumage and colour pattern. Single and pea comb types were found in both native and exotic types of chicken. A prominent yellow colour marking on a red comb was a unique feature in Ceylon Jungle fowl. The presence of white spots in red earlobes was a distinguishing feature of all native chicken types. Sixteen non-specific primers were used in the study, and produced 22 polymorphic bands ranging from 500 to 1960 bp. Genetic similarity indices ranged from 0.5 to 1.1 in average genetic distance scale, indicating a broad genetic base in the samples studied. Cluster analysis revealed a clear separation of Ceylon Jungle Fowl from all other types studied, indicating that contribution in data analysis, and the Director and staff, National Zoological Gardens, Sri Lanka, for their help in sampling Ceylon Jungle Fowl. (author)

'Cold shock' increases the frequency of homology directed repair gene editing in induced pluripotent stem cells.

Science.gov (United States)

Guo, Q; Mintier, G; Ma-Edmonds, M; Storton, D; Wang, X; Xiao, X; Kienzle, B; Zhao, D; Feder, John N

2018-02-01

Using CRISPR/Cas9 delivered as a RNA modality in conjunction with a lipid specifically formulated for large RNA molecules, we demonstrate that homology directed repair (HDR) rates between 20-40% can be achieved in induced pluripotent stem cells (iPSC). Furthermore, low HDR rates (between 1-20%) can be enhanced two- to ten-fold in both iPSCs and HEK293 cells by 'cold shocking' cells at 32 °C for 24-48 hours following transfection. This method can also increases the proportion of loci that have undergone complete sequence conversion across the donor sequence, or 'perfect HDR', as opposed to partial sequence conversion where nucleotides more distal to the CRISPR cut site are less efficiently incorporated ('partial HDR'). We demonstrate that the structure of the single-stranded DNA oligo donor can influence the fidelity of HDR, with oligos symmetric with respect to the CRISPR cleavage site and complementary to the target strand being more efficient at directing 'perfect HDR' compared to asymmetric non-target strand complementary oligos. Our protocol represents an efficient method for making CRISPR-mediated, specific DNA sequence changes within the genome that will facilitate the rapid generation of genetic models of human disease in iPSCs as well as other genome engineered cell lines.

Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification.

Science.gov (United States)

Moretti, Alessandra; Caron, Leslie; Nakano, Atsushi; Lam, Jason T; Bernshausen, Alexandra; Chen, Yinhong; Qyang, Yibing; Bu, Lei; Sasaki, Mika; Martin-Puig, Silvia; Sun, Yunfu; Evans, Sylvia M; Laugwitz, Karl-Ludwig; Chien, Kenneth R

2006-12-15

Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1(+) precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1(+) cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1(+)/Nkx2.5(+)/flk1(+) defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1(+) cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.

Targeting the autolysis loop of urokinase-type plasminogen activator with conformation-specific monoclonal antibodies

DEFF Research Database (Denmark)

Bøtkjær, Kenneth Alrø; Fogh, Sarah; Bekes, Erin C

2011-01-01

Tight regulation of serine proteases is essential for their physiological function, and unbalanced states of protease activity have been implicated in a variety of human diseases. One key example is the presence of uPA (urokinase-type plasminogen activator) in different human cancer types......, demonstrating a direct link between conformational changes of the autolysis loop and the creation of a catalytically mature active site. All three antibodies are potent inhibitors of uPA activity, the two pro-uPA-specific ones by inhibiting conversion of pro-uPA to active uPA and the active u......PA-specific antibody by shielding the access of plasminogen to the active site. Furthermore, using immunofluorescence, the conformation-specific antibodies mAb-112 and mAb-12E6B10 enabled us to selectively stain pro-uPA or active uPA on the surface of cultured cells. Moreover, in various independent model systems...

T cell reactivity with allergoids: influence of the type of APC.

Science.gov (United States)

Kahlert, H; Grage-Griebenow, E; Stüwe, H T; Cromwell, O; Fiebig, H

2000-08-15

The use of allergoids for allergen-specific immunotherapy has been established for many years. The characteristic features of these chemically modified allergens are their strongly reduced IgE binding activity compared with the native form and the retained immunogenicity. T cell reactivity of chemically modified allergens is documented in animals, but in humans indirect evidence of reactivity has been concluded from the induction of allergen-specific IgG during immunotherapy. Direct evidence of T cell reactivity was obtained recently using isolated human T cells. To obtain further insight into the mechanism of action of allergoids, we compared the Ag-presenting capacity of different APC types, including DC and macrophages, generated from CD14+ precursor cells from the blood of grass pollen allergic subjects, autologous PBMC, and B cells. These APC were used in experiments together with Phl p 5-specific T cell clones under stimulation with grass pollen allergen extract, rPhl p 5b, and the respective allergoids. Using DC and macrophages, allergoids exhibited a pronounced and reproducible T cell-stimulating capacity. Responses were superior to those with PBMC, and isolated B cells failed to present allergoids. Considerable IL-12 production was observed only when using the DC for Ag presentation of both allergens and allergoids. The amount of IL-10 in supernatants was dependent on the phenotype of the respective T cell clone. High IL-10 production was associated with suppressed IL-12 production from the DC in most cases. In conclusion, the reactivity of Th cells with allergoids is dependent on the type of the APC.

Direct and maternal genetic effects for birth weight in dorper and ...

African Journals Online (AJOL)

Variance components for birth (BWT) in Dorper and Mutton Merino sheep were estimated by Average Information Restricted Maximum Likelihood (AIREML). Animal model was fitted allowing for genetic maternal effects and a genetic covariance between direct and maternal effects. Estimates of heritability for direct genetic ...

A synthetic glycan microarray enables epitope mapping of plant cell wall glycan-directed antibodies

DEFF Research Database (Denmark)

Ruprecht, Colin; Bartetzko, Max P; Senf, Deborah

2017-01-01

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories world-wide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types......, and developmental stages. Despite their importance and broad use, the precise binding epitope for only a few of these antibodies has been determined. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies....... Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall...

Sex-specific genetic effects in physical activity: results from a quantitative genetic analysis.

Science.gov (United States)

Diego, Vincent P; de Chaves, Raquel Nichele; Blangero, John; de Souza, Michele Caroline; Santos, Daniel; Gomes, Thayse Natacha; dos Santos, Fernanda Karina; Garganta, Rui; Katzmarzyk, Peter T; Maia, José A R

2015-08-01

The objective of this study is to present a model to estimate sex-specific genetic effects on physical activity (PA) levels and sedentary behaviour (SB) using three generation families. The sample consisted of 100 families covering three generations from Portugal. PA and SB were assessed via the International Physical Activity Questionnaire short form (IPAQ-SF). Sex-specific effects were assessed by genotype-by-sex interaction (GSI) models and sex-specific heritabilities. GSI effects and heterogeneity were tested in the residual environmental variance. SPSS 17 and SOLAR v. 4.1 were used in all computations. The genetic component for PA and SB domains varied from low to moderate (11% to 46%), when analyzing both genders combined. We found GSI effects for vigorous PA (p = 0.02) and time spent watching television (WT) (p < 0.001) that showed significantly higher additive genetic variance estimates in males. The heterogeneity in the residual environmental variance was significant for moderate PA (p = 0.02), vigorous PA (p = 0.006) and total PA (p = 0.001). Sex-specific heritability estimates were significantly higher in males only for WT, with a male-to-female difference in heritability of 42.5 (95% confidence interval: 6.4, 70.4). Low to moderate genetic effects on PA and SB traits were found. Results from the GSI model show that there are sex-specific effects in two phenotypes, VPA and WT with a stronger genetic influence in males.

Highly efficient reprogramming to pluripotency and directed differentiation of human cells using synthetic modified mRNA

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Warren, Luigi; Manos, Philip D.; Ahfeldt, Tim; Loh, Yuin-Han; Li, Hu; Lau, Frank; Ebina, Wataru; Mandal, Pankaj; Smith, Zachary D.; Meissner, Alexander; Daley, George Q.; Brack, Andrew S.; Collins, James J.; Cowan, Chad; Schlaeger, Thorsten M.

2010-01-01

Clinical application of induced pluripotent stem (iPS) cells is limited by the low efficiency of iPS derivation and the fact that most protocols modify the genome to effect cellular reprogramming. Moreover, safe and effective means of directing the fate of patient-specific iPS cells towards clinically useful cell types are lacking. Here we describe a simple, non-integrating strategy for reprogramming cell fate based on administration of synthetic mRNA modified to overcome innate anti-viral re...

Genetic Engineering of Mesenchymal Stem Cells for Regenerative Medicine.

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Nowakowski, Adam; Walczak, Piotr; Janowski, Miroslaw; Lukomska, Barbara

2015-10-01

Mesenchymal stem cells (MSCs), which can be obtained from various organs and easily propagated in vitro, are one of the most extensively used types of stem cells and have been shown to be efficacious in a broad set of diseases. The unique and highly desirable properties of MSCs include high migratory capacities toward injured areas, immunomodulatory features, and the natural ability to differentiate into connective tissue phenotypes. These phenotypes include bone and cartilage, and these properties predispose MSCs to be therapeutically useful. In addition, MSCs elicit their therapeutic effects by paracrine actions, in which the metabolism of target tissues is modulated. Genetic engineering methods can greatly amplify these properties and broaden the therapeutic capabilities of MSCs, including transdifferentiation toward diverse cell lineages. However, cell engineering can also affect safety and increase the cost of therapy based on MSCs; thus, the advantages and disadvantages of these procedures should be discussed. In this review, the latest applications of genetic engineering methods for MSCs with regenerative medicine purposes are presented.

Drug delivery systems--2. Site-specific drug delivery utilizing monoclonal antibodies.

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Ranade, V V

1989-10-01

Monoclonal antibodies (MAbs) are purified antibodies produced by a single clone of cells. They are engineered to recognize and bind to a single specific antigen. Accordingly, when administered, MAbs home in on a particular circulating protein or on cells that bear the correct antigenic signature on their surfaces. It is the specificity of MAbs that has made them valuable tools for health professions. Following the discovery of Kohler and Milstein regarding the method of somatic cell hybridization, a number of investigators have successfully adopted this technique to obtain T-lymphocyte hybrid cell lines by fusion of activated T (thymus derived) lymphocytes with a T lymphoma cell line leading to an immortalization of a specific differentiated function. The hybrids thus obtained were subsequently shown to produce homogeneous effector molecules with a wide variety of immune functions such as enhancement or suppression of antibody responses, generation of helper T cells, suppressor T cells and cytotoxic T cells. Study of these regulatory molecules has been further shown to provide a greater insight into the genetic, biochemical and molecular mechanisms responsible for cellular development, and the interaction and triggering of various cell types. The successful application of hybridoma technology has now resulted into several advances in the understanding the mechanism and treatment of diseases, especially cancer and development of vaccines, promotion of organ transplantation and therapy against parasites as well. Since monoclonal antibodies could be made in unlimited supply, they have been used in genetic studies such as mRNA and gene isolation, chromosomal isolation of specific genes, immunoglobulin structure, detection of new or rare immunoglobulin gene products, structural studies of enzymes and other proteins and structural and population studies of protein polymorphisms. In some instances, the monoclonal antibodies have been found to replace conventional antisera

Derivation of novel genetically diverse human embryonic stem cell lines.

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Stefanova, Valentina T; Grifo, James A; Hansis, Christoph

2012-06-10

Human embryonic stem cells (hESCs) have the potential to revolutionize many biomedical fields ranging from basic research to disease modeling, regenerative medicine, drug discovery, and toxicity testing. A multitude of hESC lines have been derived worldwide since the first 5 lines by Thomson et al. 13 years ago, but many of these are poorly characterized, unavailable, or do not represent desired traits, thus making them unsuitable for application purposes. In order to provide the scientific community with better options, we have derived 12 new hESC lines at New York University from discarded genetically normal and abnormal embryos using the latest techniques. We examined the genetic status of the NYUES lines in detail as well as their molecular and cellular features and DNA fingerprinting profile. Furthermore, we differentiated our hESCs into the tissues most affected by a specific condition or into clinically desired cell types. To our knowledge, a number of characteristics of our hESCs have not been previously reported, for example, mutation for alpha thalassemia X-linked mental retardation syndrome, linkage to conditions with a genetic component such as asthma or poor sperm morphology, and novel combinations of ethnic backgrounds. Importantly, all of our undifferentiated euploid female lines tested to date did not show X chromosome inactivation, believed to result in superior potency. We continue to derive new hESC lines and add them to the NIH registry and other registries. This should facilitate the use of our hESCs and lead to advancements for patient-benefitting applications.

Genetic modification of stem cells for transplantation.

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Phillips, M Ian; Tang, Yao Liang

2008-01-14

Gene modification of cells prior to their transplantation, especially stem cells, enhances their survival and increases their function in cell therapy. Like the Trojan horse, the gene-modified cell has to gain entrance inside the host's walls and survive and deliver its transgene products. Using cellular, molecular and gene manipulation techniques the transplanted cell can be protected in a hostile environment from immune rejection, inflammation, hypoxia and apoptosis. Genetic engineering to modify cells involves constructing modules of functional gene sequences. They can be simple reporter genes or complex cassettes with gene switches, cell specific promoters and multiple transgenes. We discuss methods to deliver and construct gene cassettes with viral and non-viral delivery, siRNA, and conditional Cre/Lox P. We review the current uses of gene-modified stem cells in cardiovascular disease, diabetes, neurological diseases, (including Parkinson's, Alzheimer's and spinal cord injury repair), bone defects, hemophilia, and cancer.

Type II NKT-TFH cells against Gaucher lipids regulate B-cell immunity and inflammation.

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Nair, Shiny; Boddupalli, Chandra Sekhar; Verma, Rakesh; Liu, Jun; Yang, Ruhua; Pastores, Gregory M; Mistry, Pramod K; Dhodapkar, Madhav V

2015-02-19

Chronic inflammation including B-cell activation is commonly observed in both inherited (Gaucher disease [GD]) and acquired disorders of lipid metabolism. However, the cellular mechanisms underlying B-cell activation in these settings remain to be elucidated. Here, we report that β-glucosylceramide 22:0 (βGL1-22) and glucosylsphingosine (LGL1), 2 major sphingolipids accumulated in GD, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT) cells. Human βGL1-22- and LGL1-reactive CD1d tetramer-positive T cells have a distinct T-cell receptor usage and genomic and cytokine profiles compared with the classical type I NKT cells. In contrast to type I NKT cells, βGL1-22- and LGL1-specific NKT cells constitutively express T-follicular helper (TFH) phenotype. Injection of these lipids leads to an increase in respective lipid-specific type II NKT cells in vivo and downstream induction of germinal center B cells, hypergammaglobulinemia, and production of antilipid antibodies. Human βGL1-22- and LGL1-specific NKT cells can provide efficient cognate help to B cells in vitro. Frequency of LGL1-specific T cells in GD mouse models and patients correlates with disease activity and therapeutic response. Our studies identify a novel type II NKT-mediated pathway for glucosphingolipid-mediated dysregulation of humoral immunity and increased risk of B-cell malignancy observed in metabolic lipid disorders. © 2015 by The American Society of Hematology.

Direct evidence for a chronic CD8+-T-cell-mediated immune reaction to tax within the muscle of a human T-cell leukemia/lymphoma virus type 1-infected patient with sporadic inclusion body myositis.

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Ozden, Simona; Cochet, Madeleine; Mikol, Jacqueline; Teixeira, Antonio; Gessain, Antoine; Pique, Claudine

2004-10-01

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) infection can lead to the development of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), concomitantly with or without other inflammatory disorders such as myositis. These pathologies are considered immune-mediated diseases, and it is assumed that migration within tissues of both HTLV-1-infected CD4(+) T cells and anti-HTLV-1 cytotoxic T cells represents a pivotal event. However, although HTLV-1-infected T cells were found in inflamed lesions, the antigenic specificity of coinfiltrated CD8(+) T cells remains to be determined. In this study, we performed both ex vivo and in situ analyses using muscle biopsies obtained from an HTLV-1-infected patient with HAM/TSP and sporadic inclusion body myositis. We found that both HTLV-1-infected CD4(+) T cells and CD8(+) T cells directed to the dominant Tax antigen can be amplified from muscle cell cultures. Moreover, we were able to detect in two successive muscle biopsies both tax mRNA-positive mononuclear cells and T cells recognized by the Tax11-19/HLA-A*02 tetramer and positive for perforin. These findings provide the first direct demonstration that anti-Tax cytotoxic T cells are chronically recruited within inflamed tissues of an HTLV-1 infected patient, which validates the cytotoxic immune reaction model for the pathogenesis of HTLV-1-associated inflammatory disease.

RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell.

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Ryan Chong

Full Text Available Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin

Therapeutic T cells induce tumor-directed chemotaxis of innate immune cells through tumor-specific secretion of chemokines and stimulation of B16BL6 melanoma to secrete chemokines

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Fox Bernard A

2007-11-01

Full Text Available Abstract Background The mechanisms by which tumor-specific T cells induce regression of established metastases are not fully characterized. In using the poorly immunogenic B16BL6-D5 (D5 melanoma model we reported that T cell-mediated tumor regression can occur independently of perforin, IFN-γ or the combination of both. Characterization of regressing pulmonary metastases identified macrophages as a major component of the cells infiltrating the tumor after adoptive transfer of effector T cells. This led us to hypothesize that macrophages played a central role in tumor regression following T-cell transfer. Here, we sought to determine the factors responsible for the infiltration of macrophages at the tumor site. Methods These studies used the poorly immunogenic D5 melanoma model. Tumor-specific effector T cells, generated from tumor vaccine-draining lymph nodes (TVDLN, were used for adoptive immunotherapy and in vitro analysis of chemokine expression. Cellular infiltrates into pulmonary metastases were determined by immunohistochemistry. Chemokine expression by the D5 melanoma following co-culture with T cells, IFN-γ or TNF-α was determined by RT-PCR and ELISA. Functional activity of chemokines was confirmed using a macrophage migration assay. T cell activation of macrophages to release nitric oxide (NO was determined using GRIES reagent. Results We observed that tumor-specific T cells with a type 1 cytokine profile also expressed message for and secreted RANTES, MIP-1α and MIP-1β following stimulation with specific tumor. Unexpectedly, D5 melanoma cells cultured with IFN-γ or TNF-α, two type 1 cytokines expressed by therapeutic T cells, secreted Keratinocyte Chemoattractant (KC, MCP-1, IP-10 and RANTES and expressed mRNA for MIG. The chemokines released by T cells and cytokine-stimulated tumor cells were functional and induced migration of the DJ2PM macrophage cell line. Additionally, tumor-specific stimulation of wt or perforin

Amniotic Fluid Stem Cells: A Novel Source for Modeling of Human Genetic Diseases

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Ivana Antonucci

2016-04-01

Full Text Available In recent years, great interest has been devoted to the use of Induced Pluripotent Stem cells (iPS for modeling of human genetic diseases, due to the possibility of reprogramming somatic cells of affected patients into pluripotent cells, enabling differentiation into several cell types, and allowing investigations into the molecular mechanisms of the disease. However, the protocol of iPS generation still suffers from technical limitations, showing low efficiency, being expensive and time consuming. Amniotic Fluid Stem cells (AFS represent a potential alternative novel source of stem cells for modeling of human genetic diseases. In fact, by means of prenatal diagnosis, a number of fetuses affected by chromosomal or Mendelian diseases can be identified, and the amniotic fluid collected for genetic testing can be used, after diagnosis, for the isolation, culture and differentiation of AFS cells. This can provide a useful stem cell model for the investigation of the molecular basis of the diagnosed disease without the necessity of producing iPS, since AFS cells show some features of pluripotency and are able to differentiate in cells derived from all three germ layers “in vitro”. In this article, we describe the potential benefits provided by using AFS cells in the modeling of human genetic diseases.

The role of monocytes and monocyte-derived dendritic cells in type 1 diabetes mellitus and autoimmune thyroid disease

NARCIS (Netherlands)

W.K. Lam-Tse

2003-01-01

textabstractType 1 diabetes mellitus (DM1) and autoimmune thyroid disease (AITD) are organ specific autoimmune diseases in which the immune system is directed against the ß cells and the thyrocytes respectively. The etio-pathogenesis of organ-specific or endocrine autoimmune diseases is complex,

Revisit the Candidacy of Brain Cell Types as the Cell(s of Origin for Human High-Grade Glioma

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Fangjie Shao

2018-02-01

Full Text Available High-grade glioma, particularly, glioblastoma, is the most aggressive cancer of the central nervous system (CNS in adults. Due to its heterogeneous nature, glioblastoma almost inevitably relapses after surgical resection and radio-/chemotherapy, and is thus highly lethal and associated with a dismal prognosis. Identifying the cell of origin has been considered an important aspect in understanding tumor heterogeneity, thereby holding great promise in designing novel therapeutic strategies for glioblastoma. Taking advantage of genetic lineage-tracing techniques, performed mainly on genetically engineered mouse models (GEMMs, multiple cell types in the CNS have been suggested as potential cells of origin for glioblastoma, among which adult neural stem cells (NSCs and oligodendrocyte precursor cells (OPCs are the major candidates. However, it remains highly debated whether these cell types are equally capable of transforming in patients, given that in the human brain, some cell types divide so slowly, therefore may never have a chance to transform. With the recent advances in studying adult NSCs and OPCs, particularly from the perspective of comparative biology, we now realize that notable differences exist among mammalian species. These differences have critical impacts on shaping our understanding of the cell of origin of glioma in humans. In this perspective, we update the current progress in this field and clarify some misconceptions with inputs from important findings about the biology of adult NSCs and OPCs. We propose to re-evaluate the cellular origin candidacy of these cells, with an emphasis on comparative studies between animal models and humans.

Bioengineering a non-genotoxic vector for genetic modification of mesenchymal stem cells.

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Chen, Xuguang; Nomani, Alireza; Patel, Niket; Nouri, Faranak S; Hatefi, Arash

2018-01-01

Vectors used for stem cell transfection must be non-genotoxic, in addition to possessing high efficiency, because they could potentially transform normal stem cells into cancer-initiating cells. The objective of this research was to bioengineer an efficient vector that can be used for genetic modification of stem cells without any negative somatic or genetic impact. Two types of multifunctional vectors, namely targeted and non-targeted were genetically engineered and purified from E. coli. The targeted vectors were designed to enter stem cells via overexpressed receptors. The non-targeted vectors were equipped with MPG and Pep1 cell penetrating peptides. A series of commercial synthetic non-viral vectors and an adenoviral vector were used as controls. All vectors were evaluated for their efficiency and impact on metabolic activity, cell membrane integrity, chromosomal aberrations (micronuclei formation), gene dysregulation, and differentiation ability of stem cells. The results of this study showed that the bioengineered vector utilizing VEGFR-1 receptors for cellular entry could transfect mesenchymal stem cells with high efficiency without inducing genotoxicity, negative impact on gene function, or ability to differentiate. Overall, the vectors that utilized receptors as ports for cellular entry (viral and non-viral) showed considerably better somato- and genosafety profiles in comparison to those that entered through electrostatic interaction with cellular membrane. The genetically engineered vector in this study demonstrated that it can be safely and efficiently used to genetically modify stem cells with potential applications in tissue engineering and cancer therapy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Direct imaging of APP proteolysis in living cells

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Niccoló Parenti

2017-04-01

Full Text Available Alzheimer’s disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of Aβ peptide is widely accepted as being one of the main key events triggering the development of Alzheimer’s disease. Aβ peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP. Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro. By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP, respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β-secretase BACE1, or the α-secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the

Direct imaging of APP proteolysis in living cells.

Science.gov (United States)

Parenti, Niccoló; Del Grosso, Ambra; Antoni, Claudia; Cecchini, Marco; Corradetti, Renato; Pavone, Francesco S; Calamai, Martino

2017-01-01

Alzheimer's disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of A β peptide is widely accepted as being one of the main key events triggering the development of Alzheimer's disease. A β peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro . By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP), respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β -secretase BACE1, or the α -secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing

Cell type-specific response to high intracellular loading of polyacrylic acid-coated magnetic nanoparticles

Science.gov (United States)

Lojk, Jasna; Bregar, Vladimir B; Rajh, Maruša; Miš, Katarina; Kreft, Mateja Erdani; Pirkmajer, Sergej; Veranič, Peter; Pavlin, Mojca

2015-01-01

Magnetic nanoparticles (NPs) are a special type of NP with a ferromagnetic, electron-dense core that enables several applications such as cell tracking, hyperthermia, and magnetic separation, as well as multimodality. So far, superparamagnetic iron oxide NPs (SPIONs) are the only clinically approved type of metal oxide NPs, but cobalt ferrite NPs have properties suitable for biomedical applications as well. In this study, we analyzed the cellular responses to magnetic cobalt ferrite NPs coated with polyacrylic acid (PAA) in three cell types: Chinese Hamster Ovary (CHO), mouse melanoma (B16) cell line, and primary human myoblasts (MYO). We compared the internalization pathway, intracellular trafficking, and intracellular fate of our NPs using fluorescence and transmission electron microscopy (TEM) as well as quantified NP uptake and analyzed uptake dynamics. We determined cell viability after 24 or 96 hours’ exposure to increasing concentrations of NPs, and quantified the generation of reactive oxygen species (ROS) upon 24 and 48 hours’ exposure. Our NPs have been shown to readily enter and accumulate in cells in high quantities using the same two endocytic pathways; mostly by macropinocytosis and partially by clathrin-mediated endocytosis. The cell types differed in their uptake rate, the dynamics of intracellular trafficking, and the uptake capacity, as well as in their response to higher concentrations of internalized NPs. The observed differences in cell responses stress the importance of evaluation of NP–cell interactions on several different cell types for better prediction of possible toxic effects on different cell and tissue types in vivo. PMID:25733835

Mutant PIK3CA Induces EMT in a Cell Type Specific Manner.

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Divya Bhagirath

Full Text Available Breast cancer is characterized into different molecular subtypes, and each subtype is characterized by differential gene expression that are associated with distinct survival outcomes in patients. PIK3CA mutations are commonly associated with most breast cancer subtypes. More recently PIK3CA mutations have been shown to induce tumor heterogeneity and are associated with activation of EGFR-signaling and reduced relapse free survival in basal subtype of breast cancer. Thus, understanding what determines PIK3CA induced heterogeneity and oncogenesis, is an important area of investigation. In this study, we assessed the effect of mutant PIK3CA together with mutant Ras plus mutant p53 on oncogenic behavior of two distinct stem/progenitor breast cell lines, designated as K5+/K19- and K5+/K19+. Constructs were ectopically overexpressed in K5+/K19- and K5+/K19+ stem/progenitor cells, followed by various in-vitro and in-vivo analyses. Oncogene combination m-Ras/m-p53/m-PIK3CA efficiently transformed both K5+/K19- and K5+/K19+ cell lines in-vitro, as assessed by anchorage-independent soft agar colony formation assay. Significantly, while this oncogene combination induced a complete epithelial-to-mesenchymal transition (EMT in K5+/K19- cell line, mostly epithelial phenotype with minor EMT component was seen in K5+/K19+ cell line. However, both K5+/K19- and K5+/K19+ transformed cells exhibited increased invasion and migration abilities. Analyses of CD44 and CD24 expression showed both cell lines had tumor-initiating CD44+/CD24low cell population, however transformed K5+/K19- cells had more proportion of these cells. Significantly, both cell types exhibited in-vivo tumorigenesis, and maintained their EMT and epithelial nature in-vivo in mice tumors. Notably, while both cell types exhibited increase in tumor-initiating cell population, differential EMT phenotype was observed in these cell lines. These results suggest that EMT is a cell type dependent

Impaired quality of the hepatitis B virus (HBV)-specific T-cell response in human immunodeficiency virus type 1-HBV coinfection.

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Chang, J Judy; Sirivichayakul, Sunee; Avihingsanon, Anchalee; Thompson, Alex J V; Revill, Peter; Iser, David; Slavin, John; Buranapraditkun, Supranee; Marks, Pip; Matthews, Gail; Cooper, David A; Kent, Stephen J; Cameron, Paul U; Sasadeusz, Joe; Desmond, Paul; Locarnini, Stephen; Dore, Gregory J; Ruxrungtham, Kiat; Lewin, Sharon R

2009-08-01

Hepatitis B virus (HBV)-specific T cells play a key role both in the control of HBV replication and in the pathogenesis of liver disease. Human immunodeficiency virus type 1 (HIV-1) coinfection and the presence or absence of HBV e (precore) antigen (HBeAg) significantly alter the natural history of chronic HBV infection. We examined the HBV-specific T-cell responses in treatment-naïve HBeAg-positive and HBeAg-negative HIV-1-HBV-coinfected (n = 24) and HBV-monoinfected (n = 39) Asian patients. Peripheral blood was stimulated with an overlapping peptide library for the whole HBV genome, and tumor necrosis factor alpha and gamma interferon cytokine expression in CD8+ T cells was measured by intracellular cytokine staining and flow cytometry. There was no difference in the overall magnitude of the HBV-specific T-cell responses, but the quality of the response was significantly impaired in HIV-1-HBV-coinfected patients compared with monoinfected patients. In coinfected patients, HBV-specific T cells rarely produced more than one cytokine and responded to fewer HBV proteins than in monoinfected patients. Overall, the frequency and quality of the HBV-specific T-cell responses increased with a higher CD4+ T-cell count (P = 0.018 and 0.032, respectively). There was no relationship between circulating HBV-specific T cells and liver damage as measured by activity and fibrosis scores, and the HBV-specific T-cell responses were not significantly different in patients with either HBeAg-positive or HBeAg-negative disease. The quality of the HBV-specific T-cell response is impaired in the setting of HIV-1-HBV coinfection and is related to the CD4+ T-cell count.

Counseling Customers: Emerging Roles for Genetic Counselors in the Direct-to-Consumer Genetic Testing Market

NARCIS (Netherlands)

Harris, A.; Kelly, S.; Wyatt, S.

2013-01-01

Individuals now have access to an increasing number of internet resources offering personal genomics services. As the direct-to-consumer genetic testing (DTC GT) industry expands, critics have called for pre- and post-test genetic counseling to be included with the product. Several genetic testing

The "where" of social attention: Head and body direction aftereffects arise from representations specific to cue type and not direction alone.

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Lawson, Rebecca P; Calder, Andrew J

2016-01-01

Human beings have remarkable social attention skills. From the initial processing of cues, such as eye gaze, head direction, and body orientation, we perceive where other people are attending, allowing us to draw inferences about the intentions, desires, and dispositions of others. But before we can infer why someone is attending to something in the world we must first accurately represent where they are attending. Here we investigate the "where" of social attention perception, and employ adaptation paradigms to ascertain how head and body orientation are visually represented in the human brain. Across two experiments we show that the representation of two cues to social attention (head and body orientation) exists at the category-specific level. This suggests that aftereffects do not arise from "social attention cells" discovered in macaques or from abstract representations of "leftness" or "rightness."

Cell-type-specific activation of mitogen-activated protein kinases in PAN-induced progressive renal disease in rats

International Nuclear Information System (INIS)

Park, Sang-Joon; Jeong, Kyu-Shik

2004-01-01

We examined the time-course activation and the cell-type specific role of MAP kinases in puromycin aminonucleoside (PAN)-induced renal disease. The maximal activation of c-Jun-NH 2 -terminal kinase (JNK), extracellular signal regulated kinase (ERK), and p38 MAP kinase was detected on Days 52, 38, and 38 after PAN-treatment, respectively. p-JNK was localized in mesangial and proximal tubular cells at the early renal injury. It was expressed, therefore, in the inflammatory cells of tubulointerstitial lesions. While, p-ERK was markedly increased in the glomerular regions and macrophages p-p38 was observed in glomerular endothelial cells, tubular cells, and some inflammatory cells. The results show that the activation of MAP kinases in the early renal injury by PAN-treatment involves cellular changes such as cell proliferation or apoptosis in renal native cells. The activation of MAP kinases in infiltrated inflammatory cells and fibrotic cells plays an important role in destructive events such as glomerulosclerosis and tubulointerstitial fibrosis

Chimeric antigen receptor (CAR-specific monoclonal antibody to detect CD19-specific T cells in clinical trials.

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Bipulendu Jena

Full Text Available Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63. We describe a novel anti-idiotype monoclonal antibody (mAb to detect CD19-specific CAR(+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1 was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19(+ tumor targets. This clone can be used to detect CD19-specific CAR(+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1 will be useful to investigators implementing CD19-specific CAR(+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy.

Interspecific sex in grass smuts and the genetic diversity of their pheromone-receptor system.

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Ronny Kellner

2011-12-01

Full Text Available The grass smuts comprise a speciose group of biotrophic plant parasites, so-called Ustilaginaceae, which are specifically adapted to hosts of sweet grasses, the Poaceae family. Mating takes a central role in their life cycle, as it initiates parasitism by a morphological and physiological transition from saprobic yeast cells to pathogenic filaments. As in other fungi, sexual identity is determined by specific genomic regions encoding allelic variants of a pheromone-receptor (PR system and heterodimerising transcription factors. Both operate in a biphasic mating process that starts with PR-triggered recognition, directed growth of conjugation hyphae, and plasmogamy of compatible mating partners. So far, studies on the PR system of grass smuts revealed diverse interspecific compatibility and mating type determination. However, many questions concerning the specificity and evolutionary origin of the PR system remain unanswered. Combining comparative genetics and biological approaches, we report on the specificity of the PR system and its genetic diversity in 10 species spanning about 100 million years of mating type evolution. We show that three highly syntenic PR alleles are prevalent among members of the Ustilaginaceae, favouring a triallelic determination as the plesiomorphic characteristic of this group. Furthermore, the analysis of PR loci revealed increased genetic diversity of single PR locus genes compared to genes of flanking regions. Performing interspecies sex tests, we detected a high potential for hybridisation that is directly linked to pheromone signalling as known from intraspecies sex. Although the PR system seems to be optimised for intraspecific compatibility, the observed functional plasticity of the PR system increases the potential for interspecific sex, which might allow the hybrid-based genesis of newly combined host specificities.

Effects of UV radiation on genetic recombination

International Nuclear Information System (INIS)

Vlahovic, K.; Zahradka, D.; Petranovic, M.; Petranovic, D.

1996-01-01

We have used the model consisting of Escherichia coli cells and l phage to study the effects of UV radiation on genetic recombination. We found two radiation induced processes that reduce or inhibit genetic recombination. One such process leads to the inability of prophage to excise itself from the irradiated bacterial chromosome by the site-specific recombination. The other process was shown to inhibit a type of general recombination by which the prophage transfers one of its genetic markers to the infecting homologous phage. Loss of the prophage ability to take part in both site-specific and general recombination was shown to develop in recB + but not in recB cells. From this we infer that the loss of prophage recombinogenicity in irradiated cells is a consequence of one process in which RecBCD enzyme (the product of recB, recC and recD genes) plays an essential role. (author)

Identification of cell surface targets for HIV-1 therapeutics using genetic screens

International Nuclear Information System (INIS)

Dunn, Stephen J.; Khan, Imran H.; Chan, Ursula A.; Scearce, Robin L.; Melara, Claudia L.; Paul, Amber M.; Sharma, Vikram; Bih, Fong-Yih; Holzmayer, Tanya A.; Luciw, Paul A.; Abo, Arie

2004-01-01

Human immunodeficiency virus (HIV) drugs designed to interfere with obligatory utilization of certain host cell factors by virus are less likely to encounter development of resistant strains than drugs directed against viral components. Several cellular genes required for productive infection by HIV were identified by the use of genetic suppressor element (GSE) technology as potential targets for anti-HIV drug development. Fragmented cDNA libraries from various pools of human peripheral blood mononuclear cells (PBMC) were expressed in vitro in human immunodeficiency virus type 1 (HIV-1)-susceptible cell lines and subjected to genetic screens to identify GSEs that interfered with viral replication. After three rounds of selection, more than 15 000 GSEs were sequenced, and the cognate genes were identified. The GSEs that inhibited the virus were derived from a diverse set of genes including cell surface receptors, cytokines, signaling proteins, transcription factors, as well as genes with unknown function. Approximately 2.5% of the identified genes were previously shown to play a role in the HIV-1 life cycle; this finding supports the biological relevance of the assay. GSEs were derived from the following 12 cell surface proteins: CXCR4, CCR4, CCR7, CD11C, CD44, CD47, CD68, CD69, CD74, CSF3R, GABBR1, and TNFR2. Requirement of some of these genes for viral infection was also investigated by using RNA interference (RNAi) technology; accordingly, 10 genes were implicated in early events of the viral life cycle, before viral DNA synthesis. Thus, these cell surface proteins represent novel targets for the development of therapeutics against HIV-1 infection and AIDS

Cell type-specific neuroprotective activity of untranslocated prion protein.

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Elena Restelli

2010-10-01

Full Text Available A key pathogenic role in prion diseases was proposed for a cytosolic form of the prion protein (PrP. However, it is not clear how cytosolic PrP localization influences neuronal viability, with either cytotoxic or anti-apoptotic effects reported in different studies. The cellular mechanism by which PrP is delivered to the cytosol of neurons is also debated, and either retrograde transport from the endoplasmic reticulum or inefficient translocation during biosynthesis has been proposed. We investigated cytosolic PrP biogenesis and effect on cell viability in primary neuronal cultures from different mouse brain regions.Mild proteasome inhibition induced accumulation of an untranslocated form of cytosolic PrP in cortical and hippocampal cells, but not in cerebellar granules. A cyclopeptolide that interferes with the correct insertion of the PrP signal sequence into the translocon increased the amount of untranslocated PrP in cortical and hippocampal cells, and induced its synthesis in cerebellar neurons. Untranslocated PrP boosted the resistance of cortical and hippocampal neurons to apoptotic insults but had no effect on cerebellar cells.These results indicate cell type-dependent differences in the efficiency of PrP translocation, and argue that cytosolic PrP targeting might serve a physiological neuroprotective function.

Genetic variation and DNA fingerprinting of durian types in Malaysia using simple sequence repeat (SSR) markers.

Science.gov (United States)

Siew, Ging Yang; Ng, Wei Lun; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Tan, Soon Guan; Yeap, Swee Keong

2018-01-01

Durian ( Durio zibethinus ) is one of the most popular tropical fruits in Asia. To date, 126 durian types have been registered with the Department of Agriculture in Malaysia based on phenotypic characteristics. Classification based on morphology is convenient, easy, and fast but it suffers from phenotypic plasticity as a direct result of environmental factors and age. To overcome the limitation of morphological classification, there is a need to carry out genetic characterization of the various durian types. Such data is important for the evaluation and management of durian genetic resources in producing countries. In this study, simple sequence repeat (SSR) markers were used to study the genetic variation in 27 durian types from the germplasm collection of Universiti Putra Malaysia. Based on DNA sequences deposited in Genbank, seven pairs of primers were successfully designed to amplify SSR regions in the durian DNA samples. High levels of variation among the 27 durian types were observed (expected heterozygosity, H E  = 0.35). The DNA fingerprinting power of SSR markers revealed by the combined probability of identity (PI) of all loci was 2.3×10 -3 . Unique DNA fingerprints were generated for 21 out of 27 durian types using five polymorphic SSR markers (the other two SSR markers were monomorphic). We further tested the utility of these markers by evaluating the clonal status of shared durian types from different germplasm collection sites, and found that some were not clones. The findings in this preliminary study not only shows the feasibility of using SSR markers for DNA fingerprinting of durian types, but also challenges the current classification of durian types, e.g., on whether the different types should be called "clones", "varieties", or "cultivars". Such matters have a direct impact on the regulation and management of durian genetic resources in the region.

ABO alleles are linked with haplotypes of an erythroid cell-specific regulatory element in intron 1 with a few exceptions attributable to genetic recombination.

Science.gov (United States)

Nakajima, T; Sano, R; Takahashi, Y; Watanabe, K; Kubo, R; Kobayashi, M; Takahashi, K; Takeshita, H; Kominato, Y

2016-01-01

Recent investigation of transcriptional regulation of the ABO genes has identified a candidate erythroid cell-specific regulatory element, named the +5·8-kb site, in the first intron of ABO. Six haplotypes of the site have been reported previously. The present genetic population study demonstrated that each haplotype was mostly linked with specific ABO alleles with a few exceptions, possibly as a result of hybrid formation between common ABO alleles. Thus, investigation of these haplotypes could provide a clue to further elucidation of ABO alleles. © 2015 International Society of Blood Transfusion.

β-cell-specific IL-2 therapy increases islet Foxp3+Treg and suppresses type 1 diabetes in NOD mice.

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Johnson, Mark C; Garland, Alaina L; Nicolson, Sarah C; Li, Chengwen; Samulski, R Jude; Wang, Bo; Tisch, Roland

2013-11-01

Interleukin-2 (IL-2) is a critical cytokine for the homeostasis and function of forkhead box p3-expressing regulatory T cells (Foxp3(+)Tregs). Dysregulation of the IL-2-IL-2 receptor axis is associated with aberrant Foxp3(+)Tregs and T cell-mediated autoimmune diseases such as type 1 diabetes. Treatment with recombinant IL-2 has been reported to enhance Foxp3(+)Tregs and suppress different models of autoimmunity. However, efficacy of IL-2 therapy is dependent on achieving sufficient levels of IL-2 to boost tissue-resident Foxp3(+)Tregs while avoiding the potential toxic effects of systemic IL-2. With this in mind, adeno-associated virus (AAV) vector gene delivery was used to localize IL-2 expression to the islets of NOD mice. Injection of a double-stranded AAV vector encoding IL-2 driven by a mouse insulin promoter (dsAAVmIP-IL2) increased Foxp3(+)Tregs in the islets but not the draining pancreatic lymph nodes. Islet Foxp3(+)Tregs in dsAAVmIP-IL2-treated NOD mice exhibited enhanced fitness marked by increased expression of Bcl-2, proliferation, and suppressor function. In contrast, ectopic IL-2 had no significant effect on conventional islet-infiltrating effector T cells. Notably, β-cell-specific IL-2 expression suppressed late preclinical type 1 diabetes in NOD mice. Collectively, these findings demonstrate that β-cell-specific IL-2 expands an islet-resident Foxp3(+)Tregs pool that effectively suppresses ongoing type 1 diabetes long term.

The genetics of hair-cell function in zebrafish.

Science.gov (United States)

Nicolson, Teresa

2017-09-01

Our ears are remarkable sensory organs, providing the important senses of balance and hearing. The complex structure of the inner ear, or 'labyrinth', along with the assorted neuroepithelia, have evolved to detect head movements and sounds with impressive sensitivity. The rub is that the inner ear is highly vulnerable to genetic lesions and environmental insults. According to National Institute of Health estimates, hearing loss is one of the most commonly inherited or acquired sensorineural diseases. To understand the causes of deafness and balance disorders, it is imperative to understand the underlying biology of the inner ear, especially the inner workings of the sensory receptors. These receptors, which are termed hair cells, are particularly susceptible to genetic mutations - more than two dozen genes are associated with defects in this cell type in humans. Over the past decade, a substantial amount of progress has been made in working out the molecular basis of hair-cell function using vertebrate animal models. Given the transparency of the inner ear and the genetic tools that are available, zebrafish have become an increasingly popular animal model for the study of deafness and vestibular dysfunction. Mutagenesis screens for larval defects in hearing and balance have been fruitful in finding key components, many of which have been implicated in human deafness. This review will focus on the genes that are required for hair-cell function in zebrafish, with a particular emphasis on mechanotransduction. In addition, the generation of new tools available for the characterization of zebrafish hair-cell mutants will be discussed.

Cell-type specific oxytocin gene expression from AAV delivered promoter deletion constructs into the rat supraoptic nucleus in vivo.

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Raymond L Fields

Full Text Available The magnocellular neurons (MCNs in the hypothalamus selectively express either oxytocin (OXT or vasopressin (AVP neuropeptide genes, a property that defines their phenotypes. Here we examine the molecular basis of this selectivity in the OXT MCNs by stereotaxic microinjections of adeno-associated virus (AAV vectors that contain various OXT gene promoter deletion constructs using EGFP as the reporter into the rat supraoptic nucleus (SON. Two weeks following injection of the AAVs, immunohistochemical assays of EGFP expression from these constructs were done to determine whether the EGFP reporter co-localizes with either the OXT- or AVP-immunoreactivity in the MCNs. The results show that the key elements in the OT gene promoter that regulate the cell-type specific expression the SON are located -216 to -100 bp upstream of the transcription start site. We hypothesize that within this 116 bp domain a repressor exists that inhibits expression specifically in AVP MCNs, thereby leading to the cell-type specific expression of the OXT gene only in the OXT MCNs.