Imaging Voltage in Genetically Defined Neuronal Subpopulations with a Cre Recombinase-Targeted Hybrid Voltage Sensor.

PubMed

Bayguinov, Peter O; Ma, Yihe; Gao, Yu; Zhao, Xinyu; Jackson, Meyer B

2017-09-20

Genetically encoded voltage indicators create an opportunity to monitor electrical activity in defined sets of neurons as they participate in the complex patterns of coordinated electrical activity that underlie nervous system function. Taking full advantage of genetically encoded voltage indicators requires a generalized strategy for targeting the probe to genetically defined populations of cells. To this end, we have generated a mouse line with an optimized hybrid voltage sensor (hVOS) probe within a locus designed for efficient Cre recombinase-dependent expression. Crossing this mouse with Cre drivers generated double transgenics expressing hVOS probe in GABAergic, parvalbumin, and calretinin interneurons, as well as hilar mossy cells, new adult-born neurons, and recently active neurons. In each case, imaging in brain slices from male or female animals revealed electrically evoked optical signals from multiple individual neurons in single trials. These imaging experiments revealed action potentials, dynamic aspects of dendritic integration, and trial-to-trial fluctuations in response latency. The rapid time response of hVOS imaging revealed action potentials with high temporal fidelity, and enabled accurate measurements of spike half-widths characteristic of each cell type. Simultaneous recording of rapid voltage changes in multiple neurons with a common genetic signature offers a powerful approach to the study of neural circuit function and the investigation of how neural networks encode, process, and store information. SIGNIFICANCE STATEMENT Genetically encoded voltage indicators hold great promise in the study of neural circuitry, but realizing their full potential depends on targeting the sensor to distinct cell types. Here we present a new mouse line that expresses a hybrid optical voltage sensor under the control of Cre recombinase. Crossing this line with Cre drivers generated double-transgenic mice, which express this sensor in targeted cell types. In brain slices from these animals, single-trial hybrid optical voltage sensor recordings revealed voltage changes with submillisecond resolution in multiple neurons simultaneously. This imaging tool will allow for the study of the emergent properties of neural circuits and permit experimental tests of the roles of specific types of neurons in complex circuit activity. Copyright © 2017 the authors 0270-6474/17/379305-15$15.00/0.

Milan hypertensive rat as a model for studying cation transport abnormality in genetic hypertension

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

Ferrari, P.; Barber, B.R.; Torielli, L.

1987-11-01

Environmental factors, genetic polymorphisms, and different experimental designs have been the main impediments to evaluating a genetic association between cell membrane cation transport abnormalities and human essential or genetic hypertension. We review the results obtained in the Milan hypertensive strain of rats (MHS) and in its appropriate control normotensive strain (MNS) to illustrate our approach to defining the role of cation transport abnormality in a type of genetic hypertension. Before the development of a difference in blood pressure between the two strains, the comparison of kidney and erythrocyte functions showed that MHS had an increased glomerular filtration rate and urinarymore » output, and lower plasma renin and urine osmolality. Kidney cross-transplantation between the strains showed that hypertension is transplanted with the kidney. Proximal tubular cell volume and sodium content were lower in MHS while sodium transport across the brush border membrane vesicles of MHS was faster. Erythrocytes in MHS were smaller and had lower sodium concentration, and Na+-K+ cotransport and passive permeability were faster. The differences in volume, sodium content, and Na+-K+ cotransport between erythrocytes of the two strains persisted after transplantation of bone marrow to irradiated F1 (MHS X MNS) hybrids. Moreover, in normal segregating F2 hybrid populations there was a positive correlation between blood pressure and Na+-K+ cotransport. These results suggest a genetic and functional link in MHS between cell membrane cation transport abnormalities and hypertension. Thus, erythrocyte cell membrane may be used for approaching the problem of defining the genetically determined molecular mechanism underlying the development of a type of essential hypertension. 35 references.« less

Molecular and Genomic Alterations in Glioblastoma Multiforme.

PubMed

Crespo, Ines; Vital, Ana Louisa; Gonzalez-Tablas, María; Patino, María del Carmen; Otero, Alvaro; Lopes, María Celeste; de Oliveira, Catarina; Domingues, Patricia; Orfao, Alberto; Tabernero, Maria Dolores

2015-07-01

In recent years, important advances have been achieved in the understanding of the molecular biology of glioblastoma multiforme (GBM); thus, complex genetic alterations and genomic profiles, which recurrently involve multiple signaling pathways, have been defined, leading to the first molecular/genetic classification of the disease. In this regard, different genetic alterations and genetic pathways appear to distinguish primary (eg, EGFR amplification) versus secondary (eg, IDH1/2 or TP53 mutation) GBM. Such genetic alterations target distinct combinations of the growth factor receptor-ras signaling pathways, as well as the phosphatidylinositol 3-kinase/phosphatase and tensin homolog/AKT, retinoblastoma/cyclin-dependent kinase (CDK) N2A-p16(INK4A), and TP53/mouse double minute (MDM) 2/MDM4/CDKN2A-p14(ARF) pathways, in cells that present features associated with key stages of normal neurogenesis and (normal) central nervous system cell types. This translates into well-defined genomic profiles that have been recently classified by The Cancer Genome Atlas Consortium into four subtypes: classic, mesenchymal, proneural, and neural GBM. Herein, we review the most relevant genetic alterations of primary versus secondary GBM, the specific signaling pathways involved, and the overall genomic profile of this genetically heterogeneous group of malignant tumors. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Integrated single-cell genetic and transcriptional analysis suggests novel drivers of chronic lymphocytic leukemia

PubMed Central

Wang, Lili; Fan, Jean; Francis, Joshua M.; Georghiou, George; Hergert, Sarah; Li, Shuqiang; Gambe, Rutendo; Zhou, Chensheng W.; Yang, Chunxiao; Xiao, Sheng; Cin, Paola Dal; Bowden, Michaela; Kotliar, Dylan; Shukla, Sachet A.; Brown, Jennifer R.; Neuberg, Donna; Alessi, Dario R.; Zhang, Cheng-Zhong; Kharchenko, Peter V.; Livak, Kenneth J.; Wu, Catherine J.

2017-01-01

Intra-tumoral genetic heterogeneity has been characterized across cancers by genome sequencing of bulk tumors, including chronic lymphocytic leukemia (CLL). In order to more accurately identify subclones, define phylogenetic relationships, and probe genotype–phenotype relationships, we developed methods for targeted mutation detection in DNA and RNA isolated from thousands of single cells from five CLL samples. By clearly resolving phylogenic relationships, we uncovered mutated LCP1 and WNK1 as novel CLL drivers, supported by functional evidence demonstrating their impact on CLL pathways. Integrative analysis of somatic mutations with transcriptional states prompts the idea that convergent evolution generates phenotypically similar cells in distinct genetic branches, thus creating a cohesive expression profile in each CLL sample despite the presence of genetic heterogeneity. Our study highlights the potential for single-cell RNA-based targeted analysis to sensitively determine transcriptional and mutational profiles of individual cancer cells, leading to increased understanding of driving events in malignancy. PMID:28679620

Select phytochemicals suppress human T-lymphocytes and mouse splenocytes suggesting their use in autoimmunity and transplantation

PubMed Central

Hushmendy, Shazaan; Jayakumar, Lalithapriya; Hahn, Amy B.; Bhoiwala, Devang; Bhoiwala, Dipti L.; Crawford, Dana R.

2009-01-01

We have considered a novel “rational” gene targeting approach for treating pathologies whose genetic bases are defined using select phytochemicals. We reason that one such potential application of this approach would be conditions requiring immunosuppression such as autoimmune disease and transplantation, where the genetic target is clearly defined; i.e., interleukin-2 and associated T-cell activation. Therefore, we hypothesized that select phytochemicals can suppress T-lymphocyte proliferation both in vitro and in vivo. The immunosuppressive effects of berry extract, curcumin, quercetin, sulforaphane, epigallocatechin gallate (EGCG), resveratrol, α-tocopherol, vitamin C and sucrose were tested on anti-CD3 plus anti-CD28-activated primary human T-lymphocytes in culture. Curcumin, sulforaphane, quercetin, berry extract and EGCG all significantly inhibited T-cell proliferation, and this effect was not due to toxicity. IL-2 production was also reduced by these agents, implicating this important T-cell cytokine in proliferation suppression. Except for berry extract, these same agents also inhibited mouse splenic T-cell proliferation and IL-2 production. Subsequent in vivo studies revealed that quercetin (but not sulforaphane) modestly suppressed mouse splenocyte proliferation following supplementation of BALB/c mice diets. This effect was especially prominent if corrected for the loss of supplement “recall” as observed in cultured T-cells. These results suggest the potential use of these select phytochemicals for treating autoimmune and transplant patients, and support our strategy of using select phytochemicals to treat genetically-defined pathologies, an approach that we believe is simple, healthy, and cost-effective. PMID:19761891

Bone Cell Bioenergetics and Skeletal Energy Homeostasis

PubMed Central

Riddle, Ryan C.; Clemens, Thomas L.

2017-01-01

The rising incidence of metabolic diseases worldwide has prompted renewed interest in the study of intermediary metabolism and cellular bioenergetics. The application of modern biochemical methods for quantitating fuel substrate metabolism with advanced mouse genetic approaches has greatly increased understanding of the mechanisms that integrate energy metabolism in the whole organism. Examination of the intermediary metabolism of skeletal cells has been sparked by a series of unanticipated observations in genetically modified mice that suggest the existence of novel endocrine pathways through which bone cells communicate their energy status to other centers of metabolic control. The recognition of this expanded role of the skeleton has in turn led to new lines of inquiry directed at defining the fuel requirements and bioenergetic properties of bone cells. This article provides a comprehensive review of historical and contemporary studies on the metabolic properties of bone cells and the mechanisms that control energy substrate utilization and bioenergetics. Special attention is devoted to identifying gaps in our current understanding of this new area of skeletal biology that will require additional research to better define the physiological significance of skeletal cell bioenergetics in human health and disease. PMID:28202599

The impact of ex vivo clinical grade activation protocols on human T-cell phenotype and function for the generation of genetically modified cells for adoptive cell transfer therapy.

PubMed

Tumeh, Paul C; Koya, Richard C; Chodon, Thinle; Graham, Nicholas A; Graeber, Thomas G; Comin-Anduix, Begoña; Ribas, Antoni

2010-10-01

Optimized conditions for the ex vivo activation, genetic manipulation, and expansion of human lymphocytes for adoptive cell therapy may lead to protocols that maximize their in vivo function. We analyzed the effects of 4 clinical grade activation and expansion protocols over 3 weeks on cell proliferative rate, immunophenotype, cell metabolism, and transduction efficiency of human peripheral blood mononuclear cells (PBMCs). Peak lentiviral transduction efficiency was early (days 2 to 4), at a time when cells showed a larger size, maximal uptake of metabolic substrates, and the highest level of proximal T-cell receptor signaling engagement. Anti-CD2/3/28 activation beads induced greater proliferation rate and skewed PBMCs early on to a CD4 phenotype when compared with the cells cultured in OKT3. Multicolor surface phenotyping demonstrated that changes in T-cell surface markers that define T-cell functional phenotypes were dependent on the time spent in culture as opposed to the particular activation protocol. In conclusion, ex vivo activation of human PBMCs for adoptive cell therapy demonstrate defined immunophenotypic and functional signatures over time, with cells early on showing larger sizes, higher transduction efficiency, maximal metabolic activity, and zeta-chain-associated protein-70 activation.

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities

PubMed Central

Vizeacoumar, Franco J; Arnold, Roland; Vizeacoumar, Frederick S; Chandrashekhar, Megha; Buzina, Alla; Young, Jordan T F; Kwan, Julian H M; Sayad, Azin; Mero, Patricia; Lawo, Steffen; Tanaka, Hiromasa; Brown, Kevin R; Baryshnikova, Anastasia; Mak, Anthony B; Fedyshyn, Yaroslav; Wang, Yadong; Brito, Glauber C; Kasimer, Dahlia; Makhnevych, Taras; Ketela, Troy; Datti, Alessandro; Babu, Mohan; Emili, Andrew; Pelletier, Laurence; Wrana, Jeff; Wainberg, Zev; Kim, Philip M; Rottapel, Robert; O'Brien, Catherine A; Andrews, Brenda; Boone, Charles; Moffat, Jason

2013-01-01

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN−/− DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model. PMID:24104479

Presence of a non-HLA B cell antigen in rheumatic fever patients and their families as defined by a monoclonal antibody.

PubMed Central

Khanna, A K; Buskirk, D R; Williams, R C; Gibofsky, A; Crow, M K; Menon, A; Fotino, M; Reid, H M; Poon-King, T; Rubinstein, P

1989-01-01

Numerous investigators have suspected that there is a genetic predisposition to rheumatic fever (RF). In this context we have recently produced a series of monoclonal antibodies directed against B cells obtained from RF patients one of which, labeled D8/17, identifies a B cell antigen present in 100% of all RF patients studied. While the highest percentage of positive cells were exhibited by RF probands (33.5% +/- SE), the percentage of cells in unaffected siblings and parents was 14.6 and 13%, respectively. The percentage of positive cells in APSGN probands, unaffected siblings, and parents was 2.96, 3.86, and 2.8%, respectively. A low level of B cells (5-7%) bearing the D8/17 marker was seen in control patients. The segregation pattern of the phenotypes defined by the percentage of D8/17 positive cells within HLA-typed RF families are consistent with an autosomal recessive mode of inheritance not associated with the human MHC system. We postulate that these phenotypes indicate the presence of at least one necessary genetic factor for susceptibility to RF. PMID:2785121

The impact of ex vivo clinical grade activation protocols on human T cell phenotype and function for the generation of genetically modified cells for adoptive cell transfer therapy

PubMed Central

Tumeh, Paul C.; Koya, Richard C.; Chodon, Thinle; Graham, Nicholas A.; Graeber, Thomas G.; Comin-Anduix, Begoña; Ribas, Antoni

2011-01-01

Optimized conditions for the ex vivo activation, genetic manipulation, and expansion of human lymphocytes for adoptive cell therapy (ACT) may lead to protocols that maximize their in vivo function. We analyzed the effects of four clinical grade activation and expansion protocols over three weeks on cell proliferative rate, immunophenotype, cell metabolism, and transduction efficiency of human peripheral blood mononuclear cells (PBMCs). Peak lentiviral transduction efficiency was early (days 2 to 4), at a time when cells demonstrated a larger size, maximal uptake of metabolic substrates, and the highest level of proximal TCR signaling engagement. Anti-CD2/3/28 activation beads induced greater proliferation rate and skewed PBMCs early on to a CD4 phenotype when compared to the cells cultured in OKT3. Multicolor surface phenotyping demonstrated that changes in T cell surface markers that define T cell functional phenotypes were dependent on the time spent in culture as opposed to the particular activation protocol. In conclusion, ex vivo activation of human PBMCs for ACT demonstrate defined immunophenotypic and functional signatures over time, with cells early on showing larger sizes, higher transduction efficiency, maximal metabolic activity and ZAP-70 activation. PMID:20842061

A versatile modular vector system for rapid combinatorial mammalian genetics.

PubMed

Albers, Joachim; Danzer, Claudia; Rechsteiner, Markus; Lehmann, Holger; Brandt, Laura P; Hejhal, Tomas; Catalano, Antonella; Busenhart, Philipp; Gonçalves, Ana Filipa; Brandt, Simone; Bode, Peter K; Bode-Lesniewska, Beata; Wild, Peter J; Frew, Ian J

2015-04-01

Here, we describe the multiple lentiviral expression (MuLE) system that allows multiple genetic alterations to be introduced simultaneously into mammalian cells. We created a toolbox of MuLE vectors that constitute a flexible, modular system for the rapid engineering of complex polycistronic lentiviruses, allowing combinatorial gene overexpression, gene knockdown, Cre-mediated gene deletion, or CRISPR/Cas9-mediated (where CRISPR indicates clustered regularly interspaced short palindromic repeats) gene mutation, together with expression of fluorescent or enzymatic reporters for cellular assays and animal imaging. Examples of tumor engineering were used to illustrate the speed and versatility of performing combinatorial genetics using the MuLE system. By transducing cultured primary mouse cells with single MuLE lentiviruses, we engineered tumors containing up to 5 different genetic alterations, identified genetic dependencies of molecularly defined tumors, conducted genetic interaction screens, and induced the simultaneous CRISPR/Cas9-mediated knockout of 3 tumor-suppressor genes. Intramuscular injection of MuLE viruses expressing oncogenic H-RasG12V together with combinations of knockdowns of the tumor suppressors cyclin-dependent kinase inhibitor 2A (Cdkn2a), transformation-related protein 53 (Trp53), and phosphatase and tensin homolog (Pten) allowed the generation of 3 murine sarcoma models, demonstrating that genetically defined autochthonous tumors can be rapidly generated and quantitatively monitored via direct injection of polycistronic MuLE lentiviruses into mouse tissues. Together, our results demonstrate that the MuLE system provides genetic power for the systematic investigation of the molecular mechanisms that underlie human diseases.

Genetic reprogramming of host cells by bacterial pathogens.

PubMed

Tran Van Nhieu, Guy; Arbibe, Laurence

2009-10-29

During the course of infection, pathogens often induce changes in gene expression in host cells and these changes can be long lasting and global or transient and of limited amplitude. Defining how, when, and why bacterial pathogens reprogram host cells represents an exciting challenge that opens up the opportunity to grasp the essence of pathogenesis and its molecular details.

Cell wall chitosan is necessary for virulence in the opportunistic pathogen Cryptococcus neoformans.

PubMed

Baker, Lorina G; Specht, Charles A; Lodge, Jennifer K

2011-09-01

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Its cell wall is composed of glucans, proteins, chitin, and chitosan. Multiple genetic approaches have defined a chitosan-deficient syndrome that includes slow growth and decreased cell integrity. Here we demonstrate chitosan is necessary for virulence and persistence in the mammalian host.

Minireview: Genetic basis of heterogeneity and severity in sickle cell disease

PubMed Central

Habara, Alawi

2016-01-01

Sickle cell disease, a common single gene disorder, has a complex pathophysiology that at its root is initiated by the polymerization of deoxy sickle hemoglobin. Sickle vasoocclusion and hemolytic anemia drive the development of disease complications. In this review, we focus on the genetic modifiers of disease heterogeneity. The phenotypic heterogeneity of disease is only partially explained by genetic variability of fetal hemoglobin gene expression and co-inheritance of α thalassemia. Given the complexity of pathophysiology, many different definitions of severity are possible complicating a full understanding of its genetic foundation. The pathophysiological complexity and the interlocking nature of the biological processes underpinning disease severity are becoming better understood. Nevertheless, useful genetic signatures of severity, regardless of how this is defined, are insufficiently developed to be used for treatment decisions and for counseling. PMID:26936084

Wnt addiction of genetically defined cancers reversed by PORCN inhibition.

PubMed

Madan, B; Ke, Z; Harmston, N; Ho, S Y; Frois, A O; Alam, J; Jeyaraj, D A; Pendharkar, V; Ghosh, K; Virshup, I H; Manoharan, V; Ong, E H Q; Sangthongpitag, K; Hill, J; Petretto, E; Keller, T H; Lee, M A; Matter, A; Virshup, D M

2016-04-28

Enhanced sensitivity to Wnts is an emerging hallmark of a subset of cancers, defined in part by mutations regulating the abundance of their receptors. Whether these mutations identify a clinical opportunity is an important question. Inhibition of Wnt secretion by blocking an essential post-translational modification, palmitoleation, provides a useful therapeutic intervention. We developed a novel potent, orally available PORCN inhibitor, ETC-1922159 (henceforth called ETC-159) that blocks the secretion and activity of all Wnts. ETC-159 is remarkably effective in treating RSPO-translocation bearing colorectal cancer (CRC) patient-derived xenografts. This is the first example of effective targeted therapy for this subset of CRC. Consistent with a central role of Wnt signaling in regulation of gene expression, inhibition of PORCN in RSPO3-translocated cancers causes a marked remodeling of the transcriptome, with loss of cell cycle, stem cell and proliferation genes, and an increase in differentiation markers. Inhibition of Wnt signaling by PORCN inhibition holds promise as differentiation therapy in genetically defined human cancers.

Caenorhabditis elegans in regenerative medicine: a simple model for a complex discipline.

PubMed

Aitlhadj, Layla; Stürzenbaum, Stephen R

2014-06-01

Stem cell research is a major focus of regenerative medicine, which amalgamates diverse disciplines ranging from developmental cell biology to chemical and genetic therapy. Although embryonic stem cells have provided the foundation of stem cell therapy, they offer an in vitro study system that might not provide the best insight into mechanisms and behaviour of cells within living organisms. Caenorhabditis elegans is a well defined model organism with highly conserved cell development and signalling processes that specify cell fate. Its genetic amenability coupled with its chemical screening applicability make the nematode well suited as an in vivo system in which regenerative therapy and stem cell processes can be explored. Here, we describe some of the major advances in stem cell research from the worm's perspective. Copyright © 2014 Elsevier Ltd. All rights reserved.

Using measures of single-cell physiology and physiological state to understand organismic aging.

PubMed

Mendenhall, Alexander; Driscoll, Monica; Brent, Roger

2016-02-01

Genetically identical organisms in homogeneous environments have different lifespans and healthspans. These differences are often attributed to stochastic events, such as mutations and 'epimutations', changes in DNA methylation and chromatin that change gene function and expression. But work in the last 10 years has revealed differences in lifespan- and health-related phenotypes that are not caused by lasting changes in DNA or identified by modifications to DNA or chromatin. This work has demonstrated persistent differences in single-cell and whole-organism physiological states operationally defined by values of reporter gene signals in living cells. While some single-cell states, for example, responses to oxygen deprivation, were defined previously, others, such as a generally heightened ability to make proteins, were, revealed by direct experiment only recently, and are not well understood. Here, we review technical progress that promises to greatly increase the number of these measurable single-cell physiological variables and measureable states. We discuss concepts that facilitate use of single-cell measurements to provide insight into physiological states and state transitions. We assert that researchers will use this information to relate cell level physiological readouts to whole-organism outcomes, to stratify aging populations into groups based on different physiologies, to define biomarkers predictive of outcomes, and to shed light on the molecular processes that bring about different individual physiologies. For these reasons, quantitative study of single-cell physiological variables and state transitions should provide a valuable complement to genetic and molecular explanations of how organisms age. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Dynamic neuroanatomy at subcellular resolution in the zebrafish.

PubMed

Faucherre, Adèle; López-Schier, Hernán

2014-01-01

Genetic means to visualize and manipulate neuronal circuits in the intact animal have revolutionized neurobiology. "Dynamic neuroanatomy" defines a range of approaches aimed at quantifying the architecture or subcellular organization of neurons over time during their development, regeneration, or degeneration. A general feature of these approaches is their reliance on the optical isolation of defined neurons in toto by genetically expressing markers in one or few cells. Here we use the afferent neurons of the lateral line as an example to describe a simple method for the dynamic neuroanatomical study of axon terminals in the zebrafish by laser-scanning confocal microscopy.

An Epigenetic Gateway to Brain Tumor Cell Identity

PubMed Central

Mack, Stephen C.; Hubert, Christopher G.; Miller, Tyler E.; Taylor, Michael D.; Rich, Jeremy N.

2017-01-01

Precise targeting of genetic lesions alone has been insufficient to extend brain tumor patient survival. Brain cancer cells are diverse in their genetic, metabolic, and microenvironmental compositions, accounting for their phenotypic heterogeneity and disparate responses to therapy. These factors converge at the level of the epigenome, representing a unified node that can be disrupted by pharmacologic inhibition. Aberrant epigenomes define many childhood and adult brain cancers, as demonstrated by widespread changes to DNA methylation patterns, redistribution of histone marks, and disruption of chromatin structure. In this review, we describe the convergence of genetic, metabolic, and micro-environmental factors upon mechanisms of epigenetic deregulation in brain cancer. We discuss how aberrant epigenetic pathways identified in brain tumors affect cell identity, cell state, and neoplastic transformation, in addition to the potential to exploit these alterations as novel therapeutic strategies for the treatment of brain cancer. PMID:26713744

Stem Cell Pathology.

PubMed

Fu, Dah-Jiun; Miller, Andrew D; Southard, Teresa L; Flesken-Nikitin, Andrea; Ellenson, Lora H; Nikitin, Alexander Yu

2018-01-24

Rapid advances in stem cell biology and regenerative medicine have opened new opportunities for better understanding disease pathogenesis and the development of new diagnostic, prognostic, and treatment approaches. Many stem cell niches are well defined anatomically, thereby allowing their routine pathological evaluation during disease initiation and progression. Evaluation of the consequences of genetic manipulations in stem cells and investigation of the roles of stem cells in regenerative medicine and pathogenesis of various diseases such as cancer require significant expertise in pathology for accurate interpretation of novel findings. Therefore, there is an urgent need for developing stem cell pathology as a discipline to facilitate stem cell research and regenerative medicine. This review provides examples of anatomically defined niches suitable for evaluation by diagnostic pathologists, describes neoplastic lesions associated with them, and discusses further directions of stem cell pathology.

Cell Wall Chitosan Is Necessary for Virulence in the Opportunistic Pathogen Cryptococcus neoformans ▿

PubMed Central

Baker, Lorina G.; Specht, Charles A.; Lodge, Jennifer K.

2011-01-01

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Its cell wall is composed of glucans, proteins, chitin, and chitosan. Multiple genetic approaches have defined a chitosan-deficient syndrome that includes slow growth and decreased cell integrity. Here we demonstrate chitosan is necessary for virulence and persistence in the mammalian host. PMID:21784998

Manipulation of cells with laser microbeam scissors and optical tweezers: a review

NASA Astrophysics Data System (ADS)

Greulich, Karl Otto

2017-02-01

The use of laser microbeams and optical tweezers in a wide field of biological applications from genomic to immunology is discussed. Microperforation is used to introduce a well-defined amount of molecules into cells for genetic engineering and optical imaging. The microwelding of two cells induced by a laser microbeam combines their genetic outfit. Microdissection allows specific regions of genomes to be isolated from a whole set of chromosomes. Handling the cells with optical tweezers supports investigation on the attack of immune systems against diseased or cancerous cells. With the help of laser microbeams, heart infarction can be simulated, and optical tweezers support studies on the heartbeat. Finally, laser microbeams are used to induce DNA damage in living cells for studies on cancer and ageing.

The Epimmunity Theory: The Single Cell Defenses against Infectious and Genetic Diseases.

PubMed

Barghouthi, Sameer A

2017-01-01

Single cell defense against diseases defines "epimmunity." Epimmunity is complementary to the immune system and can neither be substituted by innate nor by acquired immunity. Epimmunity, the proposed new branch of immunity, is further explored and analyzed for enucleated mature mammalian erythrocytes and nucleated erythrocytes of non-mammalian vertebrates leading to the development of "The Epimmunity Theory." Enucleation of mammalian erythroblast and inactivation of nuclei in erythrocytes of non-mammalian vertebrates are major contributors to the collective immunity: epimmunity, innate, and acquired. The fact that diseases of mature erythrocytes (MEs) are rare supports the notion that a single cell can resist microbial and genetic diseases; MEs are refractory to malaria and cancer. Nucleated cells, such as B-cells, T-cells, hepatocytes, and cell developmental stages are susceptible to genetic and specific microbial diseases depending on their nuclear activities and the receptors they express; such cells show lower epimmunity relative to MEs. Epimmunity is important as a disease insulator that prevents the spread of diseases from an infected tissue to the majority of other tissues. Breakdown of epimmunity may lead to disease development.

An Advanced Preclinical Mouse Model for Acute Myeloid Leukemia Using Patients' Cells of Various Genetic Subgroups and In Vivo Bioluminescence Imaging

PubMed Central

Vick, Binje; Rothenberg, Maja; Sandhöfer, Nadine; Carlet, Michela; Finkenzeller, Cornelia; Krupka, Christina; Grunert, Michaela; Trumpp, Andreas; Corbacioglu, Selim; Ebinger, Martin; André, Maya C.; Hiddemann, Wolfgang; Schneider, Stephanie; Subklewe, Marion; Metzeler, Klaus H.; Spiekermann, Karsten; Jeremias, Irmela

2015-01-01

Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Adequate model systems are required for preclinical studies to improve understanding of AML biology and to develop novel, rational treatment approaches. Xenografts in immunodeficient mice allow performing functional studies on patient-derived AML cells. We have established an improved model system that integrates serial retransplantation of patient-derived xenograft (PDX) cells in mice, genetic manipulation by lentiviral transduction, and essential quality controls by immunophenotyping and targeted resequencing of driver genes. 17/29 samples showed primary engraftment, 10/17 samples could be retransplanted and some of them allowed virtually indefinite serial transplantation. 5/6 samples were successfully transduced using lentiviruses. Neither serial transplantation nor genetic engineering markedly altered sample characteristics analyzed. Transgene expression was stable in PDX AML cells. Example given, recombinant luciferase enabled bioluminescence in vivo imaging and highly sensitive and reliable disease monitoring; imaging visualized minimal disease at 1 PDX cell in 10000 mouse bone marrow cells and facilitated quantifying leukemia initiating cells. We conclude that serial expansion, genetic engineering and imaging represent valuable tools to improve the individualized xenograft mouse model of AML. Prospectively, these advancements enable repetitive, clinically relevant studies on AML biology and preclinical treatment trials on genetically defined and heterogeneous subgroups. PMID:25793878

Monolayer culturing and cloning of human pluripotent stem cells on laminin-521-based matrices under xeno-free and chemically defined conditions.

PubMed

Rodin, Sergey; Antonsson, Liselotte; Hovatta, Outi; Tryggvason, Karl

2014-10-01

A robust method for culturing human pluripotent stem (hPS) cells under chemically defined and xeno-free conditions is an important tool for stem cell research and for the development of regenerative medicine. Here, we describe a protocol for monolayer culturing of Oct-4-positive hPS cells on a specific laminin-521 (LN-521) isoform, under xeno-free and chemically defined conditions. The cells are dispersed into single-cell suspension and then plated on LN-521 isoform at densities higher than 5,000 cells per cm², where they attach, migrate and survive by forming small monolayer cell groups. The cells avidly divide and expand horizontally until the entire dish is covered by a confluent monolayer. LN-521, in combination with E-cadherin, allows cloning of individual hPS cells in separate wells of 96-well plates without the presence of rho-associated protein kinase (ROCK) inhibitors or any other inhibitors of anoikis. Characterization of cells maintained for several months in culture reveals pluripotency with a minimal degree of genetic abnormalities.

Acute chest syndrome is associated with single nucleotide polymorphism-defined beta globin cluster haplotype in children with sickle cell anaemia

PubMed Central

Bean, Christopher J.; Boulet, Sheree L.; Yang, Genyan; Payne, Amanda B.; Ghaji, Nafisa; Pyle, Meredith E.; Hooper, W. Craig; Bhatnagar, Pallav; Keefer, Jeffrey; Barron-Casella, Emily A.; Casella, James F.; DeBaun, Michael R.

2013-01-01

Summary Genetic diversity at the human β-globin locus has been implicated as a modifier of sickle cell anaemia (SCA) severity. However, haplotypes defined by restriction fragment length polymorphism sites across the β-globin locus have not been consistently associated with clinical phenotypes. To define the genetic structure at the β-globin locus more thoroughly, we performed high-density single nucleotide polymorphism (SNP) mapping in 820 children who were homozygous for the sickle cell mutation (HbSS). Genotyping results revealed very high linkage disequilibrium across a large region spanning the locus control region and the HBB (β-globin gene) cluster. We identified three predominant haplotypes accounting for 96% of the βS-carrying chromosomes in this population that could be distinguished using a minimal set of common SNPs. Consistent with previous studies, fetal haemoglobin level was significantly associated with βS-haplotypes. After controlling for covariates, an association was detected between haplotype and rate of hospitalization for acute chest syndrome (ACS) (incidence rate ratio 0.51, 95% confidence interval 0.29–0.89) but not incidence rate of vaso-occlusive pain or presence of silent cerebral infarct (SCI). Our results suggest that these SNP-defined βS-haplotypes may be associated with ACS, but not pain or SCI in a study population of children with SCA. PMID:23952145

Organization of the Drosophila circadian control circuit.

PubMed

Nitabach, Michael N; Taghert, Paul H

2008-01-22

Molecular genetics has revealed the identities of several components of the fundamental circadian molecular oscillator - an evolutionarily conserved molecular mechanism of transcription and translation that can operate in a cell-autonomous manner. Therefore, it was surprising when studies of circadian rhythmic behavior in the fruit fly Drosophila suggested that the normal operations of circadian clock cells, which house the molecular oscillator, in fact depend on non-cell-autonomous effects - interactions between the clock cells themselves. Here we review several genetic analyses that broadly extend that viewpoint. They support a model whereby the approximately 150 circadian clock cells in the brain of the fly are sub-divided into functionally discrete rhythmic centers. These centers alternatively cooperate or compete to control the different episodes of rhythmic behavior that define the fly's daily activity profile.

[Isolation of circulating tumor cells in blood by means of "Isolation by SizE of Tumor cells (ISET)"].

PubMed

Liadov, V K; Skrypnikova, M A; Popova, O P

2014-01-01

There is evidence of the importance of circulating tumor cells in bloodstream as a factor of poor prognosis of cancer. The optimum method for isolating and studying of these cells is not defined. The most common methods are either based on the isolation of tumor genetic material from blood or on immune-mediated isolation of epithelial tumor cells. The first group of methods is characterized by a lack of specificity, while the latter do not allow identifying a pool of cells undergone in bloodstream epithelial-mesenchymal transformation. There is presented an overview of results of clinical trials of a new technique of isolation of tumor cells from bloodstream based on the patients' blood filtration through a membrane with defined pore sizes (ISET-Isolation by SizE of Tumor cells).

Replicative senescence of T cells: does the Hayflick Limit lead to immune exhaustion?

PubMed

Effros, R B; Pawelec, G

1997-09-01

Extensive in vitro research on fibroblasts has defined numerous genetic and phenotypic changes associated with replicative senescence. Identification of T-cell replicative senescence as a feature of human immunodeficiency virus (HIV) disease and ageing suggests this phenomenon merits more careful consideration by immunologists, especially with regard to chronic infection, memory and adoptive immunotherapy.

Krox20 defines a subpopulation of cardiac neural crest cells contributing to arterial valves and bicuspid aortic valve.

PubMed

Odelin, Gaëlle; Faure, Emilie; Coulpier, Fanny; Di Bonito, Maria; Bajolle, Fanny; Studer, Michèle; Avierinos, Jean-François; Charnay, Patrick; Topilko, Piotr; Zaffran, Stéphane

2018-01-03

Although cardiac neural crest cells are required at early stages of arterial valve development, their contribution during valvular leaflet maturation remains poorly understood. Here, we show in mouse that neural crest cells from pre-otic and post-otic regions make distinct contributions to the arterial valve leaflets. Genetic fate-mapping analysis of Krox20-expressing neural crest cells shows a large contribution to the borders and the interleaflet triangles of the arterial valves. Loss of Krox20 function results in hyperplastic aortic valve and partially penetrant bicuspid aortic valve formation. Similar defects are observed in neural crest Krox20 -deficient embryos. Genetic lineage tracing in Krox20 -/- mutant mice shows that endothelial-derived cells are normal, whereas neural crest-derived cells are abnormally increased in number and misplaced in the valve leaflets. In contrast, genetic ablation of Krox20 -expressing cells is not sufficient to cause an aortic valve defect, suggesting that adjacent cells can compensate this depletion. Our findings demonstrate a crucial role for Krox20 in arterial valve development and reveal that an excess of neural crest cells may be associated with bicuspid aortic valve. © 2018. Published by The Company of Biologists Ltd.

Identification of possible genetic alterations in the breast cancer cell line MCF-7 using high-density SNP genotyping microarray

PubMed Central

Wang, Hui-Yun; Greenawalt, Danielle; Cui, Xiangfeng; Tereshchenko, Irina V; Luo, Minjie; Yang, Qifeng; Azaro, Marco A; Hu, Guohong; Chu, Yi; Li, James Y; Shen, Li; Lin, Yong; Zhang, Lianjun

2009-01-01

Context: Cancer cell lines are used extensively in various research. Knowledge of genetic alterations in these lines is important for understanding mechanisms underlying their biology. However, since paired normal tissues are usually unavailable for comparison, precisely determining genetic alterations in cancer cell lines is difficult. To address this issue, a highly efficient and reliable method is developed. Aims: Establishing a highly efficient and reliable experimental system for genetic profiling of cell lines. Materials and Methods: A widely used breast cancer cell line, MCF-7, was genetically profiled with 4,396 single nucleotide polymorphisms (SNPs) spanning 11 whole chromosomes and two other small regions using a newly developed high-throughput multiplex genotyping approach. Results: The fractions of homozygous SNPs in MCF-7 (13.3%) were significantly lower than those in the control cell line and in 24 normal human individuals (25.1% and 27.4%, respectively). Homozygous SNPs in MCF-7 were found in clusters. The sizes of these clusters were significantly larger than the expected based on random allelic combination. Fourteen such regions were found on chromosomes 1p, 1q, 2q, 6q, 13, 15q, 16q, 17q and 18p in MCF-7 and two in the small regions. Conclusions: These results are generally concordant with those obtained using different approaches but are better in defining their chromosomal positions. The used approach provides a reliable way to detecting possible genetic alterations in cancer cell lines without paired normal tissues. PMID:19439911

PLURIPOTENT STEM CELL APPLICATIONS FOR REGENERATIVE MEDICINE

PubMed Central

Angelos, Mathew G.; Kaufman, Dan S.

2015-01-01

Purpose of Review In this review, we summarize the current status of clinical trials using therapeutic cells produced from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We also discuss combined cell and gene therapy via correction of defined mutations in human pluripotent stem cells and provide commentary on key obstacles facing wide-scale clinical adoption of pluripotent stem cell-based therapy. Recent Findings Initial data suggest hESC/hiPSC-derived cell products used for retinal repair and spinal cord injury are safe for human use. Early stage studies for treatment of cardiac injury and diabetes are also in progress. However, there remain key concerns regarding the safety and efficacy of these cells that need to be addressed in additional well-designed clinical trials. Advances using the CRISPR/Cas9 gene-editing system offer an improved tool for more rapid and on-target gene correction of genetic diseases. Combined gene and cell therapy using human pluripotent stem cells may provide an additional curative approach for disabling or lethal genetic and degenerative diseases where there are currently limited therapeutic opportunities. Summary Human pluripotent stem cells are emerging as a promising tool to produce cells and tissues suitable for regenerative therapy for a variety of genetic and degenerative diseases. PMID:26536430

The touch dome defines an epidermal niche specialized for mechanosensory signaling

PubMed Central

Doucet, Yanne S.; Woo, Seung-Hyun; Ruiz, Marlon E.; Owens, David M.

2013-01-01

Summary In mammalian skin, Merkel cells are mechanoreceptor cells that are required for the perception of gentle touch. Recent evidence indicates that mature Merkel cells descend from the proliferative layer of skin epidermis; however, the stem cell niche for Merkel cell homeostasis has not been reported. Here, we provide the first genetic evidence for maintenance of mature Merkel cells during homeostasis by Krt17+ stem cells located in epidermal touch domes of hairy skin and in the tips of the rete ridges of glabrous skin. Lineage tracing analysis indicated that the entire pool of mature Merkel cells is turned over every 7–8 weeks in adult epidermis and that Krt17+ stem cells also maintain squamous differentiation in the touch dome and in glabrous skin. Finally, selective genetic ablation of Krt17+ touch dome keratinocytes indicates that these cells, and not mature Merkel cells, are primarily responsible for maintaining innervation of the Merkel cell-neurite complex. PMID:23727240

Embryonic mammary signature subsets are activated in Brca1-/- and basal-like breast cancers

PubMed Central

2013-01-01

Introduction Cancer is often suggested to result from development gone awry. Links between normal embryonic development and cancer biology have been postulated, but no defined genetic basis has been established. We recently published the first transcriptomic analysis of embryonic mammary cell populations. Embryonic mammary epithelial cells are an immature progenitor cell population, lacking differentiation markers, which is reflected in their very distinct genetic profiles when compared with those of their postnatal descendents. Methods We defined an embryonic mammary epithelial signature that incorporates the most highly expressed genes from embryonic mammary epithelium when compared with the postnatal mammary epithelial cells. We looked for activation of the embryonic mammary epithelial signature in mouse mammary tumors that formed in mice in which Brca1 had been conditionally deleted from the mammary epithelium and in human breast cancers to determine whether any genetic links exist between embryonic mammary cells and breast cancers. Results Small subsets of the embryonic mammary epithelial signature were consistently activated in mouse Brca1-/- tumors and human basal-like breast cancers, which encoded predominantly transcriptional regulators, cell-cycle, and actin cytoskeleton components. Other embryonic gene subsets were found activated in non-basal-like tumor subtypes and repressed in basal-like tumors, including regulators of neuronal differentiation, transcription, and cell biosynthesis. Several embryonic genes showed significant upregulation in estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and/or grade 3 breast cancers. Among them, the transcription factor, SOX11, a progenitor cell and lineage regulator of nonmammary cell types, is found highly expressed in some Brca1-/- mammary tumors. By using RNA interference to silence SOX11 expression in breast cancer cells, we found evidence that SOX11 regulates breast cancer cell proliferation and cell survival. Conclusions Specific subsets of embryonic mammary genes, rather than the entire embryonic development transcriptomic program, are activated in tumorigenesis. Genes involved in embryonic mammary development are consistently upregulated in some breast cancers and warrant further investigation, potentially in drug-discovery research endeavors. PMID:23506684

Genetic Engineering of Optical Properties of Biomaterials

NASA Astrophysics Data System (ADS)

Gourley, Paul; Naviaux, Robert; Yaffe, Michael

2008-03-01

Baker's yeast cells are easily cultured and can be manipulated genetically to produce large numbers of bioparticles (cells and mitochondria) with controllable size and optical properties. We have recently employed nanolaser spectroscopy to study the refractive index of individual cells and isolated mitochondria from two mutant strains. Results show that biomolecular changes induced by mutation can produce bioparticles with radical changes in refractive index. Wild-type mitochondria exhibit a distribution with a well-defined mean and small variance. In striking contrast, mitochondria from one mutant strain produced a histogram that is highly collapsed with a ten-fold decrease in the mean and standard deviation. In a second mutant strain we observed an opposite effect with the mean nearly unchanged but the variance increased nearly a thousand-fold. Both histograms could be self-consistently modeled with a single, log-normal distribution. The strains were further examined by 2-dimensional gel electrophoresis to measure changes in protein composition. All of these data show that genetic manipulation of cells represents a new approach to engineering optical properties of bioparticles.

Stem cell potency and the ability to contribute to chimeric organisms.

PubMed

Polejaeva, Irina; Mitalipov, Shoukhrat

2013-03-01

Mouse embryonic chimeras are a well-established tool for studying cell lineage commitment and pluripotency. Experimental chimeras were successfully produced by combining two or more preimplantation embryos or by introducing into host embryo cultured pluripotent embryonic stem cells (ESCs). Chimera production using genetically modified ESCs became the method of choice for the generation of knockout or knockin mice. Although the derivation of ESCs or ESC-like cells has been reported for other species, only mouse and rat pluripotent stem cells have been shown to contribute to germline-competent chimeras, which is the defining feature of ESCs. Herein, we describe different approaches employed for the generation of embryonic chimeras, define chimera-competent cell types, and describe cases of spontaneous chimerism in humans. We also review the current state of derivation of pluripotent stem cells in several species and discuss outcomes of various chimera studies when such cells are used.

Spinal schwannomatosis in the absence of neurofibromatosis: A very rare condition

PubMed Central

Landi, A.; Dugoni, D.E.; Marotta, N.; Mancarella, C.; Delfini, R.

2010-01-01

Schwannomatosis is defined as an extremely rare tumors syndrome characterized by the presence of multiple schwannomas in the absence of typical signs of NF1 and NF2 syndromes. The genetic and molecular analysis performed on these tumors makes it possible to name schwannomatosis as distinct clinical and genetic syndrome. The treatment in the case of symptomatic lesions is surgical removal; if the lesions are asymptomatic it is better to perform serial MRI studies. Given the high incidence of developing additional lesions in patients with schwannomatosis, it remains imperative to perform serial brain and spinal cord MRI studies during follow-up. The differential diagnosis is important including clinical and radiological criteria plus molecular genetic analysis of tumor cells and lymphocyte DNA. We report a rare case of spinal schwannomatosis in which genetic analysis performed on surgical samples showed two different mutations in the cells of the two lesions. PMID:22096683

Spinal schwannomatosis in the absence of neurofibromatosis: A very rare condition.

PubMed

Landi, A; Dugoni, D E; Marotta, N; Mancarella, C; Delfini, R

2011-01-01

Schwannomatosis is defined as an extremely rare tumors syndrome characterized by the presence of multiple schwannomas in the absence of typical signs of NF1 and NF2 syndromes. The genetic and molecular analysis performed on these tumors makes it possible to name schwannomatosis as distinct clinical and genetic syndrome. The treatment in the case of symptomatic lesions is surgical removal; if the lesions are asymptomatic it is better to perform serial MRI studies. Given the high incidence of developing additional lesions in patients with schwannomatosis, it remains imperative to perform serial brain and spinal cord MRI studies during follow-up. The differential diagnosis is important including clinical and radiological criteria plus molecular genetic analysis of tumor cells and lymphocyte DNA. We report a rare case of spinal schwannomatosis in which genetic analysis performed on surgical samples showed two different mutations in the cells of the two lesions.

Signals that regulate the oncogenic fate of neural stem cells and progenitors

PubMed Central

Swartling, Fredrik J.; Bolin, Sara; Phillips, Joanna J.; Persson, Anders I.

2013-01-01

Brain tumors have frequently been associated with a neural stem cell (NSC) origin and contain stem-like tumor cells, so-called brain tumor stem cells (BTSCs) that share many features with normal NSCs. A stem cell state of BTSCs confers resistance to radiotherapy and treatment with alkylating agents. It is also a hallmark of aggressive brain tumors and is maintained by transcriptional networks that are also active in embryonic stem cells. Advances in reprogramming of somatic cells into induced pluripotent stem (iPS) cells have further identified genes that drive stemness. In this review, we will highlight the possible drivers of stemness in medulloblastoma and glioma, the most frequent types of primary malignant brain cancer in children and adults, respectively. Signals that drive expansion of developmentally defined neural precursor cells are also active in corresponding brain tumors. Transcriptomal subgroups of human medulloblastoma and glioma match features of NSCs but also more restricted progenitors. Lessons from genetically-engineered mouse (GEM) models show that temporally and regionally defined NSCs can give rise to distinct subgroups of medulloblastoma and glioma. We will further discuss how acquisition of stem cell features may drive brain tumorigenesis from a non-NSC origin. Genetic alterations, signaling pathways, and therapy-induced changes in the tumor microenvironment can drive reprogramming networks and induce stemness in brain tumors. Finally, we propose a model where dysregulation of microRNAs (miRNAs) that normally provide barriers against reprogramming plays an integral role in promoting stemness in brain tumors. PMID:23376224

Genetic fine-mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci

PubMed Central

Mahajan, Anubha; Locke, Adam; Rayner, N William; Robertson, Neil; Scott, Robert A; Prokopenko, Inga; Scott, Laura J; Green, Todd; Sparso, Thomas; Thuillier, Dorothee; Yengo, Loic; Grallert, Harald; Wahl, Simone; Frånberg, Mattias; Strawbridge, Rona J; Kestler, Hans; Chheda, Himanshu; Eisele, Lewin; Gustafsson, Stefan; Steinthorsdottir, Valgerdur; Thorleifsson, Gudmar; Qi, Lu; Karssen, Lennart C; van Leeuwen, Elisabeth M; Willems, Sara M; Li, Man; Chen, Han; Fuchsberger, Christian; Kwan, Phoenix; Ma, Clement; Linderman, Michael; Lu, Yingchang; Thomsen, Soren K; Rundle, Jana K; Beer, Nicola L; van de Bunt, Martijn; Chalisey, Anil; Kang, Hyun Min; Voight, Benjamin F; Abecasis, Goncalo R; Almgren, Peter; Baldassarre, Damiano; Balkau, Beverley; Benediktsson, Rafn; Blüher, Matthias; Boeing, Heiner; Bonnycastle, Lori L; Borringer, Erwin P; Burtt, Noël P; Carey, Jason; Charpentier, Guillaume; Chines, Peter S; Cornelis, Marilyn C; Couper, David J; Crenshaw, Andrew T; van Dam, Rob M; Doney, Alex SF; Dorkhan, Mozhgan; Edkins, Sarah; Eriksson, Johan G; Esko, Tonu; Eury, Elodie; Fadista, João; Flannick, Jason; Fontanillas, Pierre; Fox, Caroline; Franks, Paul W; Gertow, Karl; Gieger, Christian; Gigante, Bruna; Gottesman, Omri; Grant, George B; Grarup, Niels; Groves, Christopher J; Hassinen, Maija; Have, Christian T; Herder, Christian; Holmen, Oddgeir L; Hreidarsson, Astradur B; Humphries, Steve E; Hunter, David J; Jackson, Anne U; Jonsson, Anna; Jørgensen, Marit E; Jørgensen, Torben; Kerrison, Nicola D; Kinnunen, Leena; Klopp, Norman; Kong, Augustine; Kovacs, Peter; Kraft, Peter; Kravic, Jasmina; Langford, Cordelia; Leander, Karin; Liang, Liming; Lichtner, Peter; Lindgren, Cecilia M; Lindholm, Eero; Linneberg, Allan; Liu, Ching-Ti; Lobbens, Stéphane; Luan, Jian’an; Lyssenko, Valeriya; Männistö, Satu; McLeod, Olga; Meyer, Julia; Mihailov, Evelin; Mirza, Ghazala; Mühleisen, Thomas W; Müller-Nurasyid, Martina; Navarro, Carmen; Nöthen, Markus M; Oskolkov, Nikolay N; Owen, Katharine R; Palli, Domenico; Pechlivanis, Sonali; Perry, John RB; Platou, Carl GP; Roden, Michael; Ruderfer, Douglas; Rybin, Denis; van der Schouw, Yvonne T; Sennblad, Bengt; Sigurðsson, Gunnar; Stančáková, Alena; Steinbach, Gerald; Storm, Petter; Strauch, Konstantin; Stringham, Heather M; Sun, Qi; Thorand, Barbara; Tikkanen, Emmi; Tonjes, Anke; Trakalo, Joseph; Tremoli, Elena; Tuomi, Tiinamaija; Wennauer, Roman; Wood, Andrew R; Zeggini, Eleftheria; Dunham, Ian; Birney, Ewan; Pasquali, Lorenzo; Ferrer, Jorge; Loos, Ruth JF; Dupuis, Josée; Florez, Jose C; Boerwinkle, Eric; Pankow, James S; van Duijn, Cornelia; Sijbrands, Eric; Meigs, James B; Hu, Frank B; Thorsteinsdottir, Unnur; Stefansson, Kari; Lakka, Timo A; Rauramaa, Rainer; Stumvoll, Michael; Pedersen, Nancy L; Lind, Lars; Keinanen-Kiukaanniemi, Sirkka M; Korpi-Hyövälti, Eeva; Saaristo, Timo E; Saltevo, Juha; Kuusisto, Johanna; Laakso, Markku; Metspalu, Andres; Erbel, Raimund; Jöckel, Karl-Heinz; Moebus, Susanne; Ripatti, Samuli; Salomaa, Veikko; Ingelsson, Erik; Boehm, Bernhard O; Bergman, Richard N; Collins, Francis S; Mohlke, Karen L; Koistinen, Heikki; Tuomilehto, Jaakko; Hveem, Kristian; Njølstad, Inger; Deloukas, Panagiotis; Donnelly, Peter J; Frayling, Timothy M; Hattersley, Andrew T; de Faire, Ulf; Hamsten, Anders; Illig, Thomas; Peters, Annette; Cauchi, Stephane; Sladek, Rob; Froguel, Philippe; Hansen, Torben; Pedersen, Oluf; Morris, Andrew D; Palmer, Collin NA; Kathiresan, Sekar; Melander, Olle; Nilsson, Peter M; Groop, Leif C; Barroso, Inês; Langenberg, Claudia; Wareham, Nicholas J; O’Callaghan, Christopher A; Gloyn, Anna L; Altshuler, David; Boehnke, Michael; Teslovich, Tanya M; McCarthy, Mark I; Morris, Andrew P

2015-01-01

We performed fine-mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in/near KCNQ1. “Credible sets” of variants most likely to drive each distinct signal mapped predominantly to non-coding sequence, implying that T2D association is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine-mapping implicated rs10830963 as driving T2D association. We confirmed that this T2D-risk allele increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D-risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease. PMID:26551672

Defining the Regulation of Telomerase Through Identification of Mammary-Specific Telomerase Interacting Proteins

DTIC Science & Technology

2007-06-01

Pharmacology and Toxicology , Virginia Commonwealth University, Richmond, VA 2002-present Member, Molecular Biology and Genetics Program, Virginia...Studying Telomeres and Telomerase. Zebrafish , 1:349-355. Jones,K.R., L.W.Elmore, L.Povirk, S.E.Holt, and D.A.Gewirtz. 2005. Reciprocal regulation... Zebrafish Blastula Cell Line on Rainbow Trout Stromal Cells and Subsequent Development under Feeder-Free Conditions into a Cell Line, ZEB2J. Zebrafish 5: 49

Induction of genetic changes in Saccharomyces cerevisiae by partial drying in air of constant relative humidity and by UV.

PubMed

Hieda, K

1981-11-01

It was investigated whether there was a critical degree of dryness for induction of genetic changes by drying. Saccharomyces cerevisiae cells were dried in air of 0, 33, 53 and 76% relative humidity (RH). The frequencies of mitotic recombination at ade2, of gene conversion at leu1, and of gene mutation at can1 were measured in X2447, XS1473 and S288C strains, respectively. After the cells had been dried at 0% RH for 4 h the frequencies of the genetic changes at ade2, leu1 and can1 were, respectively, 56, 7 and 3.5 times higher than each spontaneous frequency. Induction rates, defined as the frequencies of the induced genetic changes per unit time (1 h) of drying, were greatly decreased with increase in RH. Partial drying in air of 76% RH up to 4 and 8 h induced no genetic change at ade2 and leu1, respectively. It was concluded, therefore, that drying at a certain RH between 53 and 76% gave the critical degree of dryness of cells for the induction of the genetic changes. The water contents of cells (g water per g dry material) were 12% at 53% RH and 21% at 76% RH, whereas the water content of native cells was 212%. Removal of a large amount of cellular water had no effect on the induction of the genetic changes. UV sensitivity of partially dried cells of X2447 for the induction of the genetic change at ade2 drastically increased with decrease in RH between 76 and 53%. The drastic change in the UV sensitivity suggested that photochemical reactivity of DNA of chromosome XV, in which the ade2 locus is located, changed between 76 and 53% RH. It seems that the genetic changes were induced only in the low RH region where DNA in vivo had a different photochemical reactivity.

De novo formed satellite DNA-based mammalian artificial chromosomes and their possible applications.

PubMed

Katona, Robert L

2015-02-01

Mammalian artificial chromosomes (MACs) are non-integrating, autonomously replicating natural chromosome-based vectors that may carry a vast amount of genetic material, which in turn enable potentially prolonged, safe, and regulated therapeutic transgene expression and render MACs as attractive genetic vectors for "gene replacement" or for controlling differentiation pathways in target cells. Satellite-DNA-based artificial chromosomes (SATACs) can be made by induced de novo chromosome formation in cells of different mammalian and plant species. These artificially generated accessory chromosomes are composed of predictable DNA sequences, and they contain defined genetic information. SATACs have already passed a number of obstacles crucial to their further development as gene therapy vectors, including large-scale purification, transfer of purified artificial chromosomes into different cells and embryos, generation of transgenic animals and germline transmission with purified SATACs, and the tissue-specific expression of a therapeutic gene from an artificial chromosome in the milk of transgenic animals. SATACs could be used in cell therapy protocols. For these methods, the most versatile target cell would be one that was pluripotent and self-renewing to address multiple disease target cell types, thus making multilineage stem cells, such as adult derived early progenitor cells and embryonic stem cells, as attractive universal host cells.

Programmable full-adder computations in communicating three-dimensional cell cultures.

PubMed

Ausländer, David; Ausländer, Simon; Pierrat, Xavier; Hellmann, Leon; Rachid, Leila; Fussenegger, Martin

2018-01-01

Synthetic biologists have advanced the design of trigger-inducible gene switches and their assembly into input-programmable circuits that enable engineered human cells to perform arithmetic calculations reminiscent of electronic circuits. By designing a versatile plug-and-play molecular-computation platform, we have engineered nine different cell populations with genetic programs, each of which encodes a defined computational instruction. When assembled into 3D cultures, these engineered cell consortia execute programmable multicellular full-adder logics in response to three trigger compounds.

Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System.

PubMed

Xue, Haipeng; Wu, Jianbo; Li, Shenglan; Rao, Mahendra S; Liu, Ying

2016-01-01

Genetic modification is an indispensable tool to study gene function in normal development and disease. The recent breakthrough of creating human induced pluripotent stem cells (iPSCs) by defined factors (Takahashi et al., Cell 131:861-872, 2007) provides a renewable source of patient autologous cells that not only retain identical genetic information but also give rise to many cell types of the body including neurons and glia. Meanwhile, the rapid advancement of genome modification tools such as gene targeting by homologous recombination (Capecchi, Nat Rev Genet 6:507-512, 2005) and genome editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, TALENs (Transcription activator-like effector nucleases), and ZFNs (Zinc finger nucleases) (Wang et al., Cell 153:910-918, 2013; Mali et al., Science 339:823-826, 2013; Hwang et al., Nat Biotechnol 31:227-229, 2013; Friedland et al., Nat Methods 10(8):741-743, 2013; DiCarlo et al., Nucleic Acids Res 41:4336-4343, 2013; Cong et al., Science 339:819-823, 2013) has greatly accelerated the development of human genome manipulation at the molecular level. This chapter describes the protocols for making neural lineage reporter lines using homologous recombination and the CRISPR/Cas system-mediated genome editing, including construction of targeting vectors, guide RNAs, transfection into hPSCs, and selection and verification of successfully targeted clones. This method can be applied to various needs of hPSC genetic engineering at high efficiency and high reliability.

Embryonic and somatic cell cloning.

PubMed

Wilmut, I; Young, L; Campbell, K H

1998-01-01

Revolutionary opportunities in biology, medicine and agriculture arise from the observation that offspring are obtained after nuclear transfer if somatic donor cells are induced to become quiescent. Exploitation of many of these opportunities will depend upon optimizing procedures for nuclear transfer. This may come about through an understanding of the means by which factors in the oocyte cytoplasm act upon the DNA of the transferred nucleus to regulate gene expression. Similarly, research will extend the procedure to other species. This technology may be used for embryo production, the introduction of genetic change and the derivation of cells needed to treat human diseases. Groups of genetically identical animals will be used in research to control genetic variation and to allow transfer of cells between individuals. In agriculture, production of a small number of clones will separate genetic and environmental effects, whereas production of larger numbers of offspring will disseminate genetic improvement from nucleus herds. Precise genetic modification will be achieved by site specific recombination in the donor cells before nuclear transfer. In all mammals it will become possible to define the role of any gene product and to analyse the mechanisms that regulate gene expression. Medical uses of these techniques will include the production of proteins needed to treat disease and the supply of organs such as hearts, livers and kidneys from pigs. As genome mapping projects identify loci associated with traits of commercial importance in agriculture then gene targeting will be used to study this effect. Finally, cells capable of differentiation into any of the tissues of a patient will provide treatment for diseases reflecting damage to a specific cell population that neither repairs nor replaces itself.

Production of knock-in mice in a single generation from embryonic stem cells.

PubMed

Ukai, Hideki; Kiyonari, Hiroshi; Ueda, Hiroki R

2017-12-01

The system-level identification and analysis of molecular networks in mammals can be accelerated by 'next-generation' genetics, defined as genetics that does not require crossing of multiple generations of animals in order to achieve the desired genetic makeup. We have established a highly efficient procedure for producing knock-in (KI) mice within a single generation, by optimizing the genome-editing protocol for KI embryonic stem (ES) cells and the protocol for the generation of fully ES-cell-derived mice (ES mice). Using this protocol, the production of chimeric mice is eliminated, and, therefore, there is no requirement for the crossing of chimeric mice to produce mice that carry the KI gene in all cells of the body. Our procedure thus shortens the time required to produce KI ES mice from about a year to ∼3 months. Various kinds of KI ES mice can be produced with a minimized amount of work, facilitating the elucidation of organism-level phenomena using a systems biology approach. In this report, we describe the basic technologies and protocols for this procedure, and discuss the current challenges for next-generation mammalian genetics in organism-level systems biology studies.

Murine genetically engineered and human xenograft models of chronic lymphocytic leukemia.

PubMed

Chen, Shih-Shih; Chiorazzi, Nicholas

2014-07-01

Chronic lymphocytic leukemia (CLL) is a genetically complex disease, with multiple factors having an impact on onset, progression, and response to therapy. Genetic differences/abnormalities have been found in hematopoietic stem cells from patients, as well as in B lymphocytes of individuals with monoclonal B-cell lymphocytosis who may develop the disease. Furthermore, after the onset of CLL, additional genetic alterations occur over time, often causing disease worsening and altering patient outcomes. Therefore, being able to genetically engineer mouse models that mimic CLL or at least certain aspects of the disease will help us understand disease mechanisms and improve treatments. This notwithstanding, because neither the genetic aberrations responsible for leukemogenesis and progression nor the promoting factors that support these are likely identical in character or influences for all patients, genetically engineered mouse models will only completely mimic CLL when all of these factors are precisely defined. In addition, multiple genetically engineered models may be required because of the heterogeneity in susceptibility genes among patients that can have an effect on genetic and environmental characteristics influencing disease development and outcome. For these reasons, we review the major murine genetically engineered and human xenograft models in use at the present time, aiming to report the advantages and disadvantages of each. Copyright © 2014 Elsevier Inc. All rights reserved.

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

NASA Astrophysics Data System (ADS)

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

2014-06-01

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

Application of bacterial artificial chromosome array-based comparative genomic hybridization and spectral karyotyping to the analysis of glioblastoma multiforme.

PubMed

Cowell, John K; Matsui, Sei-Ichi; Wang, Yong D; LaDuca, Jeffrey; Conroy, Jeffrey; McQuaid, Devin; Nowak, Norma J

2004-05-01

Identification of genetic losses and gains is valuable in analysis of brain tumors. Locus-by-locus analyses have revealed correlations between prognosis and response to chemotherapy and loss or gain of specific genes and loci. These approaches are labor intensive and do not provide a global view of the genetic changes within the tumor cells. Bacterial artificial chromosome (BAC) arrays, which cover the genome with an average resolution of less than 1 MbP, allow defining the sum total of these genetic changes in a single comparative genomic hybridization (CGH) experiment. These changes are directly overlaid on the human genome sequence, thus providing the extent of the amplification or deletion, reflected by a megabase position, and gene content of the abnormal region. Although this array-based CGH approach (CGHa) seems to detect the extent of the genetic changes in tumors reliably, it has not been robustly tested. We compared genetic changes in four newly derived, early-passage glioma cell lines, using spectral karyotyping (SKY) and CGHa. Chromosome changes seen in cell lines under SKY analysis were also detected with CGHa. In addition, CGHa detected cryptic genetic gains and losses and resolved the nature of subtle marker chromosomes that could not be resolved with SKY, thus providing distinct advantages over previous technologies. There was remarkable general concordance between the CGHa results comparing the cell lines to the original tumor, except that the magnitude of the changes seen in the tumor sample was generally suppressed compared with the cell lines, a consequence of normal cells contaminating the tumor sample. CGHa revealed changes in cell lines that were not present in the original tumors and vice versa, even when analyzed at the earliest passage possible, which highlights the adaptation of the cells to in vitro culture. CGHa proved to be highly accurate and efficient for identifying genetic changes in tumor cells. This approach can accurately identify subtle, novel genetic abnormalities in tumors directly linked to the human genome sequence. CGHa far surpasses the resolution and information provided by conventional metaphase CGH, without relying on in vitro culture of tumors for metaphase spreads.

Genome Editing in Human Pluripotent Stem Cells.

PubMed

Carlson-Stevermer, Jared; Saha, Krishanu

2017-01-01

Genome editing in human pluripotent stem cells (hPSCs) enables the generation of reporter lines and knockout cell lines. Zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 technology have recently increased the efficiency of proper gene editing by creating double strand breaks (DSB) at defined sequences in the human genome. These systems typically use plasmids to transiently transcribe nucleases within the cell. Here, we describe the process for preparing hPSCs for transient expression of nucleases via electroporation and subsequent analysis to create genetically modified stem cell lines.

Cell-Free and In Vivo Characterization of Lux, Las, and Rpa Quorum Activation Systems in E. coli.

PubMed

Halleran, Andrew D; Murray, Richard M

2018-02-16

Synthetic biologists have turned toward quorum systems as a path for building sophisticated microbial consortia that exhibit group decision making. Currently, however, even the most complex consortium circuits rely on only one or two quorum sensing systems, greatly restricting the available design space. High-throughput characterization of available quorum sensing systems is useful for finding compatible sets of systems that are suitable for a defined circuit architecture. Recently, cell-free systems have gained popularity as a test-bed for rapid prototyping of genetic circuitry. We take advantage of the transcription-translation cell-free system to characterize three commonly used Lux-type quorum activators, Lux, Las, and Rpa. We then compare the cell-free characterization to results obtained in vivo. We find significant genetic crosstalk in both the Las and Rpa systems and substantial signal crosstalk in Lux activation. We show that cell-free characterization predicts crosstalk observed in vivo.

Exploiting genomics and natural genetic variation to decode macrophage enhancers

PubMed Central

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

2015-01-01

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

Defining Differential Genetic Signatures in CXCR4- and the CCR5-Utilizing HIV-1 Co-Linear Sequences

PubMed Central

Aiamkitsumrit, Benjamas; Dampier, Will; Martin-Garcia, Julio; Nonnemacher, Michael R.; Pirrone, Vanessa; Ivanova, Tatyana; Zhong, Wen; Kilareski, Evelyn; Aldigun, Hazeez; Frantz, Brian; Rimbey, Matthew; Wojno, Adam; Passic, Shendra; Williams, Jean W.; Shah, Sonia; Blakey, Brandon; Parikh, Nirzari; Jacobson, Jeffrey M.; Moldover, Brian; Wigdahl, Brian

2014-01-01

The adaptation of human immunodeficiency virus type-1 (HIV-1) to an array of physiologic niches is advantaged by the plasticity of the viral genome, encoded proteins, and promoter. CXCR4-utilizing (X4) viruses preferentially, but not universally, infect CD4+ T cells, generating high levels of virus within activated HIV-1-infected T cells that can be detected in regional lymph nodes and peripheral blood. By comparison, the CCR5-utilizing (R5) viruses have a greater preference for cells of the monocyte-macrophage lineage; however, while R5 viruses also display a propensity to enter and replicate in T cells, they infect a smaller percentage of CD4+ T cells in comparison to X4 viruses. Additionally, R5 viruses have been associated with viral transmission and CNS disease and are also more prevalent during HIV-1 disease. Specific adaptive changes associated with X4 and R5 viruses were identified in co-linear viral sequences beyond the Env-V3. The in silico position-specific scoring matrix (PSSM) algorithm was used to define distinct groups of X4 and R5 sequences based solely on sequences in Env-V3. Bioinformatic tools were used to identify genetic signatures involving specific protein domains or long terminal repeat (LTR) transcription factor sites within co-linear viral protein R (Vpr), trans-activator of transcription (Tat), or LTR sequences that were preferentially associated with X4 or R5 Env-V3 sequences. A number of differential amino acid and nucleotide changes were identified across the co-linear Vpr, Tat, and LTR sequences, suggesting the presence of specific genetic signatures that preferentially associate with X4 or R5 viruses. Investigation of the genetic relatedness between X4 and R5 viruses utilizing phylogenetic analyses of complete sequences could not be used to definitively and uniquely identify groups of R5 or X4 sequences; in contrast, differences in the genetic diversities between X4 and R5 were readily identified within these co-linear sequences in HIV-1-infected patients. PMID:25265194

Models of ovarian cancer metastasis: Murine models

PubMed Central

Šale, Sanja; Orsulic, Sandra

2008-01-01

Mice have mainly been used in ovarian cancer research as immunodeficient hosts for cell lines derived from the primary tumors and ascites of ovarian cancer patients. These xenograft models have provided a valuable system for pre-clinical trials, however, the genetic complexity of human tumors has precluded the understanding of key events that drive metastatic dissemination. Recently developed immunocompetent, genetically defined mouse models of epithelial ovarian cancer represent significant improvements in the modeling of metastatic disease. PMID:19337569

Factors affecting the electrofusion of mouse and ferret oocytes with ferret somatic cells.

PubMed

Li, Ziyi; Sun, Xingshen; Chen, Juan; Leno, Gregory H; Engelhardt, John F

2005-09-01

The domestic ferret, Mustela putorius furos, holds great promise as a genetic model for human lung disease, provided that key technologies for somatic cell nuclear transfer (SCNT) are developed. In this report, we extend our understanding of SCNT in this species by defining conditions for efficient cell fusion by electrical pulse. Two experimental systems were employed in this study. First, in vivo-matured mouse oocytes and ferret somatic cells were used to establish general parameters for fusion. One fibroblast, or cumulus cell, was agglutinated to nucleate, zona pellucida-free, mouse oocytes, and subjected to an electrical pulse. Similar electrical pulse conditions were also tested with 1 or 2 somatic cells inserted into the perivitelline space (PVS) of intact mouse oocytes. The fusion rate for a single fibroblast with a zona-free oocyte was 80.2%, significantly higher (P < 0.05) than that observed for 1, or 2, fibroblasts placed in the PVS (52.0% and 63.8%, respectively). The fusion rate (44.1%) following insertion of two cumulus cells was significantly higher (P < 0.05) than that following insertion of one cumulus cell (25.1%). Second, in vitro-matured ferret oocytes were enucleated, and one to three fibroblasts or cumulus cells were inserted into the PVS. Zona pellucida-free ferret oocytes were fragile and excluded from the study. The fusion rates with two or three fibroblasts were 71.4% and 76.8%, respectively; significantly higher (P < 0.05) than that for one fibroblast (48.6%). This cell number-dependent difference in fusion efficiency was also observed with cumulus cells. Fusion-derived (ferret-ferret) NT embryos cleaved, formed blastocysts in vitro, and underwent early-stage fetal development following embryo transfer. The rate of development was cell type-independent, in contrast to the cell type-dependent differences observed in fusion efficiency. In conclusion, fibroblasts fused more efficiently than cumulus cells and the efficiency of single cell fusions was improved when two or more cells were inserted into the PVS. These studies define conditions for efficient cell fusion with ferret oocytes and should facilitate SCNT and the development of genetically defined animal models in this species.

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing.

PubMed

Tang, Qin; Iyer, Sowmya; Lobbardi, Riadh; Moore, John C; Chen, Huidong; Lareau, Caleb; Hebert, Christine; Shaw, McKenzie L; Neftel, Cyril; Suva, Mario L; Ceol, Craig J; Bernards, Andre; Aryee, Martin; Pinello, Luca; Drummond, Iain A; Langenau, David M

2017-10-02

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA-protein kinase catalytic subunit ( prkdc ), interleukin-2 receptor γ a ( il2rga ), and double-homozygous-mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish. © 2017 Tang et al.

Dissecting hematopoietic and renal cell heterogeneity in adult zebrafish at single-cell resolution using RNA sequencing

PubMed Central

Iyer, Sowmya; Lobbardi, Riadh; Chen, Huidong; Hebert, Christine; Shaw, McKenzie L.; Neftel, Cyril; Suva, Mario L.; Bernards, Andre; Aryee, Martin; Drummond, Iain A.

2017-01-01

Recent advances in single-cell, transcriptomic profiling have provided unprecedented access to investigate cell heterogeneity during tissue and organ development. In this study, we used massively parallel, single-cell RNA sequencing to define cell heterogeneity within the zebrafish kidney marrow, constructing a comprehensive molecular atlas of definitive hematopoiesis and functionally distinct renal cells found in adult zebrafish. Because our method analyzed blood and kidney cells in an unbiased manner, our approach was useful in characterizing immune-cell deficiencies within DNA–protein kinase catalytic subunit (prkdc), interleukin-2 receptor γ a (il2rga), and double-homozygous–mutant fish, identifying blood cell losses in T, B, and natural killer cells within specific genetic mutants. Our analysis also uncovered novel cell types, including two classes of natural killer immune cells, classically defined and erythroid-primed hematopoietic stem and progenitor cells, mucin-secreting kidney cells, and kidney stem/progenitor cells. In total, our work provides the first, comprehensive, single-cell, transcriptomic analysis of kidney and marrow cells in the adult zebrafish. PMID:28878000

Guiding plant virus particles to integrin-displaying cells

NASA Astrophysics Data System (ADS)

Hovlid, Marisa L.; Steinmetz, Nicole F.; Laufer, Burkhardt; Lau, Jolene L.; Kuzelka, Jane; Wang, Qian; Hyypiä, Timo; Nemerow, Glen R.; Kessler, Horst; Manchester, Marianne; Finn, M. G.

2012-05-01

Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors. Electronic supplementary information (ESI) available: Synthetic procedures and compound characterization data; assay procedures; additional confocal micrographs at different time points. See DOI: 10.1039/c2nr30571b

Photocrosslinking approaches to interactome mapping

PubMed Central

Pham, Nam D.; Parker, Randy B.; Kohler, Jennifer J.

2012-01-01

Photocrosslinking approaches can be used to map interactome networks within the context of living cells. Photocrosslinking methods rely on use of metabolic engineering or genetic code expansion to incorporate photocrosslinking analogs of amino acids or sugars into cellular biomolecules. Immunological and mass spectrometry techniques are used to analyze crosslinked complexes, thereby defining specific interactomes. Because photocrosslinking can be conducted in native, cellular settings, it can be used to define context-dependent interactions. Photocrosslinking methods are also ideally suited for determining interactome dynamics, mapping interaction interfaces, and identifying transient interactions in which intrinsically disordered proteins and glycoproteins engage. Here we discuss the application of cell-based photocrosslinking to the study of specific problems in immune cell signaling, transcription, membrane protein dynamics, nucleocytoplasmic transport, and chaperone-assisted protein folding. PMID:23149092

Genetic dissection of eosinophilic esophagitis provides insight into disease pathogenesis and treatment strategies

PubMed Central

Sherrill, Joseph D.; Rothenberg, Marc E.

2011-01-01

Eosinophilic esophagitis (EoE) is a chronic inflammatory disorder of the esophagus that is compounded by both genetic predisposition and aberrant responses to environmental antigens, particularly those that are food-derived. Data have indicated a unique transcriptional response in vivo that defines EoE and which is partially attributable to the Th2 cytokine interleukin 13 (IL-13). Moreover, a number of genetic risk variants in pro-inflammatory and epithelial cell genes associate with EoE susceptibility, demonstrating novel heritable mechanisms that contribute to disease risk. Here, we discuss recent advances in our understanding of the intrinsic (genetic) and extrinsic (environmental) components that illustrate the complex nature of EoE. PMID:21570716

Synthetic Genetic Arrays: Automation of Yeast Genetics.

PubMed

Kuzmin, Elena; Costanzo, Michael; Andrews, Brenda; Boone, Charles

2016-04-01

Genome-sequencing efforts have led to great strides in the annotation of protein-coding genes and other genomic elements. The current challenge is to understand the functional role of each gene and how genes work together to modulate cellular processes. Genetic interactions define phenotypic relationships between genes and reveal the functional organization of a cell. Synthetic genetic array (SGA) methodology automates yeast genetics and enables large-scale and systematic mapping of genetic interaction networks in the budding yeast,Saccharomyces cerevisiae SGA facilitates construction of an output array of double mutants from an input array of single mutants through a series of replica pinning steps. Subsequent analysis of genetic interactions from SGA-derived mutants relies on accurate quantification of colony size, which serves as a proxy for fitness. Since its development, SGA has given rise to a variety of other experimental approaches for functional profiling of the yeast genome and has been applied in a multitude of other contexts, such as genome-wide screens for synthetic dosage lethality and integration with high-content screening for systematic assessment of morphology defects. SGA-like strategies can also be implemented similarly in a number of other cell types and organisms, includingSchizosaccharomyces pombe,Escherichia coli, Caenorhabditis elegans, and human cancer cell lines. The genetic networks emerging from these studies not only generate functional wiring diagrams but may also play a key role in our understanding of the complex relationship between genotype and phenotype. © 2016 Cold Spring Harbor Laboratory Press.

Atlas of prostate cancer heritability in European and African-American men pinpoints tissue-specific regulation.

PubMed

Gusev, Alexander; Shi, Huwenbo; Kichaev, Gleb; Pomerantz, Mark; Li, Fugen; Long, Henry W; Ingles, Sue A; Kittles, Rick A; Strom, Sara S; Rybicki, Benjamin A; Nemesure, Barbara; Isaacs, William B; Zheng, Wei; Pettaway, Curtis A; Yeboah, Edward D; Tettey, Yao; Biritwum, Richard B; Adjei, Andrew A; Tay, Evelyn; Truelove, Ann; Niwa, Shelley; Chokkalingam, Anand P; John, Esther M; Murphy, Adam B; Signorello, Lisa B; Carpten, John; Leske, M Cristina; Wu, Suh-Yuh; Hennis, Anslem J M; Neslund-Dudas, Christine; Hsing, Ann W; Chu, Lisa; Goodman, Phyllis J; Klein, Eric A; Witte, John S; Casey, Graham; Kaggwa, Sam; Cook, Michael B; Stram, Daniel O; Blot, William J; Eeles, Rosalind A; Easton, Douglas; Kote-Jarai, Zsofia; Al Olama, Ali Amin; Benlloch, Sara; Muir, Kenneth; Giles, Graham G; Southey, Melissa C; Fitzgerald, Liesel M; Gronberg, Henrik; Wiklund, Fredrik; Aly, Markus; Henderson, Brian E; Schleutker, Johanna; Wahlfors, Tiina; Tammela, Teuvo L J; Nordestgaard, Børge G; Key, Tim J; Travis, Ruth C; Neal, David E; Donovan, Jenny L; Hamdy, Freddie C; Pharoah, Paul; Pashayan, Nora; Khaw, Kay-Tee; Stanford, Janet L; Thibodeau, Stephen N; McDonnell, Shannon K; Schaid, Daniel J; Maier, Christiane; Vogel, Walther; Luedeke, Manuel; Herkommer, Kathleen; Kibel, Adam S; Cybulski, Cezary; Wokolorczyk, Dominika; Kluzniak, Wojciech; Cannon-Albright, Lisa; Teerlink, Craig; Brenner, Hermann; Dieffenbach, Aida K; Arndt, Volker; Park, Jong Y; Sellers, Thomas A; Lin, Hui-Yi; Slavov, Chavdar; Kaneva, Radka; Mitev, Vanio; Batra, Jyotsna; Spurdle, Amanda; Clements, Judith A; Teixeira, Manuel R; Pandha, Hardev; Michael, Agnieszka; Paulo, Paula; Maia, Sofia; Kierzek, Andrzej; Conti, David V; Albanes, Demetrius; Berg, Christine; Berndt, Sonja I; Campa, Daniele; Crawford, E David; Diver, W Ryan; Gapstur, Susan M; Gaziano, J Michael; Giovannucci, Edward; Hoover, Robert; Hunter, David J; Johansson, Mattias; Kraft, Peter; Le Marchand, Loic; Lindström, Sara; Navarro, Carmen; Overvad, Kim; Riboli, Elio; Siddiq, Afshan; Stevens, Victoria L; Trichopoulos, Dimitrios; Vineis, Paolo; Yeager, Meredith; Trynka, Gosia; Raychaudhuri, Soumya; Schumacher, Frederick R; Price, Alkes L; Freedman, Matthew L; Haiman, Christopher A; Pasaniuc, Bogdan

2016-04-07

Although genome-wide association studies have identified over 100 risk loci that explain ∼33% of familial risk for prostate cancer (PrCa), their functional effects on risk remain largely unknown. Here we use genotype data from 59,089 men of European and African American ancestries combined with cell-type-specific epigenetic data to build a genomic atlas of single-nucleotide polymorphism (SNP) heritability in PrCa. We find significant differences in heritability between variants in prostate-relevant epigenetic marks defined in normal versus tumour tissue as well as between tissue and cell lines. The majority of SNP heritability lies in regions marked by H3k27 acetylation in prostate adenoc7arcinoma cell line (LNCaP) or by DNaseI hypersensitive sites in cancer cell lines. We find a high degree of similarity between European and African American ancestries suggesting a similar genetic architecture from common variation underlying PrCa risk. Our findings showcase the power of integrating functional annotation with genetic data to understand the genetic basis of PrCa.

Bridging the Gap: Towards a Cell-Type Specific Understanding of Neural Circuits Underlying Fear Behaviors

PubMed Central

McCullough, KM; Morrison, FG; Ressler, KJ

2016-01-01

Fear and anxiety-related disorders are remarkably common and debilitating, and are often characterized by dysregulated fear responses. Rodent models of fear learning and memory have taken great strides towards elucidating the specific neuronal circuitries underlying the learning of fear responses. The present review addresses recent research utilizing optogenetic approaches to parse circuitries underlying fear behaviors. It also highlights the powerful advances made when optogenetic techniques are utilized in a genetically defined, cell-type specific, manner. The application of next-generation genetic and sequencing approaches in a cell-type specific context will be essential for a mechanistic understanding of the neural circuitry underlying fear behavior and for the rational design of targeted, circuit specific, pharmacologic interventions for the treatment and prevention of fear-related disorders. PMID:27470092

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

PubMed Central

2013-01-01

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. PMID:23324445

mTOR Complex 1 Signaling Regulates the Generation and Function of Central and Effector Foxp3+ Regulatory T Cells.

PubMed

Sun, Im-Hong; Oh, Min-Hee; Zhao, Liang; Patel, Chirag H; Arwood, Matthew L; Xu, Wei; Tam, Ada J; Blosser, Richard L; Wen, Jiayu; Powell, Jonathan D

2018-06-08

The mechanistic/mammalian target of rapamycin (mTOR) has emerged as a critical integrator of signals from the immune microenvironment capable of regulating T cell activation, differentiation, and function. The precise role of mTOR in the control of regulatory T cell (Treg) differentiation and function is complex. Pharmacologic inhibition and genetic deletion of mTOR promotes the generation of Tregs even under conditions that would normally promote generation of effector T cells. Alternatively, mTOR activity has been observed to be increased in Tregs, and the genetic deletion of the mTOR complex 1 (mTORC1)-scaffold protein Raptor inhibits Treg function. In this study, by employing both pharmacologic inhibitors and genetically altered T cells, we seek to clarify the role of mTOR in Tregs. Our studies demonstrate that inhibition of mTOR during T cell activation promotes the generation of long-lived central Tregs with a memory-like phenotype in mice. Metabolically, these central memory Tregs possess enhanced spare respiratory capacity, similar to CD8 + memory cells. Alternatively, the generation of effector Tregs (eTregs) requires mTOR function. Indeed, genetic deletion of Rptor leads to the decreased expression of ICOS and PD-1 on the eTregs. Overall, our studies define a subset of mTORC1 hi eTregs and mTORC1 lo central Tregs. Copyright © 2018 by The American Association of Immunologists, Inc.

Dclk1 Defines Quiescent Pancreatic Progenitors that Promote Injury-Induced Regeneration and Tumorigenesis.

PubMed

Westphalen, C Benedikt; Takemoto, Yoshihiro; Tanaka, Takayuki; Macchini, Marina; Jiang, Zhengyu; Renz, Bernhard W; Chen, Xiaowei; Ormanns, Steffen; Nagar, Karan; Tailor, Yagnesh; May, Randal; Cho, Youngjin; Asfaha, Samuel; Worthley, Daniel L; Hayakawa, Yoku; Urbanska, Aleksandra M; Quante, Michael; Reichert, Maximilian; Broyde, Joshua; Subramaniam, Prem S; Remotti, Helen; Su, Gloria H; Rustgi, Anil K; Friedman, Richard A; Honig, Barry; Califano, Andrea; Houchen, Courtney W; Olive, Kenneth P; Wang, Timothy C

2016-04-07

The existence of adult pancreatic progenitor cells has been debated. While some favor the concept of facultative progenitors involved in homeostasis and repair, neither a location nor markers for such cells have been defined. Using genetic lineage tracing, we show that Doublecortin-like kinase-1 (Dclk1) labels a rare population of long-lived, quiescent pancreatic cells. In vitro, Dclk1+ cells proliferate readily and sustain pancreatic organoid growth. In vivo, Dclk1+ cells are necessary for pancreatic regeneration following injury and chronic inflammation. Accordingly, their loss has detrimental effects after cerulein-induced pancreatitis. Expression of mutant Kras in Dclk1+ cells does not affect their quiescence or longevity. However, experimental pancreatitis converts Kras mutant Dclk1+ cells into potent cancer-initiating cells. As a potential effector of Kras, Dclk1 contributes functionally to the pathogenesis of pancreatic cancer. Taken together, these observations indicate that Dclk1 marks quiescent pancreatic progenitors that are candidates for the origin of pancreatic cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Metastasis genetics, epigenetics, and the tumor microenvironment

USDA-ARS?s Scientific Manuscript database

KISS1 is a member of a family of genes known as metastasis suppressors, defined by their ability to block metastasis without blocking primary tumor development and growth. KISS1 re-expression in multiple metastatic cell lines of diverse cellular origin suppresses metastasis; yet, still allows comple...

CDC42 inhibition suppresses progression of incipient intestinal tumors

USDA-ARS?s Scientific Manuscript database

Mutations in the APC or Beta-catenin genes are well-established initiators of colorectal cancer, yet modifiers that facilitate the survival and progression of nascent tumor cells are not well defined. Using genetic and pharmacologic approaches in mouse colorectal cancer and human colorectal cancer x...

Towards Defining Nutrient Conditions Encountered by the Rice Blast Fungus during Host Infection

PubMed Central

Wilson, Richard A.; Fernandez, Jessie; Quispe, Cristian F.; Gradnigo, Julien; Seng, Anya; Moriyama, Etsuko; Wright, Janet D.

2012-01-01

Fungal diseases cause enormous crop losses, but defining the nutrient conditions encountered by the pathogen remains elusive. Here, we generated a mutant strain of the devastating rice pathogen Magnaporthe oryzae impaired for de novo methionine biosynthesis. The resulting methionine-requiring strain grew strongly on synthetic minimal media supplemented with methionine, aspartate or complex mixtures of partially digested proteins, but could not establish disease in rice leaves. Live-cell-imaging showed the mutant could produce normal appressoria and enter host cells but failed to develop, indicating the availability or accessibility of aspartate and methionine is limited in the plant. This is the first report to demonstrate the utility of combining biochemical genetics, plate growth tests and live-cell-imaging to indicate what nutrients might not be readily available to the fungal pathogen in rice host cells. PMID:23071797

Human genetic mapping studies using single sperm typing

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

Hubert, R.S.

1993-01-01

Sperm typing is a powerful technique that uses the polymerase chain reaction (PCR) to analyze DNA sequences within single sperm cells in order to construct genetic maps. This methodology was used to estimate the recombination fraction between D3S2 and D3S2 which was found to be 0.28 (95% CI = 0.20-0.36). Pedigree analysis was unable to determine genetic distance between these two markers due to their low informativeness. We also showed that dinucleotide and tetranucleotide repeat polymorphisms can be analyzed in single cells without using radioactivity or denaturing gels. This provides a rich new source of DANA polymorphisms for genetic mappingmore » by sperm typing. In addition, an approach that uses the sperm typing methodology is described that can define the physical boundaries of meiotic recombination hotspots. The hotspot at 4p16.3 near the Huntington disease gene was localized to an interval between D4S10 and D4S126. These studies demonstrated the usefulness of sperm typing as a tool for the study of human genetic.« less

Human pluripotent stem cell models of autism spectrum disorder: emerging frontiers, opportunities, and challenges towards neuronal networks in a dish.

PubMed

Aigner, Stefan; Heckel, Tobias; Zhang, Jitao D; Andreae, Laura C; Jagasia, Ravi

2014-03-01

Autism spectrum disorder (ASD) is characterized by deficits in language development and social cognition and the manifestation of repetitive and restrictive behaviors. Despite recent major advances, our understanding of the pathophysiological mechanisms leading to ASD is limited. Although most ASD cases have unknown genetic underpinnings, animal and human cellular models of several rare, genetically defined syndromic forms of ASD have provided evidence for shared pathophysiological mechanisms that may extend to idiopathic cases. Here, we review our current knowledge of the genetic basis and molecular etiology of ASD and highlight how human pluripotent stem cell-based disease models have the potential to advance our understanding of molecular dysfunction. We summarize landmark studies in which neuronal cell populations generated from human embryonic stem cells and patient-derived induced pluripotent stem cells have served to model disease mechanisms, and we discuss recent technological advances that may ultimately allow in vitro modeling of specific human neuronal circuitry dysfunction in ASD. We propose that these advances now offer an unprecedented opportunity to help better understand ASD pathophysiology. This should ultimately enable the development of cellular models for ASD, allowing drug screening and the identification of molecular biomarkers for patient stratification.

Exploring the molecular aspects associated with testicular germ cell tumors: a review

PubMed Central

Facchini, Gaetano; Rossetti, Sabrina; Cavaliere, Carla; D’Aniello, Carmine; Di Franco, Rossella; Iovane, Gelsomina; Grimaldi, Giovanni; Piscitelli, Raffaele; Muto, Paolo; Botti, Gerardo; Perdonà, Sisto; Veneziani, Bianca Maria; Berretta, Massimiliano; Montanari, Micaela

2018-01-01

Testicular germ cell tumors (TGCTs) represent the most common solid tumors affecting young men. They constitute a distinct entity because of their embryonic origin and their unique biological behavior. Recent preclinical data regarding biological signaling machinery as well as genetic and epigenetic mechanisms associated with molecular patterns of tumors have contribute to explain the pathogenesis and the differentiation of TGCTs and to understand the mechanisms responsible for the development of resistance to treatment. In this review, we discuss the main genetic and epigenetic events associated with TGCTs development in order to better define their role in the pathogenesis of these tumors and in cisplatin-acquired resistance. PMID:29416701

Genetic doping and health damages.

PubMed

Fallahi, Aa; Ravasi, Aa; Farhud, Dd

2011-01-01

Use of genetic doping or gene transfer technology will be the newest and the lethal method of doping in future and have some unpleasant consequences for sports, athletes, and outcomes of competitions. The World Anti-Doping Agency (WADA) defines genetic doping as "the non-therapeutic use of genes, genetic elements, and/or cells that have the capacity to enhance athletic performance ". The purpose of this review is to consider genetic doping, health damages and risks of new genes if delivered in athletes. This review, which is carried out by reviewing relevant publications, is primarily based on the journals available in GOOGLE, ELSEVIER, PUBMED in fields of genetic technology, and health using a combination of keywords (e.g., genetic doping, genes, exercise, performance, athletes) until July 2010. There are several genes related to sport performance and if they are used, they will have health risks and sever damages such as cancer, autoimmunization, and heart attack.

Genetic Doping and Health Damages

PubMed Central

Fallahi, AA; Ravasi, AA; Farhud, DD

2011-01-01

Background: Use of genetic doping or gene transfer technology will be the newest and the lethal method of doping in future and have some unpleasant consequences for sports, athletes, and outcomes of competitions. The World Anti-Doping Agency (WADA) defines genetic doping as “the non-therapeutic use of genes, genetic elements, and/or cells that have the capacity to enhance athletic performance ”. The purpose of this review is to consider genetic doping, health damages and risks of new genes if delivered in athletes. Methods: This review, which is carried out by reviewing relevant publications, is primarily based on the journals available in GOOGLE, ELSEVIER, PUBMED in fields of genetic technology, and health using a combination of keywords (e.g., genetic doping, genes, exercise, performance, athletes) until July 2010. Conclusion: There are several genes related to sport performance and if they are used, they will have health risks and sever damages such as cancer, autoimmunization, and heart attack. PMID:23113049

Genetic engineering with T cell receptors.

PubMed

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.

Genetic and molecular alterations across medulloblastoma subgroups.

PubMed

Skowron, Patryk; Ramaswamy, Vijay; Taylor, Michael D

2015-10-01

Medulloblastoma is the most common malignant brain tumour diagnosed in children. Over the last few decades, advances in radiation and chemotherapy have significantly improved the odds of survival. Nevertheless, one third of all patients still succumb to their disease, and many long-term survivors are afflicted with neurocognitive sequelae. Large-scale multi-institutional efforts have provided insight into the transcriptional and genetic landscape of medulloblastoma. Four distinct subgroups of medulloblastoma have been identified, defined by distinct transcriptomes, genetics, demographics and outcomes. Integrated genomic profiling of each of these subgroups has revealed distinct genetic alterations, driving pathways and in some instances cells of origin. In this review, we highlight, in a subgroup-specific manner, our current knowledge of the genetic and molecular alterations in medulloblastoma and underscore the possible avenues for future therapeutic intervention.

Genetic heterogeneity of diffuse large B-cell lymphoma.

PubMed

Zhang, Jenny; Grubor, Vladimir; Love, Cassandra L; Banerjee, Anjishnu; Richards, Kristy L; Mieczkowski, Piotr A; Dunphy, Cherie; Choi, William; Au, Wing Yan; Srivastava, Gopesh; Lugar, Patricia L; Rizzieri, David A; Lagoo, Anand S; Bernal-Mizrachi, Leon; Mann, Karen P; Flowers, Christopher; Naresh, Kikkeri; Evens, Andrew; Gordon, Leo I; Czader, Magdalena; Gill, Javed I; Hsi, Eric D; Liu, Qingquan; Fan, Alice; Walsh, Katherine; Jima, Dereje; Smith, Lisa L; Johnson, Amy J; Byrd, John C; Luftig, Micah A; Ni, Ting; Zhu, Jun; Chadburn, Amy; Levy, Shawn; Dunson, David; Dave, Sandeep S

2013-01-22

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-κB (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.

Genetic fine mapping and genomic annotation defines causal mechanisms at type 2 diabetes susceptibility loci.

PubMed

Gaulton, Kyle J; Ferreira, Teresa; Lee, Yeji; Raimondo, Anne; Mägi, Reedik; Reschen, Michael E; Mahajan, Anubha; Locke, Adam; Rayner, N William; Robertson, Neil; Scott, Robert A; Prokopenko, Inga; Scott, Laura J; Green, Todd; Sparso, Thomas; Thuillier, Dorothee; Yengo, Loic; Grallert, Harald; Wahl, Simone; Frånberg, Mattias; Strawbridge, Rona J; Kestler, Hans; Chheda, Himanshu; Eisele, Lewin; Gustafsson, Stefan; Steinthorsdottir, Valgerdur; Thorleifsson, Gudmar; Qi, Lu; Karssen, Lennart C; van Leeuwen, Elisabeth M; Willems, Sara M; Li, Man; Chen, Han; Fuchsberger, Christian; Kwan, Phoenix; Ma, Clement; Linderman, Michael; Lu, Yingchang; Thomsen, Soren K; Rundle, Jana K; Beer, Nicola L; van de Bunt, Martijn; Chalisey, Anil; Kang, Hyun Min; Voight, Benjamin F; Abecasis, Gonçalo R; Almgren, Peter; Baldassarre, Damiano; Balkau, Beverley; Benediktsson, Rafn; Blüher, Matthias; Boeing, Heiner; Bonnycastle, Lori L; Bottinger, Erwin P; Burtt, Noël P; Carey, Jason; Charpentier, Guillaume; Chines, Peter S; Cornelis, Marilyn C; Couper, David J; Crenshaw, Andrew T; van Dam, Rob M; Doney, Alex S F; Dorkhan, Mozhgan; Edkins, Sarah; Eriksson, Johan G; Esko, Tonu; Eury, Elodie; Fadista, João; Flannick, Jason; Fontanillas, Pierre; Fox, Caroline; Franks, Paul W; Gertow, Karl; Gieger, Christian; Gigante, Bruna; Gottesman, Omri; Grant, George B; Grarup, Niels; Groves, Christopher J; Hassinen, Maija; Have, Christian T; Herder, Christian; Holmen, Oddgeir L; Hreidarsson, Astradur B; Humphries, Steve E; Hunter, David J; Jackson, Anne U; Jonsson, Anna; Jørgensen, Marit E; Jørgensen, Torben; Kao, Wen-Hong L; Kerrison, Nicola D; Kinnunen, Leena; Klopp, Norman; Kong, Augustine; Kovacs, Peter; Kraft, Peter; Kravic, Jasmina; Langford, Cordelia; Leander, Karin; Liang, Liming; Lichtner, Peter; Lindgren, Cecilia M; Lindholm, Eero; Linneberg, Allan; Liu, Ching-Ti; Lobbens, Stéphane; Luan, Jian'an; Lyssenko, Valeriya; Männistö, Satu; McLeod, Olga; Meyer, Julia; Mihailov, Evelin; Mirza, Ghazala; Mühleisen, Thomas W; Müller-Nurasyid, Martina; Navarro, Carmen; Nöthen, Markus M; Oskolkov, Nikolay N; Owen, Katharine R; Palli, Domenico; Pechlivanis, Sonali; Peltonen, Leena; Perry, John R B; Platou, Carl G P; Roden, Michael; Ruderfer, Douglas; Rybin, Denis; van der Schouw, Yvonne T; Sennblad, Bengt; Sigurðsson, Gunnar; Stančáková, Alena; Steinbach, Gerald; Storm, Petter; Strauch, Konstantin; Stringham, Heather M; Sun, Qi; Thorand, Barbara; Tikkanen, Emmi; Tonjes, Anke; Trakalo, Joseph; Tremoli, Elena; Tuomi, Tiinamaija; Wennauer, Roman; Wiltshire, Steven; Wood, Andrew R; Zeggini, Eleftheria; Dunham, Ian; Birney, Ewan; Pasquali, Lorenzo; Ferrer, Jorge; Loos, Ruth J F; Dupuis, Josée; Florez, Jose C; Boerwinkle, Eric; Pankow, James S; van Duijn, Cornelia; Sijbrands, Eric; Meigs, James B; Hu, Frank B; Thorsteinsdottir, Unnur; Stefansson, Kari; Lakka, Timo A; Rauramaa, Rainer; Stumvoll, Michael; Pedersen, Nancy L; Lind, Lars; Keinanen-Kiukaanniemi, Sirkka M; Korpi-Hyövälti, Eeva; Saaristo, Timo E; Saltevo, Juha; Kuusisto, Johanna; Laakso, Markku; Metspalu, Andres; Erbel, Raimund; Jöcke, Karl-Heinz; Moebus, Susanne; Ripatti, Samuli; Salomaa, Veikko; Ingelsson, Erik; Boehm, Bernhard O; Bergman, Richard N; Collins, Francis S; Mohlke, Karen L; Koistinen, Heikki; Tuomilehto, Jaakko; Hveem, Kristian; Njølstad, Inger; Deloukas, Panagiotis; Donnelly, Peter J; Frayling, Timothy M; Hattersley, Andrew T; de Faire, Ulf; Hamsten, Anders; Illig, Thomas; Peters, Annette; Cauchi, Stephane; Sladek, Rob; Froguel, Philippe; Hansen, Torben; Pedersen, Oluf; Morris, Andrew D; Palmer, Collin N A; Kathiresan, Sekar; Melander, Olle; Nilsson, Peter M; Groop, Leif C; Barroso, Inês; Langenberg, Claudia; Wareham, Nicholas J; O'Callaghan, Christopher A; Gloyn, Anna L; Altshuler, David; Boehnke, Michael; Teslovich, Tanya M; McCarthy, Mark I; Morris, Andrew P

2015-12-01

We performed fine mapping of 39 established type 2 diabetes (T2D) loci in 27,206 cases and 57,574 controls of European ancestry. We identified 49 distinct association signals at these loci, including five mapping in or near KCNQ1. 'Credible sets' of the variants most likely to drive each distinct signal mapped predominantly to noncoding sequence, implying that association with T2D is mediated through gene regulation. Credible set variants were enriched for overlap with FOXA2 chromatin immunoprecipitation binding sites in human islet and liver cells, including at MTNR1B, where fine mapping implicated rs10830963 as driving T2D association. We confirmed that the T2D risk allele for this SNP increases FOXA2-bound enhancer activity in islet- and liver-derived cells. We observed allele-specific differences in NEUROD1 binding in islet-derived cells, consistent with evidence that the T2D risk allele increases islet MTNR1B expression. Our study demonstrates how integration of genetic and genomic information can define molecular mechanisms through which variants underlying association signals exert their effects on disease.

Patterns of expression of position-dependent integrated transgenes in mouse embryo.

PubMed Central

Bonnerot, C; Grimber, G; Briand, P; Nicolas, J F

1990-01-01

The abilities to introduce foreign DNA into the genome of mice and to visualize gene expression at the single-cell level underlie a method for defining individual elements of a genetic program. We describe the use of an Escherichia coli lacZ reporter gene fused to the promoter of the gene for hypoxanthine phosphoribosyl transferase that is expressed in all tissues. Most transgenic mice (six of seven) obtained with this construct express the lacZ gene from the hypoxanthine phosphoribosyltransferase promoter. Unexpectedly, however, the expression is temporally and spatially regulated. Each transgenic line is characterized by a specific, highly reproducible pattern of lacZ expression. These results show that, for expression, the integrated construct must be complemented by elements of the genome. These elements exert dominant developmental control on the hypoxanthine phosphoribosyltransferase promoter. The expression patterns in some transgenic mice conform to a typological marker and in others to a subtle combination of typology and topography. These observations define discrete heterogeneities of cell types and of certain structures, particularly in the nervous system and in the mesoderm. This system opens opportunities for developmental studies by providing cellular, molecular, and genetic markers of cell types, cell states, and cells from developmental compartments. Finally this method illustrates that genes transduced or transposed to a different position in the genome acquire different spatiotemporal specificities, a result that has implications for evolution. Images PMID:1696727

PCR amplification and genetic analysis in a microwell cell culturing chip.

PubMed

Lindström, Sara; Hammond, Maria; Brismar, Hjalmar; Andersson-Svahn, Helene; Ahmadian, Afshin

2009-12-21

We have previously described a microwell chip designed for high throughput, long-term single-cell culturing and clonal analysis in individual wells providing a controlled way of studying high numbers of individual adherent or non-adherent cells. Here we present a method for the genetic analysis of cells cultured on-chip by PCR and minisequencing, demonstrated using two human adherent cell lines: one wild type and one with a single-base mutation in the p53 gene. Five wild type or mutated cells were seeded per well (in a defined set of wells, each holding 500 nL of culture medium) in a 672-microwell chip. The cell chip was incubated overnight, or cultured for up to five days, depending on the desired colony size, after which the cells were lysed and subjected to PCR directly in the wells. PCR products were detected, in the wells, using a biotinylated primer and a fluorescently labelled primer, allowing the products to be captured on streptavidin-coated magnetic beads and detected by a fluorescence microscope. In addition, to enable genetic analysis by minisequencing, the double-stranded PCR products were denatured and the immobilized strands were kept in the wells by applying a magnetic field from the bottom of the wells while the wells were washed, a minisequencing reaction mixture was added, and after incubation in appropriate conditions the expected genotypes were detected in the investigated microwells, simultaneously, by an array scanner. We anticipate that the technique could be used in mutation frequency screening, providing the ability to correlate cells' proliferative heterogeneity to their genetic heterogeneity, in hundreds of samples simultaneously. The presented method of single-cell culture and DNA amplification thus offers a potentially powerful alternative to single-cell PCR, with advantageous robustness and sensitivity.

The immunogenetics of primary biliary cirrhosis: A comprehensive review.

PubMed

Webb, G J; Siminovitch, K A; Hirschfield, G M

2015-11-01

Primary biliary cirrhosis (PBC), a classic autoimmune liver disease, is characterised by a progressive T cell predominant lymphocytic cholangitis, and a serologic pattern of reactivity in the form of specific anti-mitochondrial antibodies (AMA). CD4+ T cells are particularly implicated by PBC's cytokine signature, the presence of CD4+ T cells specific to mitochondrial auto-antigens, the expression of MHC II on injured biliary epithelial cells, and PBC's coincidence with other similar T cell mediated autoimmune conditions. CD4+ T cells are also central to current animal models of PBC, and their transfer typically also transfers disease. The importance of genetic risk to developing PBC is evidenced by a much higher concordance rate in monozygotic than dizygotic twins, increased AMA rates in asymptomatic relatives, and disproportionate rates of disease in siblings of PBC patients, PBC family members and certain genetically defined populations. Recently, high-throughput genetic studies have greatly expanded our understanding of the gene variants underpinning risk for PBC development, so linking genetics and immunology. Here we summarize genetic association data that has emerged from large scale genome-wide association studies and discuss the evidence for the potential functional significance of the individual genes and pathways identified; we particularly highlight associations in the IL-12-STAT4-Th1 pathway. HLA associations and epigenetic effects are specifically considered and individual variants are linked to clinical phenotypes where data exist. We also consider why there is a gap between calculated genetic risk and clinical data: so-called missing heritability, and how immunogenetic observations are being translated to novel therapies. Ultimately whilst genetic risk factors will only account for a proportion of disease risk, ongoing efforts to refine associations and understand biologic links to disease pathways are hoped to drive more rational therapy for patients. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Genetic interaction between two insulin-dependent diabetes susceptibility loci, Idd2 and Idd13, in determining immunoregulatory DN T cell proportion.

PubMed

Collin, Roxanne; Doyon, Kathy; Mullins-Dansereau, Victor; Karam, Martin; Chabot-Roy, Geneviève; Hillhouse, Erin E; Orthwein, Alexandre; Lesage, Sylvie

2018-04-25

Several immune regulatory cell types participate in the protection against autoimmune diseases such as autoimmune diabetes. Of these immunoregulatory cells, we and others have shown that peripheral CD4 - CD8 - double negative (DN) T cells can induce antigen-specific immune tolerance. Particularly, we have described that diabetes-prone mice exhibit a lower number of peripheral DN T cells compared to diabetes-resistant mice. Identifying the molecular pathways that influence the size of the DN T cell pool in peripheral lymphoid organs may thus be of interest for maintaining antigen-specific immune tolerance. Hence, through immunogenetic approaches, we found that two genetic loci linked to autoimmune diabetes susceptibility, namely Idd2 and Idd13, independently contribute to the partial restoration of DN T cell proportion in secondary lymphoid organs. We now extend these findings to show an interaction between the Idd2 and Idd13 loci in determining the number of DN T cells in secondary lymphoid organs. Using bioinformatics tools, we link potential biological pathways arising from interactions of genes encoded within the two loci. By focusing on cell cycle, we validate that both the Idd2 and Idd13 loci influence RAD51 expression as well as DN T cell progression through the cell cycle. Altogether, we find that genetic interactions between Idd2 and Idd13 loci modulate cell cycle progression, which contributes, at least in part, to defining the proportion of DN T cells in secondary lymphoid organs.

Recurrent candidiasis and early-onset gastric cancer in a patient with a genetically defined partial MYD88 defect.

PubMed

Vogelaar, Ingrid P; Ligtenberg, Marjolijn J L; van der Post, Rachel S; de Voer, Richarda M; Kets, C Marleen; Jansen, Trees J G; Jacobs, Liesbeth; Schreibelt, Gerty; de Vries, I Jolanda M; Netea, Mihai G; Hoogerbrugge, Nicoline

2016-04-01

Gastric cancer is caused by both genetic and environmental factors. A woman who suffered from recurrent candidiasis throughout her life developed diffuse-type gastric cancer at the age of 23 years. Using whole-exome sequencing we identified a germline homozygous missense variant in MYD88. Immunological assays on peripheral blood mononuclear cells revealed an impaired immune response upon stimulation with Candida albicans, characterized by a defective production of the cytokine interleukin-17. Our data suggest that a genetic defect in MYD88 results in an impaired immune response and may increase gastric cancer risk.

Translation of Genotype to Phenotype by a Hierarchy of Cell Subsystems.

PubMed

Yu, Michael Ku; Kramer, Michael; Dutkowski, Janusz; Srivas, Rohith; Licon, Katherine; Kreisberg, Jason; Ng, Cherie T; Krogan, Nevan; Sharan, Roded; Ideker, Trey

2016-02-24

Accurately translating genotype to phenotype requires accounting for the functional impact of genetic variation at many biological scales. Here we present a strategy for genotype-phenotype reasoning based on existing knowledge of cellular subsystems. These subsystems and their hierarchical organization are defined by the Gene Ontology or a complementary ontology inferred directly from previously published datasets. Guided by the ontology's hierarchical structure, we organize genotype data into an "ontotype," that is, a hierarchy of perturbations representing the effects of genetic variation at multiple cellular scales. The ontotype is then interpreted using logical rules generated by machine learning to predict phenotype. This approach substantially outperforms previous, non-hierarchical methods for translating yeast genotype to cell growth phenotype, and it accurately predicts the growth outcomes of two new screens of 2,503 double gene knockouts impacting DNA repair or nuclear lumen. Ontotypes also generalize to larger knockout combinations, setting the stage for interpreting the complex genetics of disease.

Receptive Field Vectors of Genetically-Identified Retinal Ganglion Cells Reveal Cell-Type-Dependent Visual Functions

PubMed Central

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

2016-01-01

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

A role for adult TLX-positive neural stem cells in learning and behaviour.

PubMed

Zhang, Chun-Li; Zou, Yuhua; He, Weimin; Gage, Fred H; Evans, Ronald M

2008-02-21

Neurogenesis persists in the adult brain and can be regulated by a plethora of external stimuli, such as learning, memory, exercise, environment and stress. Although newly generated neurons are able to migrate and preferentially incorporate into the neural network, how these cells are molecularly regulated and whether they are required for any normal brain function are unresolved questions. The adult neural stem cell pool is composed of orphan nuclear receptor TLX-positive cells. Here, using genetic approaches in mice, we demonstrate that TLX (also called NR2E1) regulates adult neural stem cell proliferation in a cell-autonomous manner by controlling a defined genetic network implicated in cell proliferation and growth. Consequently, specific removal of TLX from the adult mouse brain through inducible recombination results in a significant reduction of stem cell proliferation and a marked decrement in spatial learning. In contrast, the resulting suppression of adult neurogenesis does not affect contextual fear conditioning, locomotion or diurnal rhythmic activities, indicating a more selective contribution of newly generated neurons to specific cognitive functions.

Genetics of Inflammatory Bowel Diseases

PubMed Central

McGovern, Dermot; Kugathasan, Subra; Cho, Judy H.

2015-01-01

In this Review, we provide an update on genome-wide association studies (GWAS) in inflammatory bowel disease (IBD). In addition, we summarize progress in defining the functional consequences of associated alleles for coding and non-coding genetic variation. In the small minority of loci where major association signals correspond to non-synonymous variation, we summarize studies defining their functional effects and implications for therapeutic targeting. Importantly, the large majority of GWAS-associated loci involve non-coding variation, many of which modulate levels of gene expression. Recent expression quantitative trait loci (eQTL) studies have established that expression of the large majority of human genes is regulated by non-coding genetic variation. Significant advances in defining the epigenetic landscape have demonstrated that IBD GWAS signals are highly enriched within cell-specific active enhancer marks. Studies in European ancestry populations have dominated the landscape of IBD genetics studies, but increasingly, studies in Asian and African-American populations are being reported. Common variation accounts for only a modest fraction of the predicted heritability and the role of rare genetic variation of higher effects (i.e. odds ratios markedly deviating from one) is increasingly being identified through sequencing efforts. These sequencing studies have been particularly productive in very-early onset, more severe cases. A major challenge in IBD genetics will be harnessing the vast array of genetic discovery for clinical utility, through emerging precision medicine initiatives. We discuss the rapidly evolving area of direct to consumer genetic testing, as well as the current utility of clinical exome sequencing, especially in very early onset, severe IBD cases. We summarize recent progress in the pharmacogenetics of IBD with respect of partitioning patient responses to anti-TNF and thiopurine therapies. Highly collaborative studies across research centers and across subspecialties and disciplines will be required to fully realize the promise of genetic discovery in IBD. PMID:26255561

Extremely high genetic diversity in a single tumor points to prevalence of non-Darwinian cell evolution.

PubMed

Ling, Shaoping; Hu, Zheng; Yang, Zuyu; Yang, Fang; Li, Yawei; Lin, Pei; Chen, Ke; Dong, Lili; Cao, Lihua; Tao, Yong; Hao, Lingtong; Chen, Qingjian; Gong, Qiang; Wu, Dafei; Li, Wenjie; Zhao, Wenming; Tian, Xiuyun; Hao, Chunyi; Hungate, Eric A; Catenacci, Daniel V T; Hudson, Richard R; Li, Wen-Hsiung; Lu, Xuemei; Wu, Chung-I

2015-11-24

The prevailing view that the evolution of cells in a tumor is driven by Darwinian selection has never been rigorously tested. Because selection greatly affects the level of intratumor genetic diversity, it is important to assess whether intratumor evolution follows the Darwinian or the non-Darwinian mode of evolution. To provide the statistical power, many regions in a single tumor need to be sampled and analyzed much more extensively than has been attempted in previous intratumor studies. Here, from a hepatocellular carcinoma (HCC) tumor, we evaluated multiregional samples from the tumor, using either whole-exome sequencing (WES) (n = 23 samples) or genotyping (n = 286) under both the infinite-site and infinite-allele models of population genetics. In addition to the many single-nucleotide variations (SNVs) present in all samples, there were 35 "polymorphic" SNVs among samples. High genetic diversity was evident as the 23 WES samples defined 20 unique cell clones. With all 286 samples genotyped, clonal diversity agreed well with the non-Darwinian model with no evidence of positive Darwinian selection. Under the non-Darwinian model, MALL (the number of coding region mutations in the entire tumor) was estimated to be greater than 100 million in this tumor. DNA sequences reveal local diversities in small patches of cells and validate the estimation. In contrast, the genetic diversity under a Darwinian model would generally be orders of magnitude smaller. Because the level of genetic diversity will have implications on therapeutic resistance, non-Darwinian evolution should be heeded in cancer treatments even for microscopic tumors.

Extremely high genetic diversity in a single tumor points to prevalence of non-Darwinian cell evolution

PubMed Central

Ling, Shaoping; Hu, Zheng; Yang, Zuyu; Yang, Fang; Li, Yawei; Lin, Pei; Chen, Ke; Dong, Lili; Cao, Lihua; Tao, Yong; Hao, Lingtong; Chen, Qingjian; Gong, Qiang; Wu, Dafei; Li, Wenjie; Zhao, Wenming; Tian, Xiuyun; Hao, Chunyi; Hungate, Eric A.; Catenacci, Daniel V. T.; Hudson, Richard R.; Li, Wen-Hsiung; Lu, Xuemei; Wu, Chung-I

2015-01-01

The prevailing view that the evolution of cells in a tumor is driven by Darwinian selection has never been rigorously tested. Because selection greatly affects the level of intratumor genetic diversity, it is important to assess whether intratumor evolution follows the Darwinian or the non-Darwinian mode of evolution. To provide the statistical power, many regions in a single tumor need to be sampled and analyzed much more extensively than has been attempted in previous intratumor studies. Here, from a hepatocellular carcinoma (HCC) tumor, we evaluated multiregional samples from the tumor, using either whole-exome sequencing (WES) (n = 23 samples) or genotyping (n = 286) under both the infinite-site and infinite-allele models of population genetics. In addition to the many single-nucleotide variations (SNVs) present in all samples, there were 35 “polymorphic” SNVs among samples. High genetic diversity was evident as the 23 WES samples defined 20 unique cell clones. With all 286 samples genotyped, clonal diversity agreed well with the non-Darwinian model with no evidence of positive Darwinian selection. Under the non-Darwinian model, MALL (the number of coding region mutations in the entire tumor) was estimated to be greater than 100 million in this tumor. DNA sequences reveal local diversities in small patches of cells and validate the estimation. In contrast, the genetic diversity under a Darwinian model would generally be orders of magnitude smaller. Because the level of genetic diversity will have implications on therapeutic resistance, non-Darwinian evolution should be heeded in cancer treatments even for microscopic tumors. PMID:26561581

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

PubMed

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

2008-04-01

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

Magnetic nanoparticles: Applications in gene delivery and gene therapy.

PubMed

Majidi, Sima; Zeinali Sehrig, Fatemeh; Samiei, Mohammad; Milani, Morteza; Abbasi, Elham; Dadashzadeh, Kianoosh; Akbarzadeh, Abolfazl

2016-06-01

Gene therapy is defined as the direct transfer of genetic material to tissues or cells for the treatment of inherited disorders and acquired diseases. For gene delivery, magnetic nanoparticles (MNPs) are typically combined with a delivery platform to encapsulate the gene, and promote cell uptake. Delivery technologies that have been used with MNPs contain polymeric, viral, as well as non-viral platforms. In this review, we focus on targeted gene delivery using MNPs.

Mi Casa es Su Casa: how an intracellular symbiont manipulates host biology.

PubMed

Bhattacharya, Tamanash; Newton, Irene L G

2017-10-27

Wolbachia pipientis, the most common intracellular infection on the planet, infects 40% of insects as well as nematodes, isopods and arachnids. Wolbachia are obligately intracellular and challenging to study; there are no genetic tools for manipulating Wolbachia nor can they be cultured outside of host cells. Despite these roadblocks, the research community has defined a set of Wolbachia loci involved in host interaction: Wolbachia effectors. Through the use of Drosophila genetics, surrogate systems and biochemistry, the field has begun to define the toolkit Wolbachia use for host manipulation. Below we review recent findings identifying these Wolbachia effectors and point to potential, as yet uncharacterized, links between known phenotypes induced by Wolbachia infection and predicted effectors. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Fibrinogen Induces RUNX2 Activity and Osteogenic Development from Human Pluripotent Stem Cells

PubMed Central

Kidwai, Fahad; Edwards, Jessica; Zou, Li; Kaufman, Dan S.

2016-01-01

Pluripotent stem cells, both human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC), provide an important resource to produce specialized cells such as osteogenic cells for therapeutic applications such as repair or replacement of injured, diseased or damaged bone. hESCs and iPSCs can also be used to better define basic cellular and genetic mechanisms that regulate the earliest stages of human bone development. However, current strategies to mediate osteogenic differentiation of hESC and iPSC are typically limited by the use of xenogeneic components such as fetal bovine serum (FBS) that make defining specific agents that mediate human osteogenesis difficult. Runt-related transcription factor 2 (RUNX2) is a key regulator required for osteogenic differentiation. Here, we used a RUNX2-YFP reporter system to characterize the novel ability of fibrinogen to mediate human osteogenic development from hESC and iPSC in defined (serum-free) conditions. These studies demonstrate that fibrinogen mediates significant osteo-induction potential. Specifically, fibrinogen binds to the surface integrin (α9β1) to mediate RUNX2 gene expression through the SMAD1/5/8 signaling pathway. Additional studies characterize the fibrinogen-induced hESC/iPSC-derived osteogenic cells to demonstrate these osteogenic cells retain the capacity to express typical mature osteoblastic markers. Together, these studies define a novel fibrinogen-α9β1-SMAD1/5/8-RUNX2 signaling axis can efficiently induce osteogenic differentiation from hESCs and iPSCs. PMID:27331788

Clinical Utility of Circulating Tumor Cells in ALK-Positive Non-Small-Cell Lung Cancer.

PubMed

Faugeroux, Vincent; Pailler, Emma; Auger, Nathalie; Taylor, Melissa; Farace, Françoise

2014-01-01

The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate, progression free survival) compared to systemic therapy. However, the detection of such molecular abnormalities is complicated by the difficulty in obtaining sufficient tumor material, in terms of quantity and quality, from a biopsy. Here, we described how circulating tumor cells (CTCs) can have a clinical utility in anaplastic lymphoma kinase (ALK) positive NSCLC patients to diagnose ALK-EML4 gene rearrangement and to guide therapeutic management of these patients. The ability to detect genetic abnormalities such ALK rearrangement in CTCs shows that these cells could offer new perspectives both for the diagnosis and the monitoring of ALK-positive patients eligible for treatment with ALK inhibitors.

Clinical Utility of Circulating Tumor Cells in ALK-Positive Non-Small-Cell Lung Cancer

PubMed Central

Faugeroux, Vincent; Pailler, Emma; Auger, Nathalie; Taylor, Melissa; Farace, Françoise

2014-01-01

The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate, progression free survival) compared to systemic therapy. However, the detection of such molecular abnormalities is complicated by the difficulty in obtaining sufficient tumor material, in terms of quantity and quality, from a biopsy. Here, we described how circulating tumor cells (CTCs) can have a clinical utility in anaplastic lymphoma kinase (ALK) positive NSCLC patients to diagnose ALK-EML4 gene rearrangement and to guide therapeutic management of these patients. The ability to detect genetic abnormalities such ALK rearrangement in CTCs shows that these cells could offer new perspectives both for the diagnosis and the monitoring of ALK-positive patients eligible for treatment with ALK inhibitors. PMID:25414829

The genomic landscape of mantle cell lymphoma is related to the epigenetically determined chromatin state of normal B cells

PubMed Central

Zhang, Jenny; Jima, Dereje; Moffitt, Andrea B.; Liu, Qingquan; Czader, Magdalena; Hsi, Eric D.; Fedoriw, Yuri; Dunphy, Cherie H.; Richards, Kristy L.; Gill, Javed I.; Sun, Zhen; Love, Cassandra; Scotland, Paula; Lock, Eric; Levy, Shawn; Hsu, David S.; Dunson, David; Dave, Sandeep S.

2014-01-01

In this study, we define the genetic landscape of mantle cell lymphoma (MCL) through exome sequencing of 56 cases of MCL. We identified recurrent mutations in ATM, CCND1, MLL2, and TP53. We further identified a number of novel genes recurrently mutated in patients with MCL including RB1, WHSC1, POT1, and SMARCA4. We noted that MCLs have a distinct mutational profile compared with lymphomas from other B-cell stages. The ENCODE project has defined the chromatin structure of many cell types. However, a similar characterization of primary human mature B cells has been lacking. We defined, for the first time, the chromatin structure of primary human naïve, germinal center, and memory B cells through chromatin immunoprecipitation and sequencing for H3K4me1, H3K4me3, H3Ac, H3K36me3, H3K27me3, and PolII. We found that somatic mutations that occur more frequently in either MCLs or Burkitt lymphomas were associated with open chromatin in their respective B cells of origin, naïve B cells, and germinal center B cells. Our work thus elucidates the landscape of gene-coding mutations in MCL and the critical interplay between epigenetic alterations associated with B-cell differentiation and the acquisition of somatic mutations in cancer. PMID:24682267

Global expression analysis of gene regulatory pathways during endocrine pancreatic development.

PubMed

Gu, Guoqiang; Wells, James M; Dombkowski, David; Preffer, Fred; Aronow, Bruce; Melton, Douglas A

2004-01-01

To define genetic pathways that regulate development of the endocrine pancreas, we generated transcriptional profiles of enriched cells isolated from four biologically significant stages of endocrine pancreas development: endoderm before pancreas specification, early pancreatic progenitor cells, endocrine progenitor cells and adult islets of Langerhans. These analyses implicate new signaling pathways in endocrine pancreas development, and identified sets of known and novel genes that are temporally regulated, as well as genes that spatially define developing endocrine cells from their neighbors. The differential expression of several genes from each time point was verified by RT-PCR and in situ hybridization. Moreover, we present preliminary functional evidence suggesting that one transcription factor encoding gene (Myt1), which was identified in our screen, is expressed in endocrine progenitors and may regulate alpha, beta and delta cell development. In addition to identifying new genes that regulate endocrine cell fate, this global gene expression analysis has uncovered informative biological trends that occur during endocrine differentiation.

Successful In Vitro Expansion and Differentiation of Cord Blood Derived CD34+ Cells into Early Endothelial Progenitor Cells Reveals Highly Differential Gene Expression

PubMed Central

Topcic, Denijal; Haviv, Izhak; Merivirta, Ruusu-Maaria; Agrotis, Alexander; Leitner, Ephraem; Jowett, Jeremy B.; Bode, Christoph; Lappas, Martha; Peter, Karlheinz

2011-01-01

Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU) assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP), PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15) or pro-angiogenic (galectin-3) properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP) was the most up-regulated gene. PMID:21858032

Genetic and pharmacological intervention for treatment/prevention of hearing loss

PubMed Central

Cotanche, Douglas A.

2008-01-01

Twenty years ago it was first demonstrated that birds could regenerate their cochlear hair cells following noise damage or aminoglycoside treatment. An understanding of how this structural and functional regeneration occurred might lead to the development of therapies for treatment of sensorineural hearing loss in humans. Recent experiments have demonstrated that noise exposure and aminoglycoside treatment lead to apoptosis of the hair cells. In birds, this programmed cell death induces the adjacent supporting cells to undergo regeneration to replace the lost hair cells. Although hair cells in the mammalian cochlea undergo apoptosis in response to noise damage and ototoxic drug treatment, the supporting cells do not possess the ability to undergo regeneration. However, current experiments on genetic manipulation, gene therapy, and stem cell transplantation suggest that regeneration in the mammalian cochlea may eventually be possible and may 1 day provide a therapeutic tool for hearing loss in humans. Learning outcomes The reader should be able to: (1) Describe the anatomy of the avian and mammalian cochlea, identify the individual cell types in the organ of Corti, and distinguish major features that participate in hearing function, (2) Demonstrate a knowledge of how sound damage and aminoglycoside poisoning induce apoptosis of hair cells in the cochlea, (3) Define how hair cell loss in the avian cochlea leads to regeneration of new hair cells and distinguish this from the mammalian cochlea where there is no regeneration following damage, and (4) Interpret the potential for new approaches, such as genetic manipulation, gene therapy and stem cell transplantation, could provide a therapeutic approach to hair cell loss in the mammalian cochlea. PMID:18455177

Genetic and pharmacological intervention for treatment/prevention of hearing loss.

PubMed

Cotanche, Douglas A

2008-01-01

Twenty years ago it was first demonstrated that birds could regenerate their cochlear hair cells following noise damage or aminoglycoside treatment. An understanding of how this structural and functional regeneration occurred might lead to the development of therapies for treatment of sensorineural hearing loss in humans. Recent experiments have demonstrated that noise exposure and aminoglycoside treatment lead to apoptosis of the hair cells. In birds, this programmed cell death induces the adjacent supporting cells to undergo regeneration to replace the lost hair cells. Although hair cells in the mammalian cochlea undergo apoptosis in response to noise damage and ototoxic drug treatment, the supporting cells do not possess the ability to undergo regeneration. However, current experiments on genetic manipulation, gene therapy, and stem cell transplantation suggest that regeneration in the mammalian cochlea may eventually be possible and may 1 day provide a therapeutic tool for hearing loss in humans. The reader should be able to: (1) Describe the anatomy of the avian and mammalian cochlea, identify the individual cell types in the organ of Corti, and distinguish major features that participate in hearing function, (2) Demonstrate a knowledge of how sound damage and aminoglycoside poisoning induce apoptosis of hair cells in the cochlea, (3) Define how hair cell loss in the avian cochlea leads to regeneration of new hair cells and distinguish this from the mammalian cochlea where there is no regeneration following damage, and (4) Interpret the potential for new approaches, such as genetic manipulation, gene therapy and stem cell transplantation, could provide a therapeutic approach to hair cell loss in the mammalian cochlea.

"Atypical" Pleomorphic Lipomatous Tumor: A Clinicopathologic, Immunohistochemical and Molecular Study of 21 Cases, Emphasizing its Relationship to Atypical Spindle Cell Lipomatous Tumor and Suggesting a Morphologic Spectrum (Atypical Spindle Cell/Pleomorphic Lipomatous Tumor).

PubMed

Creytens, David; Mentzel, Thomas; Ferdinande, Liesbeth; Lecoutere, Evelyne; van Gorp, Joost; Atanesyan, Lilit; de Groot, Karel; Savola, Suvi; Van Roy, Nadine; Van Dorpe, Jo; Flucke, Uta

2017-11-01

The classification of the until recently poorly explored group of atypical adipocytic neoplasms with spindle cell features, for which recently the term atypical spindle cell lipomatous tumor (ASLT) has been proposed, remains challenging. Recent studies have proposed ASLT as a unique entity with (in at least a significant subset of cases) a specific genetic background, namely deletions/losses of 13q14, including RB1 and its flanking genes RCBTB2, DLEU1, and ITM2B. Similar genetic aberrations have been reported in pleomorphic liposarcomas (PLSs). This prompted us to investigate a series of 21 low-grade adipocytic neoplasms with a pleomorphic lipoma-like appearance, but with atypical morphologic features (including atypical spindle cells, pleomorphic [multinucleated] cells, pleomorphic lipoblasts and poor circumscription), for which we propose the term "atypical" pleomorphic lipomatous tumor (APLT). Five cases of PLS were also included in this study. We used multiplex ligation-dependent probe amplification to evaluate genetic changes of 13q14. In addition, array-based comparative genomic hybridization was performed on 4 APLTs and all PLSs. Multiplex ligation-dependent probe amplification showed consistent loss of RB1 and its flanking gene RCBTB2 in all cases of APLT. This genetic alteration was also present in all PLSs, suggesting genetic overlap, in addition to morphologic overlap, with APLTs. However, array-based comparative genomic hybridization demonstrated more complex genetic alterations with more losses and gains in PLSs compared with APLTs. APLTs arose in the subcutis (67%) more frequently than in the deep (subfascial) soft tissues (33%). With a median follow-up of 42 months, recurrences were documented in 2 of 12 APLTs for which a long follow-up was available. Herein, we also demonstrate that APLTs share obvious overlapping morphologic, immunohistochemical, genetic and clinical characteristics with the recently defined ASLT, suggesting that they are related lesions that form a spectrum (atypical spindle cell/pleomorphic lipomatous tumor).

Modeling synthetic lethality

PubMed Central

Le Meur, Nolwenn; Gentleman, Robert

2008-01-01

Background Synthetic lethality defines a genetic interaction where the combination of mutations in two or more genes leads to cell death. The implications of synthetic lethal screens have been discussed in the context of drug development as synthetic lethal pairs could be used to selectively kill cancer cells, but leave normal cells relatively unharmed. A challenge is to assess genome-wide experimental data and integrate the results to better understand the underlying biological processes. We propose statistical and computational tools that can be used to find relationships between synthetic lethality and cellular organizational units. Results In Saccharomyces cerevisiae, we identified multi-protein complexes and pairs of multi-protein complexes that share an unusually high number of synthetic genetic interactions. As previously predicted, we found that synthetic lethality can arise from subunits of an essential multi-protein complex or between pairs of multi-protein complexes. Finally, using multi-protein complexes allowed us to take into account the pleiotropic nature of the gene products. Conclusions Modeling synthetic lethality using current estimates of the yeast interactome is an efficient approach to disentangle some of the complex molecular interactions that drive a cell. Our model in conjunction with applied statistical methods and computational methods provides new tools to better characterize synthetic genetic interactions. PMID:18789146

Acute engagement of Gq-mediated signaling in the bed nucleus of the stria terminalis induces anxiety-like behavior.

PubMed

Mazzone, C M; Pati, D; Michaelides, M; DiBerto, J; Fox, J H; Tipton, G; Anderson, C; Duffy, K; McKlveen, J M; Hardaway, J A; Magness, S T; Falls, W A; Hammack, S E; McElligott, Z A; Hurd, Y L; Kash, T L

2018-01-01

The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptor (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds designer receptors exclusively activated by a designer drug (DREADD), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping to profile brain-wide network activity following activation of G q -mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area and hindbrain structures such as the locus coeruleus and parabrachial nucleus. These results highlight that G q -mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple G q -coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the G q -coupled receptor 5-HT 2C R in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT neurons that may serve as therapeutic targets for regulating anxiety states and provide a blueprint for examining how G-protein-mediated signaling in a genetically defined cell type can be used to assess behavior and brain-wide circuit function.

Synthetic Substrata to Instruct Human Pluripotent Stem Cell Fate: From Novel Ligands to Functional Biomaterials

NASA Astrophysics Data System (ADS)

Musah, Samira

Human pluripotent stem (hPS) cells have the remarkable capacity to self-renew indefinitely and differentiate into desired cell types. They can serve as a virtually unlimited supply of cells for applications ranging from drug screening to cell therapies to understanding human development. Reaping the promise of hPS cells hinges on effective defined culture and differentiation conditions. Efforts to generate chemically-defined environments for hPS cell propagation and directed differentiation have been hindered by access to only a handful of ligands to target hPS cells. Additionally, progress has been limited also by lack of knowledge regarding the relevant functional properties of the cell culture substratum. To address these problems, I first employed forward-chemical-genetics coupled with self-assembled monolayer technology to identify novel peptides that bind to hPS cell-surface receptors. I then developed a controlled synthesis of hydrogels with tailored peptide display and mechanical properties. This approach yielded synthetic hydrogels with specific mechanical properties that function in a defined medium to robustly support hPS cell self-renewal. Finally, by starting from molecular level understanding that matrix elasticity regulates developmental pathways, I generated a highly efficient hydrogel platform that restricts hPS cell differentiation to neurons, even without soluble inductive factors. These results indicate that insoluble cues can be important information conduits to guide hPS cell fate decisions. I envision that the blueprint provided by this work can be utilized to devise new materials to guide hPS cell fate.

Single-Cell RNA-Sequencing in Glioma.

PubMed

Johnson, Eli; Dickerson, Katherine L; Connolly, Ian D; Hayden Gephart, Melanie

2018-04-10

In this review, we seek to summarize the literature concerning the use of single-cell RNA-sequencing for CNS gliomas. Single-cell analysis has revealed complex tumor heterogeneity, subpopulations of proliferating stem-like cells and expanded our view of tumor microenvironment influence in the disease process. Although bulk RNA-sequencing has guided our initial understanding of glioma genetics, this method does not accurately define the heterogeneous subpopulations found within these tumors. Single-cell techniques have appealing applications in cancer research, as diverse cell types and the tumor microenvironment have important implications in therapy. High cost and difficult protocols prevent widespread use of single-cell RNA-sequencing; however, continued innovation will improve accessibility and expand our of knowledge gliomas.

Functional diversification of taste cells in vertebrates

PubMed Central

Matsumoto, Ichiro; Ohmoto, Makoto; Abe, Keiko

2012-01-01

Tastes are senses resulting from the activation of taste cells distributed in oral epithelia. Sweet, umami, bitter, sour, and salty tastes are called the five “basic” tastes, but why five, and why these five? In this review, we dissect the peripheral gustatory system in vertebrates from molecular and cellular perspectives. Recent behavioral and molecular genetic studies have revealed the nature of functional taste receptors and cells and show that different taste qualities are accounted for by the activation of different subsets of taste cells. Based on this concept, the diversity of basic tastes should be defined by the diversity of taste cells in taste buds, which varies among species. PMID:23085625

Medical hypothesis: bifunctional genetic-hormonal pathways to breast cancer.

PubMed

Davis, D L; Telang, N T; Osborne, M P; Bradlow, H L

1997-04-01

As inherited germ line mutations, such as loss of BRCA1 or AT, account for less than 5% of all breast cancer, most cases involve acquired somatic perturbations. Cumulative lifetime exposure to bioavailable estradiol links most known risk factors (except radiation) for breast cancer. Based on a series of recent experimental and epidemiologic findings, we hypothesize that the multistep process of breast carcinogenesis results from exposure to endogenous or exogenous hormones, including phytoestrogens that directly or indirectly alter estrogen metabolism. Xenohormones are defined as xenobiotic materials that modify hormonal production; they can work bifunctionally, through genetic or hormonal paths, depending on the periods and extent of exposure. As for genetic paths, xenohormones can modify DNA structure or function. As for hormonal paths, two distinct mechanisms can influence the potential for aberrant cell growth: compounds can directly bind with endogenous hormone or growth factor receptors affecting cell proliferation or compounds can modify breast cell proliferation altering the formation of hormone metabolites that influence epithelial-stromal interaction and growth regulation. Beneficial xenohormones, such as indole-3-carbinol, genistein, and other bioflavonoids, may reduce aberrant breast cell proliferation, and influence the rate of DNA repair or apoptosis and thereby influence the genetic or hormonal microenvironments. Upon validation with appropriate in vitro and in vivo studies, biologic markers of the risk for breast cancer, such as hormone metabolites, total bioavailable estradiol, and free radical generators can enhance cancer detection and prevention.

Human chimera-type galectin-3: defining the critical tail length for high-affinity glycoprotein/cell surface binding and functional competition with galectin-1 in neuroblastoma cell growth regulation.

PubMed

Kopitz, Jürgen; Vértesy, Sabine; André, Sabine; Fiedler, Sabine; Schnölzer, Martina; Gabius, Hans-Joachim

2014-09-01

Many human proteins have a modular design with receptor and structural domains. Using adhesion/growth-regulatory galectin-3 as model, we describe an interdisciplinary strategy to define the functional significance of its tail established by nine non-triple helical collagen-like repeats (I-IX) and the N-terminal peptide. Genetic engineering with sophisticated mass spectrometric product analysis provided the tools for biotesting, i.e. eight protein variants with different degrees of tail truncation. Evidently,various aspects of galectin-3 activity (cis binding and cell bridging) are affected by tail shortening in a different manner. Thus, this combined approach reveals an unsuspected complexity of structure-function relationship, encouraging further application beyond this chimera-type galectin. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Dedifferentiated Liposarcoma: Updates on Morphology, Genetics, and Therapeutic Strategies.

PubMed

Thway, Khin; Jones, Robin L; Noujaim, Jonathan; Zaidi, Shane; Miah, Aisha B; Fisher, Cyril

2016-01-01

Well-differentiated liposarcoma (WDL) and dedifferentiated liposarcoma (DDL) form the largest subgroup of liposarcomas, and represent a morphologic and behavioral spectrum of 1 disease entity, which arises typically in middle to late adult life, most frequently within the retroperitoneum or extremities. DDL is defined as nonlipogenic sarcoma that is juxtaposed to WDL, occurs as a recurrence of WDL or which can arise de novo, and typically has the appearance of undifferentiated pleomorphic or spindle cell sarcoma. DDL have a propensity for local recurrence, whereas distant metastasis is rarer, and behavior is related to anatomic site, with retroperitoneal neoplasms showing a significantly worse prognosis. Surgical resection remains the mainstay of treatment, and medical options for patients with aggressive recurrent or metastatic disease are limited. DDL share similar genetic abnormalities to WDL, with high-level amplifications of chromosome 12q14-15, including the MDM2 and CDK4 cell cycle oncogenes, and DDL harbor additional genetic changes, particularly coamplifications of 6q23 and 1p32. Novel therapies targeted at the gene products of chromosome 12 are being tested in clinical trials. We review the pathology and genetics of DDL, discussing morphologic patterns, immunohistochemical and genetic findings, the differential diagnosis, and future therapeutic strategies.

Purpose and regulation of stem cells: a systems-biology view from the Caenorhabditis elegans germ line.

PubMed

Cinquin, Olivier

2009-01-01

Stem cells are expected to play a key role in the development and maintenance of organisms, and hold great therapeutic promises. However, a number of questions must be answered to achieve an understanding of stem cells and put them to use. Here I review some of these questions, and how they relate to the model system provided by the Caenorhabditis elegans germ line, which is exceptional in its thorough genetic characterization and experimental accessibility under in vivo conditions. A fundamental question is how to define a stem cell; different definitions can be adopted that capture different features of interest. In the C. elegans germ line, stem cells can be defined by cell lineage or by cell commitment ('commitment' must itself be carefully defined). These definitions are associated with two other important questions about stem cells: their functions (which must be addressed following a systems approach, based on an evolutionary perspective) and their regulation. I review possible functions and their evolutionary groundings, including genome maintenance and powerful regulation of cell proliferation and differentiation, and possible regulatory mechanisms, including asymmetrical division and control of transit amplification by a developmental timer. I draw parallels between Drosophila and C. elegans germline stem cells; such parallels raise intriguing questions about Drosophila stem cells. I conclude by showing that the C. elegans germ line bears similarities with a number of other stem cell systems, which underscores its relevance to the understanding of stem cells.

Tumor cell dormancy: implications for the biology and treatment of breast cancer.

PubMed

Fehm, T; Mueller, V; Marches, R; Klein, G; Gueckel, B; Neubauer, H; Solomayer, E; Becker, S

2008-01-01

Despite progress made in the therapy of solid tumors such as breast cancer, the prognosis of patients even with small primary tumors is still limited by metastatic relapse often long after removal of the primary tumor. Therefore, it has been hypothesized that primary tumors shed tumor cells already at an early stage into the blood circulation. A subset of these disseminated tumor cells may persist in a state of so-called "dormancy". Based on cell culture and animal models, dormancy can occur at two different stages. Single dormant cells are defined as cells with a lack of proliferation and apoptosis with the cells undergoing cell cycle arrest. The micrometastasis model defines tumor cell dormancy as a state of balanced apoptosis and proliferation of micrometastasis resulting in no net increase of tumor mass. Mechanisms leading to a growth activation of dormant tumor cells and the outgrowth of manifest metastases are not completely understood. Genetic predisposition of the dormant cells as well as immunological and angiogenetic influences of the surrounding environment may contribute to this phenomenon. In this review, we summarize findings on different factors for tumor cell dormancy and potential therapeutic implications that should help to reduce metastatic relapse in cancer patients.

Cellular resolution maps of X chromosome inactivation: implications for neural development, function, and disease.

PubMed

Wu, Hao; Luo, Junjie; Yu, Huimin; Rattner, Amir; Mo, Alisa; Wang, Yanshu; Smallwood, Philip M; Erlanger, Bracha; Wheelan, Sarah J; Nathans, Jeremy

2014-01-08

Female eutherian mammals use X chromosome inactivation (XCI) to epigenetically regulate gene expression from ∼4% of the genome. To quantitatively map the topography of XCI for defined cell types at single cell resolution, we have generated female mice that carry X-linked, Cre-activated, and nuclear-localized fluorescent reporters--GFP on one X chromosome and tdTomato on the other. Using these reporters in combination with different Cre drivers, we have defined the topographies of XCI mosaicism for multiple CNS cell types and of retinal vascular dysfunction in a model of Norrie disease. Depending on cell type, fluctuations in the XCI mosaic are observed over a wide range of spatial scales, from neighboring cells to left versus right sides of the body. These data imply a major role for XCI in generating female-specific, genetically directed, stochastic diversity in eutherian mammals on spatial scales that would be predicted to affect CNS function within and between individuals. Copyright © 2014 Elsevier Inc. All rights reserved.

Cellular resolution maps of X-chromosome inactivation: implications for neural development, function, and disease

PubMed Central

Wu, Hao; Luo, Junjie; Yu, Huimin; Rattner, Amir; Mo, Alisa; Wang, Yanshu; Smallwood, Philip M.; Erlanger, Bracha; Wheelan, Sarah J.; Nathans, Jeremy

2014-01-01

Female eutherian mammals use X-chromosome inactivation (XCI) to epigenetically regulate gene expression from ~4% of genes. To quantitatively map the topography of XCI for defined cell types at single cell resolution, we have generated female mice that carry X-linked, Cre-activated, and nuclear-localized fluorescent reporters – GFP on one X-chromosome and tdTomato on the other. Using these reporters in combination with different Cre drivers we have defined the topographies of XCI mosaicism for multiple CNS cell types and of retinal vascular dysfunction in a model of Norrie Disease. Depending on cell type, fluctuations in the XCI mosaic are observed over a wide range of spatial scales, from neighboring cells to left vs. right sides of the body. These data imply a major role for XCI in generating female-specific, genetically directed, stochastic diversity in eutherian mammals on spatial scales that would be predicted to affect CNS function within and between individuals. PMID:24411735

Defining and redefining the scope and goals of genetic counseling.

PubMed

Resta, Robert G

2006-11-15

Many definitions of genetic counseling have been proposed since Sheldon Reed first defined the term in 1947. This study reviews selected definitions of genetic counseling including the most recent definition proposed by a committee of the National Society of Genetic Counselors. The analysis focuses on the professional background of who was formulating the definition; the reasons why the definition was created; medical, historical, and social factors; and the definer's implicit or explicit goals of genetic counseling. No definition of genetic counseling is ideal, and any definition can only reflect the values, ethics, goals, and medical practices of the person or group defining the practice of genetic counseling. (c) 2006 Wiley-Liss, Inc.

Survival of mature mouse olfactory sensory neurons labeled genetically perinatally.

PubMed

Holl, Anna-Maria

2018-04-01

The main olfactory epithelium (MOE) of an adult mouse harbors a few million mature olfactory sensory neurons (OSNs), which are traditionally defined as mature by their expression of the olfactory marker protein (OMP). Mature OSNs differentiate in situ from stem cells at the base of the MOE. The consensus view is that mature OSNs have a defined lifespan and then undergo programmed cell death, and that the adult MOE maintains homeostasis by generating new mature OSNs from stem cells. But there is also evidence for mature OSNs that are long-lived. Thus far modern genetic tools have not been applied to quantify survival of a population of OSNs that are mature at a given point in time. Here, a genetic strategy was developed to label irreversibly OMP-expressing OSNs in mice. A gene-targeted OMP-CreERT2 strain was generated in which mature OSNs express an enzymatically inactive version of the Cre recombinase. The fusion protein CreERT2 becomes transiently active when exposed to tamoxifen, and in the presence of a Cre reporter in the genome such as tdRFP, CreERT2-expressing cells become irreversibly labeled. A cohort of mice was generated with the same day of birth by in vitro fertilization and embryo transfer, and injected tamoxifen in their mothers at E18.5 of gestation. I counted RFP immunoreactive cells in the MOE and vomeronasal organ of 36 tamoxifen-exposed OMP-CreERT2 × tdRFP mice from 7 age groups: postnatal day (PD)1.5, PD3.5, PD6.5, 3 weeks, 9 weeks, 6 months, and 12 months. Approximately 7.8% of perinatally labeled cells remain at 12 months, confirming that some mature OSNs are indeed long-lived. The survival curve of the population of perinatally labeled MOE cells can be modeled with a mean half-life of 26 days for the population as a whole, excluding the long-lived cells. Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.

Genetics of Gonadal Stem Cell Renewal

PubMed Central

Greenspan, Leah Joy; de Cuevas, Margaret

2015-01-01

Stem cells are necessary for the maintenance of many adult tissues. Signals within the stem cell microenvironment, or niche, regulate the self-renewal and differentiation capability of these cells. Misregulation of these signals through mutation or damage can lead to overgrowth or depletion of different stem cell pools. In this review, we focus on the Drosophila testis and ovary, both of which contain well-defined niches, as well as the mouse testis, which has become a more approachable stem cell system with recent technical advances. We discuss the signals that regulate gonadal stem cells in their niches, how these signals mediate self-renewal and differentiation under homeostatic conditions, and how stress, whether from mutations or damage, can cause changes in cell fate and drive stem cell competition. PMID:26355592

Increased microtubule assembly rates mediate chromosomal instability in colorectal cancer cells

PubMed Central

Ertych, Norman; Stolz, Ailine; Stenzinger, Albrecht; Weichert, Wilko; Kaulfuß, Silke; Burfeind, Peter; Aigner, Achim; Wordeman, Linda

2015-01-01

Chromosomal instability (CIN) is defined as the perpetual missegregation of whole chromosomes during mitosis and represents a hallmark of human cancer. However, the mechanisms causing CIN and its consequences on tumor growth are largely unknown. We identify an increase in microtubule plus end assembly rates as a fundamental trigger for CIN in CRC cells. This trigger is mediated by overexpression of the oncogene AURKA or by loss of the tumor suppressor gene CHK2, a genetic constitution found in 73% of human colorectal cancers. Increased microtubule assembly rates are associated with transient abnormalities in mitotic spindle geometry promoting the generation of lagging chromosomes and resulting in CIN. Reconstitution of proper microtubule assembly rates by chemical or genetic means suppresses CIN and thereby, unexpectedly, accelerates tumor growth in vitro and in vivo. Thus, we identify a fundamental mechanism triggering CIN in cancer cells and reveal its adverse consequence on tumor growth. PMID:24976383

Defining Genetic Risk for GVHD and Mortality Following Allogeneic Hematopoietic Stem Cell Transplantation

PubMed Central

Hansen, John A; Chien, Jason W; Warren, Edus H; Zhao, Lue Ping; Martin, Paul J

2011-01-01

Purpose of review To explore what is known about the genetics of hematopoietic stem cell transplantation (HCT) and how genetic polymorphism affects risk of graft-versus-host disease (GVHD) and mortality. Recent findings Genetic variation found across the human genome can impact HCT outcome by 1) causing genetic disparity between patient and donor, and 2) modifying gene function. Single nucleotide polymorphisms (SNP) and structural variation can result in mismatching for cellular peptides known as histocompatibility antigens (HA). At least 25 to 30 polymorphic genes are known to encode functional HA in mismatched individuals, but their individual contribution to clinical GVHD is unclear. HCT outcome may also be affected by polymorphism in donor or recipient. Association studies have implicated several genes with GVHD and mortality, however results have been inconsistent most likely due to limited sample size, and differences in racial diversity and clinical covariates. New technologies using DNA arrays genotyping for a million or more SNPs promise genome-wide discovery of HCT associated genes, however adequate statistical power requires study populations of several thousand patient-donor pairs. Summary Available data offers strong preliminary support for the impact that genetic variation has on risk of GVHD and mortality following HCT. Definitive results however await future genome-wide studies of large multi-center HCT cohorts. PMID:20827186

Genetic heterogeneity of L-Zagreb mumps virus vaccine strain.

PubMed

Kosutic-Gulija, Tanja; Forcic, Dubravko; Santak, Maja; Ramljak, Ana; Mateljak-Lukacevic, Sanja; Mazuran, Renata

2008-07-10

The most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous. We identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo. L-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes.

Mechanisms underlying cellular responses of cells from haemopoietic tissue to low dose/low LET radiation

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

Munira A Kadhim

2010-03-05

To accurately define the risks associated with human exposure to relevant environmental doses of low LET ionizing radiation, it is necessary to completely understand the biological effects at very low doses (i.e., less than 0.1 Gy), including the lowest possible dose, that of a single electron track traversal. At such low doses, a range of studies have shown responses in biological systems which are not related to the direct interaction of radiation tracks with DNA. The role of these “non-targeted” responses in critical tissues is poorly understood and little is known regarding the underlying mechanisms. Although critical for dosimetry andmore » risk assessment, the role of individual genetic susceptibility in radiation risk is not satisfactorily defined at present. The aim of the proposed grant is to critically evaluate radiation-induced genomic instability and bystander responses in key stem cell populations from haemopoietic tissue. Using stem cells from two mouse strains (CBA/H and C57BL/6J) known to differ in their susceptibility to radiation effects, we plan to carefully dissect the role of genetic predisposition on two non-targeted radiation responses in these models; the bystander effect and genomic instability, which we believe are closely related. We will specifically focus on the effects of low doses of low LET radiation, down to doses approaching a single electron traversal. Using conventional X-ray and γ-ray sources, novel dish separation and targeted irradiation approaches, we will be able to assess the role of genetic variation under various bystander conditions at doses down to a few electron tracks. Irradiations will be carried out using facilities in routine operation for bystander targeted studies. Mechanistic studies of instability and the bystander response in different cell lineages will focus initially on the role of cytokines which have been shown to be involved in bystander signaling and the initiation of instability. These studies also aim to uncover protein mediators of the bystander responses using advanced proteomic screening of factors released from irradiated, bystander and unstable cells. Integral to these studies will be an assessment of the role of genetic susceptibility in these responses, using CBA/H and C57BL/6J mice. The relevance of in vivo interactions between stem cells and the stem cell niche will be explored in the future by re-implantation techniques of previously irradiated cells. The above studies will provide fundamental mechanistic information relating genetic predisposition to important low dose phenomena, and will aid in the development of Department of Energy policy, as well as radiation risk policy for the public and the workplace. We believe the proposed studies accurately reflect the goals of the DOE low dose program.« less

PIK3CA mutations in non-small cell lung cancer (NSCLC): genetic heterogeneity, prognostic impact and incidence of prior malignancies.

PubMed

Scheffler, Matthias; Bos, Marc; Gardizi, Masyar; König, Katharina; Michels, Sebastian; Fassunke, Jana; Heydt, Carina; Künstlinger, Helen; Ihle, Michaela; Ueckeroth, Frank; Albus, Kerstin; Serke, Monika; Gerigk, Ulrich; Schulte, Wolfgang; Töpelt, Karin; Nogova, Lucia; Zander, Thomas; Engel-Riedel, Walburga; Stoelben, Erich; Ko, Yon-Dschun; Randerath, Winfried; Kaminsky, Britta; Panse, Jens; Becker, Carolin; Hellmich, Martin; Merkelbach-Bruse, Sabine; Heukamp, Lukas C; Büttner, Reinhard; Wolf, Jürgen

2015-01-20

Somatic mutations of the PIK3CA gene have been described in non-small cell lung cancer (NSCLC), but limited data is available on their biological relevance. This study was performed to characterize PIK3CA-mutated NSCLC clinically and genetically. Tumor tissue collected consecutively from 1144 NSCLC patients within a molecular screening network between March 2010 and March 2012 was analyzed for PIK3CA mutations using dideoxy-sequencing and next-generation sequencing (NGS). Clinical, pathological, and genetic characteristics of PIK3CA-mutated patients are described and compared with a control group of PIK3CA-wildtype patients. Among the total cohort of 1144 patients we identified 42 (3.7%) patients with PIK3CA mutations in exon 9 and exon 20. These mutations were found with a higher frequency in sqamous cell carcinoma (8.9%) compared to adenocarcinoma (2.9%, p<0.001). The most common PIK3CA mutation was exon 9 E545K. The majority of patients (57.1%) had additional oncogenic driver aberrations. With the exception of EGFR-mutated patients, non of the genetically defined subgroups in this cohort had a significantly better median overall survival. Further, PIK3CA-mutated patients had a significantly higher incidence of malignancy prior to lung cancer (p<0.001). PIK3CA-mutated NSCLC represents a clinically and genetically heterogeneous subgroup in adenocarcinomas as well as in squamous cell carcinomas with a higher prevalence of these mutations in sqamous cell carcinoma. PIK3CA mutations have no negative impact on survival after surgery or systemic therapy. However, PIK3CA mutated lung cancer frequently develops in patients with prior malignancies.

Autosomal recessive PGM3 mutations link glycosylation defects to atopy, immune deficiency, autoimmunity, and neurocognitive impairment

PubMed Central

Zhang, Yu; Yu, Xiaomin; Ichikawa, Mie; Lyons, Jonathan J.; Datta, Shrimati; Lamborn, Ian T.; Jing, Huie; Kim, Emily S.; Biancalana, Matthew; Wolfe, Lynne A.; DiMaggio, Thomas; Matthews, Helen F.; Kranick, Sarah M.; Stone, Kelly D.; Holland, Steven M.; Reich, Daniel S.; Hughes, Jason D.; Mehmet, Huseyin; McElwee, Joshua; Freeman, Alexandra F.; Freeze, Hudson H.; Su, Helen C.; Milner, Joshua D.

2014-01-01

Background Identifying genetic syndromes that lead to significant atopic disease can open new pathways for investigation and intervention in allergy. Objective To define a genetic syndrome of severe atopy, elevated serum IgE, immune deficiency, autoimmunity, and motor and neurocognitive impairment. Methods Eight patients from two families who had similar syndromic features were studied. Thorough clinical evaluations, including brain MRI and sensory evoked potentials, were performed. Peripheral lymphocyte flow cytometry, antibody responses, and T cell cytokine production were measured. Whole exome sequencing was performed to identify disease-causing mutations. Immunoblotting, qRT-PCR, enzymatic assays, nucleotide sugar and sugar phosphate analyses along with MALDI-TOF mass spectrometry of glycans were used to determine the molecular consequences of the mutations. Results Marked atopy and autoimmunity were associated with increased TH2 and TH17 cytokine production by CD4+ T cells. Bacterial and viral infection susceptibility were noted along with T cell lymphopenia, particularly of CD8+ T cells, and reduced memory B cells. Apparent brain hypomyelination resulted in markedly delayed evoked potentials and likely contributed to neurological abnormalities. Disease segregated with novel autosomal recessive mutations in a single gene, phosphoglucomutase 3 (PGM3). Although PGM3 protein expression was variably diminished, impaired function was demonstrated by decreased enzyme activity and reduced UDP-GlcNAc, along with decreased O- and N-linked protein glycosylation in patients’ cells. These results define a new Congenital Disorder of Glycosylation. Conclusions Autosomal recessive, hypomorphic PGM3 mutations underlie a disorder of severe atopy, immune deficiency, autoimmunity, intellectual disability and hypomyelination. PMID:24589341

Recycling, clustering, and endocytosis jointly maintain PIN auxin carrier polarity at the plasma membrane

PubMed Central

Kleine-Vehn, Jürgen; Wabnik, Krzysztof; Martinière, Alexandre; Łangowski, Łukasz; Willig, Katrin; Naramoto, Satoshi; Leitner, Johannes; Tanaka, Hirokazu; Jakobs, Stefan; Robert, Stéphanie; Luschnig, Christian; Govaerts, Willy; W Hell, Stefan; Runions, John; Friml, Jiří

2011-01-01

Cell polarity reflected by asymmetric distribution of proteins at the plasma membrane is a fundamental feature of unicellular and multicellular organisms. It remains conceptually unclear how cell polarity is kept in cell wall-encapsulated plant cells. We have used super-resolution and semi-quantitative live-cell imaging in combination with pharmacological, genetic, and computational approaches to reveal insights into the mechanism of cell polarity maintenance in Arabidopsis thaliana. We show that polar-competent PIN transporters for the phytohormone auxin are delivered to the center of polar domains by super-polar recycling. Within the plasma membrane, PINs are recruited into non-mobile membrane clusters and their lateral diffusion is dramatically reduced, which ensures longer polar retention. At the circumventing edges of the polar domain, spatially defined internalization of escaped cargos occurs by clathrin-dependent endocytosis. Computer simulations confirm that the combination of these processes provides a robust mechanism for polarity maintenance in plant cells. Moreover, our study suggests that the regulation of lateral diffusion and spatially defined endocytosis, but not super-polar exocytosis have primary importance for PIN polarity maintenance. PMID:22027551

Microvesicles released from human renal cancer stem cells stimulate angiogenesis and formation of lung premetastatic niche.

PubMed

Grange, Cristina; Tapparo, Marta; Collino, Federica; Vitillo, Loriana; Damasco, Christian; Deregibus, Maria Chiara; Tetta, Ciro; Bussolati, Benedetta; Camussi, Giovanni

2011-08-01

Recent studies suggest that tumor-derived microvesicles (MV) act as a vehicle for exchange of genetic information between tumor and stromal cells, engendering a favorable microenvironment for cancer development. Within the tumor mass, all cell types may contribute to MV shedding, but specific contributions to tumor progression have yet to be established. Here we report that a subset of tumor-initiating cells expressing the mesenchymal stem cell marker CD105 in human renal cell carcinoma releases MVs that trigger angiogenesis and promote the formation of a premetastatic niche. MVs derived only from CD105-positive cancer stem cells conferred an activated angiogenic phenotype to normal human endothelial cells, stimulating their growth and vessel formation after in vivo implantation in immunocompromised severe combined immunodeficient (SCID) mice. Furthermore, treating SCID mice with MVs shed from CD105-positive cells greatly enhanced lung metastases induced by i.v. injection of renal carcinoma cells. Molecular characterization of CD105-positive MVs defines a set of proangiogenic mRNAs and microRNAs implicated in tumor progression and metastases. Our results define a specific source of cancer stem cell-derived MVs that contribute to triggering the angiogenic switch and coordinating metastatic diffusion during tumor progression.

Biochemical and genetic analysis of the role of the viral polymerase in enterovirus recombination.

PubMed

Woodman, Andrew; Arnold, Jamie J; Cameron, Craig E; Evans, David J

2016-08-19

Genetic recombination in single-strand, positive-sense RNA viruses is a poorly understand mechanism responsible for generating extensive genetic change and novel phenotypes. By moving a critical cis-acting replication element (CRE) from the polyprotein coding region to the 3' non-coding region we have further developed a cell-based assay (the 3'CRE-REP assay) to yield recombinants throughout the non-structural coding region of poliovirus from dually transfected cells. We have additionally developed a defined biochemical assay in which the only protein present is the poliovirus RNA dependent RNA polymerase (RdRp), which recapitulates the strand transfer events of the recombination process. We have used both assays to investigate the role of the polymerase fidelity and nucleotide turnover rates in recombination. Our results, of both poliovirus intertypic and intratypic recombination in the CRE-REP assay and using a range of polymerase variants in the biochemical assay, demonstrate that RdRp fidelity is a fundamental determinant of recombination frequency. High fidelity polymerases exhibit reduced recombination and low fidelity polymerases exhibit increased recombination in both assays. These studies provide the basis for the analysis of poliovirus recombination throughout the non-structural region of the virus genome and provide a defined biochemical assay to further dissect this important evolutionary process. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genes involved in transitory recombination between phage M13 and plasmid pHV33.

PubMed Central

Dagert, M; Ehrlich, S D

1984-01-01

Plasmid pHV33 and phage M13 combine in Escherichia coli cells to form a chimera, which decombines to regenerate two parental genomes. Combination can occur via two genetic pathways, one defined by the recBC genes, the other by recA, recF and possibly recL genes. Decombination can also occur via two pathways, one defined again by the recBC genes, the other by a gene not identified, but active only in the absence of the recL gene product. PMID:6323172

Blocks of limited haplotype diversity revealed by high-resolution scanning of human chromosome 21.

PubMed

Patil, N; Berno, A J; Hinds, D A; Barrett, W A; Doshi, J M; Hacker, C R; Kautzer, C R; Lee, D H; Marjoribanks, C; McDonough, D P; Nguyen, B T; Norris, M C; Sheehan, J B; Shen, N; Stern, D; Stokowski, R P; Thomas, D J; Trulson, M O; Vyas, K R; Frazer, K A; Fodor, S P; Cox, D R

2001-11-23

Global patterns of human DNA sequence variation (haplotypes) defined by common single nucleotide polymorphisms (SNPs) have important implications for identifying disease associations and human traits. We have used high-density oligonucleotide arrays, in combination with somatic cell genetics, to identify a large fraction of all common human chromosome 21 SNPs and to directly observe the haplotype structure defined by these SNPs. This structure reveals blocks of limited haplotype diversity in which more than 80% of a global human sample can typically be characterized by only three common haplotypes.

Whole genomic sequence analysis of Bacillus infantis: defining the genetic blueprint of strain NRRL B-14911, an emerging cardiopathogenic microbe

USDA-ARS?s Scientific Manuscript database

Background: We recently reported the identification of Bacillus sp. NRRL B-14911 that induces heart autoimmunity by generating cardiac-reactive T cells through molecular mimicry. This marine bacterium was originally isolated from the Gulf of Mexico, but no associations with human diseases were rep...

Route to genetically targeted optical electrophysiology: development and applications of voltage-sensitive fluorescent proteins

PubMed Central

Akemann, Walther; Song, Chenchen; Mutoh, Hiroki; Knöpfel, Thomas

2015-01-01

Abstract. The invention of membrane voltage protein indicators widens the reach of optical voltage imaging in cell physiology, most notably neurophysiology, by enabling membrane voltage recordings from genetically defined cell types in chronic and life-long preparations. While the last years have seen a dramatic improvement in the technical performance of these indicators, concomitant innovations in optogenetics, optical axon tracing, and high-speed digital microscopy are beginning to fulfill the age-old vision of an all-optical analysis of neuronal circuits, reaching beyond the limits of traditional electrode-based recordings. We will present our personal account of the development of protein voltage indicators from the pioneering days to the present state, including their applications in neurophysiology that has inspired our own work for more than a decade. PMID:26082930

Difficult Diagnosis between B Cell Lymphoma and Classical Hodgkin's Lymphoma.

PubMed

Rentas Torres, Yaixa; Rodríguez-López, Joshua L; Valentin, Maria; Silva, Hector

2015-01-01

Although primary mediastinal large B-cell lymphoma and classic Hodgkin lymphoma of nodular sclerosis type are distinct disease, they share several clinical characteristics and biologic features. However, there are mediastinal lymphomas that not fit in either category. These types of lymphomas are recognized as mediastinal gray zone lymphomas. Gray zone lymphomas are lymphatic tumors that cannot be assigned to a defined lymphoma entity due to morphological, clinical, or genetic reasons. In this report, we present a case of a 22 year-old-Hispanic-female diagnosed with B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Hodgkin lymphoma.

Engineered M13 bacteriophage nanocarriers for intracellular delivery of exogenous proteins to human prostate cancer cells.

PubMed

DePorter, Sandra M; McNaughton, Brian R

2014-09-17

The size, well-defined structure, and relatively high folding energies of most proteins allow them to recognize disease-relevant receptors that present a challenge to small molecule reagents. While multiple challenges must be overcome in order to fully exploit the use of protein reagents in basic research and medicine, perhaps the greatest challenge is their intracellular delivery to a particular diseased cell. Here, we describe the genetic and enzymatic manipulation of prostate cancer cell-penetrating M13 bacteriophage to generate nanocarriers for the intracellular delivery of functional exogenous proteins to a human prostate cancer cell line.

A discrete model of Drosophila eggshell patterning reveals cell-autonomous and juxtacrine effects.

PubMed

Fauré, Adrien; Vreede, Barbara M I; Sucena, Elio; Chaouiya, Claudine

2014-03-01

The Drosophila eggshell constitutes a remarkable system for the study of epithelial patterning, both experimentally and through computational modeling. Dorsal eggshell appendages arise from specific regions in the anterior follicular epithelium that covers the oocyte: two groups of cells expressing broad (roof cells) bordered by rhomboid expressing cells (floor cells). Despite the large number of genes known to participate in defining these domains and the important modeling efforts put into this developmental system, key patterning events still lack a proper mechanistic understanding and/or genetic basis, and the literature appears to conflict on some crucial points. We tackle these issues with an original, discrete framework that considers single-cell models that are integrated to construct epithelial models. We first build a phenomenological model that reproduces wild type follicular epithelial patterns, confirming EGF and BMP signaling input as sufficient to establish the major features of this patterning system within the anterior domain. Importantly, this simple model predicts an instructive juxtacrine signal linking the roof and floor domains. To explore this prediction, we define a mechanistic model that integrates the combined effects of cellular genetic networks, cell communication and network adjustment through developmental events. Moreover, we focus on the anterior competence region, and postulate that early BMP signaling participates with early EGF signaling in its specification. This model accurately simulates wild type pattern formation and is able to reproduce, with unprecedented level of precision and completeness, various published gain-of-function and loss-of-function experiments, including perturbations of the BMP pathway previously seen as conflicting results. The result is a coherent model built upon rules that may be generalized to other epithelia and developmental systems.

Comparative expression analysis reveals lineage relationships between human and murine gliomas and a dominance of glial signatures during tumor propagation in vitro.

PubMed

Henriquez, Nico V; Forshew, Tim; Tatevossian, Ruth; Ellis, Matthew; Richard-Loendt, Angela; Rogers, Hazel; Jacques, Thomas S; Reitboeck, Pablo Garcia; Pearce, Kerra; Sheer, Denise; Grundy, Richard G; Brandner, Sebastian

2013-09-15

Brain tumors are thought to originate from stem/progenitor cell populations that acquire specific genetic mutations. Although current preclinical models have relevance to human pathogenesis, most do not recapitulate the histogenesis of the human disease. Recently, a large series of human gliomas and medulloblastomas were analyzed for genetic signatures of prognosis and therapeutic response. Using a mouse model system that generates three distinct types of intrinsic brain tumors, we correlated RNA and protein expression levels with human brain tumors. A combination of genetic mutations and cellular environment during tumor propagation defined the incidence and phenotype of intrinsic murine tumors. Importantly, in vitro passage of cancer stem cells uniformly promoted a glial expression profile in culture and in brain tumors. Gene expression profiling revealed that experimental gliomas corresponded to distinct subclasses of human glioblastoma, whereas experimental supratentorial primitive neuroectodermal tumors (sPNET) correspond to atypical teratoid/rhabdoid tumor (AT/RT), a rare childhood tumor. ©2013 AACR.

Chloroplasts in anther endothecium of Zea mays (Poaceae).

PubMed

Murphy, Katherine M; Egger, Rachel L; Walbot, Virginia

2015-11-01

Although anthers of Zea mays, Oryza sativa, and Arabidopsis thaliana have been studied intensively using genetic and biochemical analyses in the past 20 years, few updates to anther anatomical and ultrastructural descriptions have been reported. For example, no transmission electron microscopy (TEM) images of the premeiotic maize anther have been published. Here we report the presence of chloroplasts in maize anthers. TEM imaging, electron acceptor photosynthesis assay, in planta photon detection, microarray analysis, and light and fluorescence microscopy were used to investigate the presence of chloroplasts in the maize anther. Most cells of the maize subepidermal endothecium have starch-containing chloroplasts that do not conduct measurable photosynthesis in vitro. The maize anther contains chloroplasts in most subepidermal, endothecial cells. Although maize anthers receive sufficient light to photosynthesize in vivo and the maize anther transcribes >96% of photosynthesis-associated genes found in the maize leaf, no photosynthetic light reaction activity was detected in vitro. The endothecial cell layer should no longer be defined as a complete circle viewed transversely in anther lobes, because chloroplasts are observed only in cells directly beneath the epidermis and not those adjacent to the connective tissue. We propose that chloroplasts be a defining characteristic of differentiated endothecial cells and that nonsubepidermal endothecial cells that lack chloroplasts be defined as a separate cell type, the interendothecium. © 2015 Botanical Society of America.

Laser-Based Propagation of Human iPS and ES Cells Generates Reproducible Cultures with Enhanced Differentiation Potential

PubMed Central

Hohenstein Elliott, Kristi A.; Peterson, Cory; Soundararajan, Anuradha; Kan, Natalia; Nelson, Brandon; Spiering, Sean; Mercola, Mark; Bright, Gary R.

2012-01-01

Proper maintenance of stem cells is essential for successful utilization of ESCs/iPSCs as tools in developmental and drug discovery studies and in regenerative medicine. Standardization is critical for all future applications of stem cells and necessary to fully understand their potential. This study reports a novel approach for the efficient, consistent expansion of human ESCs and iPSCs using laser sectioning, instead of mechanical devices or enzymes, to divide cultures into defined size clumps for propagation. Laser-mediated propagation maintained the pluripotency, quality, and genetic stability of ESCs/iPSCs and led to enhanced differentiation potential. This approach removes the variability associated with ESC/iPSC propagation, significantly reduces the expertise, labor, and time associated with manual passaging techniques and provides the basis for scalable delivery of standardized ESC/iPSC lines. Adoption of standardized protocols would allow researchers to understand the role of genetics, environment, and/or procedural effects on stem cells and would ensure reproducible production of stem cell cultures for use in clinical/therapeutic applications. PMID:22701128

Chemical Proteomics Identifies Druggable Vulnerabilities in a Genetically Defined Cancer.

PubMed

Bar-Peled, Liron; Kemper, Esther K; Suciu, Radu M; Vinogradova, Ekaterina V; Backus, Keriann M; Horning, Benjamin D; Paul, Thomas A; Ichu, Taka-Aki; Svensson, Robert U; Olucha, Jose; Chang, Max W; Kok, Bernard P; Zhu, Zhou; Ihle, Nathan T; Dix, Melissa M; Jiang, Ping; Hayward, Matthew M; Saez, Enrique; Shaw, Reuben J; Cravatt, Benjamin F

2017-10-19

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

[Genetic tests in oncology: from identification of high risk groups to therapy].

PubMed

Sgambato, Alessandro; Ripani, Maurizio; Romano Spica, Vincenzo

2010-01-01

The development of genetic epidemiology in oncology has made possible more frequent analysis of high risk groups, allowing the development of promising susceptibility indicators. The main public health implications include screening and new perspectives for pharmacogenetics and nutrigenomics. The study of genetic variants allows the evaluation of individual risk of developing a disease and has important implications in primary and secondary prevention programs. The analysis of somatic mutations present in tumour cells may contribute to selecting the optimal treatment on an individual basis and to reducing the occurrence of adverse effects of chemotherapy. The authors give a summary of the state of the art of this field and analyze the potential applications of genetic tests in oncology, from identification of high risk groups to defining individualized therapies with particular emphasis on implications for prevention.

Biomanufacturing of Therapeutic Cells: State of the Art, Current Challenges, and Future Perspectives.

PubMed

Roh, Kyung-Ho; Nerem, Robert M; Roy, Krishnendu

2016-06-07

Stem cells and other functionally defined therapeutic cells (e.g., T cells) are promising to bring hope of a permanent cure for diseases and disorders that currently cannot be cured by conventional drugs or biological molecules. This paradigm shift in modern medicine of using cells as novel therapeutics can be realized only if suitable manufacturing technologies for large-scale, cost-effective, reproducible production of high-quality cells can be developed. Here we review the state of the art in therapeutic cell manufacturing, including cell purification and isolation, activation and differentiation, genetic modification, expansion, packaging, and preservation. We identify current challenges and discuss opportunities to overcome them such that cell therapies become highly effective, safe, and predictively reproducible while at the same time becoming affordable and widely available.

Comparison of cancer-associated genetic abnormalities in columnar-lined esophagus tissues with and without goblet cells.

PubMed

Bandla, Santhoshi; Peters, Jeffrey H; Ruff, David; Chen, Shiaw-Min; Li, Chieh-Yuan; Song, Kunchang; Thoms, Kimberly; Litle, Virginia R; Watson, Thomas; Chapurin, Nikita; Lada, Michal; Pennathur, Arjun; Luketich, James D; Peterson, Derick; Dulak, Austin; Lin, Lin; Bass, Adam; Beer, David G; Godfrey, Tony E; Zhou, Zhongren

2014-07-01

To determine and compare the frequency of cancer-associated genetic abnormalities in esophageal metaplasia biopsies with and without goblet cells. Barrett's esophagus is associated with increased risk of esophageal adenocarcinoma (EAC), but the appropriate histologic definition of Barrett's esophagus is debated. Intestinal metaplasia (IM) is defined by the presence of goblet cells whereas nongoblet cell metaplasia (NGM) lacks goblet cells. Both have been implicated in EAC risk but this is controversial. Although IM is known to harbor genetic changes associated with EAC, little is known about NGM. We hypothesized that if NGM and IM infer similar EAC risk, then they would harbor similar genetic aberrations in genes associated with EAC. Ninety frozen NGM, IM, and normal tissues from 45 subjects were studied. DNA copy number abnormalities were identified using microarrays and fluorescence in situ hybridization. Targeted sequencing of all exons from 20 EAC-associated genes was performed on metaplasia biopsies using Ion AmpliSeq DNA sequencing. Frequent copy number abnormalities targeting cancer-associated genes were found in IM whereas no such changes were observed in NGM. In 1 subject, fluorescence in situ hybridization confirmed loss of CDKN2A and amplification of chromosome 8 in IM but not in a nearby NGM biopsy. Targeted sequencing revealed 11 nonsynonymous mutations in 16 IM samples and 2 mutations in 19 NGM samples. This study reports the largest and most comprehensive comparison of DNA aberrations in IM and NGM genomes. Our results show that IM has a much higher frequency of cancer-associated mutations than NGM.

Comprehensive high-resolution genomic profiling and cytogenetics of human chondrocyte cultures by GTG-banding, locus-specific FISH, SKY and SNP array.

PubMed

Wallenborn, M; Petters, O; Rudolf, D; Hantmann, H; Richter, M; Ahnert, P; Rohani, L; Smink, J J; Bulwin, G C; Krupp, W; Schulz, R M; Holland, H

2018-04-23

In the development of cell-based medicinal products, it is crucial to guarantee that the application of such an advanced therapy medicinal product (ATMP) is safe for the patients. The consensus of the European regulatory authorities is: "In conclusion, on the basis of the state of art, conventional karyotyping can be considered a valuable and useful technique to analyse chromosomal stability during preclinical studies". 408 chondrocyte samples (84 monolayers and 324 spheroids) from six patients were analysed using trypsin-Giemsa staining, spectral karyotyping and fluorescence in situ hybridisation, to evaluate the genetic stability of chondrocyte samples from non-clinical studies. Single nucleotide polymorphism (SNP) array analysis was performed on chondrocyte spheroids from five of the six donors. Applying this combination of techniques, the genetic analyses performed revealed no significant genetic instability until passage 3 in monolayer cells and interphase cells from spheroid cultures at different time points. Clonal occurrence of polyploid metaphases and endoreduplications were identified associated with prolonged cultivation time. Also, gonosomal losses were observed in chondrocyte spheroids, with increasing passage and duration of the differentiation phase. Interestingly, in one of the donors, chromosomal aberrations that are also described in extraskeletal myxoid chondrosarcoma were identified. The SNP array analysis exhibited chromosomal aberrations in two donors and copy neutral losses of heterozygosity regions in four donors. This study showed the necessity of combined genetic analyses at defined cultivation time points in quality studies within the field of cell therapy.

Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability.

PubMed

Takaki, Tohru; Montagner, Marco; Serres, Murielle P; Le Berre, Maël; Russell, Matt; Collinson, Lucy; Szuhai, Karoly; Howell, Michael; Boulton, Simon J; Sahai, Erik; Petronczki, Mark

2017-07-24

Altered nuclear shape is a defining feature of cancer cells. The mechanisms underlying nuclear dysmorphia in cancer remain poorly understood. Here we identify PPP1R12A and PPP1CB, two subunits of the myosin phosphatase complex that antagonizes actomyosin contractility, as proteins safeguarding nuclear integrity. Loss of PPP1R12A or PPP1CB causes nuclear fragmentation, nuclear envelope rupture, nuclear compartment breakdown and genome instability. Pharmacological or genetic inhibition of actomyosin contractility restores nuclear architecture and genome integrity in cells lacking PPP1R12A or PPP1CB. We detect actin filaments at nuclear envelope rupture sites and define the Rho-ROCK pathway as the driver of nuclear damage. Lamin A protects nuclei from the impact of actomyosin activity. Blocking contractility increases nuclear circularity in cultured cancer cells and suppresses deformations of xenograft nuclei in vivo. We conclude that actomyosin contractility is a major determinant of nuclear shape and that unrestrained contractility causes nuclear dysmorphia, nuclear envelope rupture and genome instability.

Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability

PubMed Central

Takaki, Tohru; Montagner, Marco; Serres, Murielle P.; Le Berre, Maël; Russell, Matt; Collinson, Lucy; Szuhai, Karoly; Howell, Michael; Boulton, Simon J.; Sahai, Erik; Petronczki, Mark

2017-01-01

Altered nuclear shape is a defining feature of cancer cells. The mechanisms underlying nuclear dysmorphia in cancer remain poorly understood. Here we identify PPP1R12A and PPP1CB, two subunits of the myosin phosphatase complex that antagonizes actomyosin contractility, as proteins safeguarding nuclear integrity. Loss of PPP1R12A or PPP1CB causes nuclear fragmentation, nuclear envelope rupture, nuclear compartment breakdown and genome instability. Pharmacological or genetic inhibition of actomyosin contractility restores nuclear architecture and genome integrity in cells lacking PPP1R12A or PPP1CB. We detect actin filaments at nuclear envelope rupture sites and define the Rho-ROCK pathway as the driver of nuclear damage. Lamin A protects nuclei from the impact of actomyosin activity. Blocking contractility increases nuclear circularity in cultured cancer cells and suppresses deformations of xenograft nuclei in vivo. We conclude that actomyosin contractility is a major determinant of nuclear shape and that unrestrained contractility causes nuclear dysmorphia, nuclear envelope rupture and genome instability. PMID:28737169

Dclk1+ small intestinal epithelial tuft cells display the hallmarks of quiescence and self-renewal

PubMed Central

Chandrakesan, Parthasarathy; May, Randal; Qu, Dongfeng; Weygant, Nathaniel; Taylor, Vivian E.; Li, James D.; Ali, Naushad; Sureban, Sripathi M.; Qante, Michael; Wang, Timothy C.; Bronze, Michael S.; Houchen, Courtney W.

2015-01-01

To date, no discrete genetic signature has been defined for isolated Dclk1+ tuft cells within the small intestine. Furthermore, recent reports on the functional significance of Dclk1+ cells in the small intestine have been inconsistent. These cells have been proposed to be fully differentiated cells, reserve stem cells, and tumor stem cells. In order to elucidate the potential function of Dclk1+ cells, we FACS-sorted Dclk1+ cells from mouse small intestinal epithelium using transgenic mice expressing YFP under the control of the Dclk1 promoter (Dclk1-CreER;Rosa26-YFP). Analysis of sorted YFP+ cells demonstrated marked enrichment (~6000 fold) for Dclk1 mRNA compared with YFP− cells. Dclk1+ population display ~6 fold enrichment for the putative quiescent stem cell marker Bmi1. We observed significantly greater expression of pluripotency genes, pro-survival genes, and quiescence markers in the Dclk1+ population. A significant increase in self-renewal capability (14-fold) was observed in in vitro isolated Dclk1+ cells. The unique genetic report presented in this manuscript suggests that Dclk1+ cells may maintain quiescence, pluripotency, and metabolic activity for survival/longevity. Functionally, these reserve characteristics manifest in vitro, with Dclk1+ cells exhibiting greater ability to self-renew. These findings indicate that quiescent stem-like functionality is a feature of Dclk1-expressing tuft cells. PMID:26362399

Genetic heterogeneity of L-Zagreb mumps virus vaccine strain

PubMed Central

Kosutic-Gulija, Tanja; Forcic, Dubravko; Šantak, Maja; Ramljak, Ana; Mateljak-Lukacevic, Sanja; Mazuran, Renata

2008-01-01

Background The most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous. Results We identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo. Conclusion L-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes. PMID:18616793

Traffic jam functions in a branched pathway from Notch activation to niche cell fate.

PubMed

Wingert, Lindsey; DiNardo, Stephen

2015-07-01

The niche directs key behaviors of its resident stem cells, and is thus crucial for tissue maintenance, repair and longevity. However, little is known about the genetic pathways that guide niche specification and development. The male germline stem cell niche in Drosophila houses two stem cell populations and is specified within the embryonic gonad, thus making it an excellent model for studying niche development. The hub cells that form the niche are specified early by Notch activation. Over the next few hours, these individual cells then cluster together and take up a defined position before expressing markers of hub cell differentiation. This timing suggests that there are other factors for niche development yet to be defined. Here, we have identified a role for the large Maf transcription factor Traffic jam (Tj) in hub cell specification downstream of Notch. Tj downregulation is the first detectable effect of Notch activation in hub cells. Furthermore, Tj depletion is sufficient to generate ectopic hub cells that can recruit stem cells. Surprisingly, ectopic niche cells in tj mutants remain dispersed in the absence of Notch activation. This led us to uncover a branched pathway downstream of Notch in which Bowl functions to direct hub cell assembly in parallel to Tj downregulation. © 2015. Published by The Company of Biologists Ltd.

A new approach to the evolution of the blastic crisis from chronic myelocytic leukemia: dynamic interplay of cellular alterations and a changing microenvironment.

PubMed Central

Grossman, Z

1986-01-01

The mechanisms responsible for the massive hyperplasia and for the blastic crisis in chronic myelocytic leukemia are poorly understood. The most generally accepted hypothesis proposes that this progression is due to the development of genetic instability in the leukemic cells. In particular, the two phases of the disease are believed to reflect different, discrete genetic events. Such events remain undefined as yet, and the causal significance of observed genetic aberrations is not clear. An alternative hypothesis is presented here. It is assumed that the feedback interactions adjust the relative probabilities of maturation and replication of the 'committed' as well as the pluripotent cells, and further that mitotic cells at all stages possess considerable phenotypic adaptability; in particular their self-renewal capacity can vary in response to changes in the cellular composition of the tissue even within a conventionally defined compartment. On this basis, it is shown that chronic leukemia can arise and evolve into the blastic crisis from a progressive decline in a single clonal characteristic--inducibility to maturation. It is shown, with the help of mathematical considerations, how an initial hereditable event in an early hemopoietic cell can cause a disturbance of the tissue which feeds back onto the individual members of the clone, resulting in a cascade of dynamic changes which can lead to blast cell dominance. PMID:3458586

The importance of detailed epigenomic profiling of different cell types within organs.

PubMed

Stueve, Theresa Ryan; Marconett, Crystal N; Zhou, Beiyun; Borok, Zea; Laird-Offringa, Ite A

2016-06-01

The human body consists of hundreds of kinds of cells specified from a single genome overlaid with cell type-specific epigenetic information. Comprehensively profiling the body's distinct epigenetic landscapes will allow researchers to verify cell types used in regenerative medicine and to determine the epigenetic effects of disease, environmental exposures and genetic variation. Key marks/factors that should be investigated include regions of nucleosome-free DNA accessible to regulatory factors, histone marks defining active enhancers and promoters, DNA methylation levels, regulatory RNAs, and factors controlling the three-dimensional conformation of the genome. Here we use the lung to illustrate the importance of investigating an organ's purified cell epigenomes, and outline the challenges and promise of realizing a comprehensive catalog of primary cell epigenomes.

Deep brain optical measurements of cell type-specific neural activity in behaving mice.

PubMed

Cui, Guohong; Jun, Sang Beom; Jin, Xin; Luo, Guoxiang; Pham, Michael D; Lovinger, David M; Vogel, Steven S; Costa, Rui M

2014-01-01

Recent advances in genetically encoded fluorescent sensors enable the monitoring of cellular events from genetically defined groups of neurons in vivo. In this protocol, we describe how to use a time-correlated single-photon counting (TCSPC)-based fiber optics system to measure the intensity, emission spectra and lifetime of fluorescent biosensors expressed in deep brain structures in freely moving mice. When combined with Cre-dependent selective expression of genetically encoded Ca(2+) indicators (GECIs), this system can be used to measure the average neural activity from a specific population of cells in mice performing complex behavioral tasks. As an example, we used viral expression of GCaMPs in striatal projection neurons (SPNs) and recorded the fluorescence changes associated with calcium spikes from mice performing a lever-pressing operant task. The whole procedure, consisting of virus injection, behavior training and optical recording, takes 3-4 weeks to complete. With minor adaptations, this protocol can also be applied to recording cellular events from other cell types in deep brain regions, such as dopaminergic neurons in the ventral tegmental area. The simultaneously recorded fluorescence signals and behavior events can be used to explore the relationship between the neural activity of specific brain circuits and behavior.

Defining functional DNA elements in the human genome

PubMed Central

Kellis, Manolis; Wold, Barbara; Snyder, Michael P.; Bernstein, Bradley E.; Kundaje, Anshul; Marinov, Georgi K.; Ward, Lucas D.; Birney, Ewan; Crawford, Gregory E.; Dekker, Job; Dunham, Ian; Elnitski, Laura L.; Farnham, Peggy J.; Feingold, Elise A.; Gerstein, Mark; Giddings, Morgan C.; Gilbert, David M.; Gingeras, Thomas R.; Green, Eric D.; Guigo, Roderic; Hubbard, Tim; Kent, Jim; Lieb, Jason D.; Myers, Richard M.; Pazin, Michael J.; Ren, Bing; Stamatoyannopoulos, John A.; Weng, Zhiping; White, Kevin P.; Hardison, Ross C.

2014-01-01

With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease. PMID:24753594

A combined gene and cell therapy approach for restoration of conduction.

PubMed

Hofshi, Anat; Itzhaki, Ilanit; Gepstein, Amira; Arbel, Gil; Gross, Gil J; Gepstein, Lior

2011-01-01

Abnormal conduction underlies both bradyarrhythmias and re-entrant tachyarrhythmias. However, no practical way exists for restoring or improving conduction in areas of conduction slowing or block. This study sought to test the feasibility of a novel strategy for conduction repair using genetically engineered cells designed to form biological "conducting cables." An in vitro model of conduction block was established using spatially separated, spontaneously contracting, nonsynchronized human embryonic stem cell-derived cardiomyocytes clusters. Immunostaining, dye transfer, intracellular recordings, and multielectrode array (MEA) studies were performed to evaluate the ability of genetically engineered HEK293 cells, expressing the SCN5A-encoded Na(+) channel, to couple with cultured cardiomyocytes and to synchronize their electrical activity. Connexin-43 immunostaining and calcein dye-transfer experiments confirmed the formation of functional gap junctions between the engineered cells and neighboring cardiomyocytes. MEA and intracellular recordings were performed to assess the ability of the engineered cells to restore conduction in the co-cultures. Synchronization was defined by establishment of fixed local activation time differences between the cardiomyocytes clusters and convergence of their activation cycle lengths. Nontransfected control cells were able to induce synchronization between cardiomyocytes clusters separated by distances up to 300 μm (n = 21). In contrast, the Na(+) channel-expressing cells synchronized contractions between clusters separated by up to 1,050 μm, the longest distance studied (n = 23). Finally, engineered cells expressing the voltage-sensitive K(v)1.3 potassium channel prevented synchronization at any distance. Genetically engineered cells, transfected to express Na(+) channels, can form biological conducting cables bridging and coupling spatially separated cardiomyocytes. This novel cell therapy approach might be useful for the development of therapeutic strategies for both bradyarrhythmias and tachyarrhythmias. Copyright © 2011 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Combined enzyme/prodrug treatment by genetically engineered AT-MSC exerts synergy and inhibits growth of MDA-MB-231 induced lung metastases.

PubMed

Matuskova, Miroslava; Kozovska, Zuzana; Toro, Lenka; Durinikova, Erika; Tyciakova, Silvia; Cierna, Zuzana; Bohovic, Roman; Kucerova, Lucia

2015-04-09

Metastatic spread of tumor cells remains a serious problem in cancer treatment. Gene-directed enzyme/prodrug therapy mediated by tumor-homing genetically engineered mesenchymal stromal cells (MSC) represents a promising therapeutic modality for elimination of disseminated cells. Efficacy of gene-directed enzyme/prodrug therapy can be improved by combination of individual systems. We aimed to define the combination effect of two systems of gene therapy mediated by MSC, and evaluate the ability of systemically administered genetically engineered mesenchymal stromal cells to inhibit the growth of experimental metastases derived from human breast adenocarcinoma cells MDA-MB-231/EGFP. Human adipose tissue-derived mesenchymal stromal cells (AT-MSC) were retrovirally transduced with fusion yeast cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT) or with Herpes simplex virus thymidine kinase (HSVtk). Engineered MSC were cocultured with tumor cells in the presence of prodrugs 5-fluorocytosin (5-FC) and ganciclovir (GCV). Combination effect of these enzyme/prodrug approaches was calculated. SCID/bg mice bearing experimental lung metastases were treated with CD::UPRT-MSC, HSVtk-MSC or both in combination in the presence of respective prodrug(s). Treatment efficiency was evaluated by EGFP-positive cell detection by flow cytometry combined with real-time PCR quantification of human cells in mouse organs. Results were confirmed by histological and immunohistochemical examination. We demonstrated various extent of synergy depending on tested cell line and experimental setup. The strongest synergism was observed on breast cancer-derived cell line MDA-MB-231/EGFP. Systemic administration of CD::UPRT-MSC and HSVtk-MSC in combination with 5-FC and GCV inhibited growth of MDA-MB-231 induced lung metastases. Combined gene-directed enzyme/prodrug therapy mediated by MSC exerted synergic cytotoxic effect and resulted in high therapeutic efficacy in vivo.

Self-targeting of TNF-releasing cancer cells in preclinical models of primary and metastatic tumors.

PubMed

Dondossola, Eleonora; Dobroff, Andrey S; Marchiò, Serena; Cardó-Vila, Marina; Hosoya, Hitomi; Libutti, Steven K; Corti, Angelo; Sidman, Richard L; Arap, Wadih; Pasqualini, Renata

2016-02-23

Circulating cancer cells can putatively colonize distant organs to form metastases or to reinfiltrate primary tumors themselves through a process termed "tumor self-seeding." Here we exploit this biological attribute to deliver tumor necrosis factor alpha (TNF), a potent antitumor cytokine, directly to primary and metastatic tumors in a mechanism that we have defined as "tumor self-targeting." For this purpose, we genetically engineered mouse mammary adenocarcinoma (TSA), melanoma (B16-F10), and Lewis lung carcinoma cells to produce and release murine TNF. In a series of intervention trials, systemic administration of TNF-expressing tumor cells was associated with reduced growth of both primary tumors and metastatic colonies in immunocompetent mice. We show that these malignant cells home to tumors, locally release TNF, damage neovascular endothelium, and induce massive cancer cell apoptosis. We also demonstrate that such tumor-cell-mediated delivery avoids or minimizes common side effects often associated with TNF-based therapy, such as acute inflammation and weight loss. Our study provides proof of concept that genetically modified circulating tumor cells may serve as targeted vectors to deliver anticancer agents. In a clinical context, this unique paradigm represents a personalized approach to be translated into applications potentially using patient-derived circulating tumor cells as self-targeted vectors for drug delivery.

Development of a targeted transgenesis strategy in highly differentiated cells: a powerful tool for functional genomic analysis.

PubMed

Puttini, Stefania; Ouvrard-Pascaud, Antoine; Palais, Gael; Beggah, Ahmed T; Gascard, Philippe; Cohen-Tannoudji, Michel; Babinet, Charles; Blot-Chabaud, Marcel; Jaisser, Frederic

2005-03-16

Functional genomic analysis is a challenging step in the so-called post-genomic field. Identification of potential targets using large-scale gene expression analysis requires functional validation to identify those that are physiologically relevant. Genetically modified cell models are often used for this purpose allowing up- or down-expression of selected targets in a well-defined and if possible highly differentiated cell type. However, the generation of such models remains time-consuming and expensive. In order to alleviate this step, we developed a strategy aimed at the rapid and efficient generation of genetically modified cell lines with conditional, inducible expression of various target genes. Efficient knock-in of various constructs, called targeted transgenesis, in a locus selected for its permissibility to the tet inducible system, was obtained through the stimulation of site-specific homologous recombination by the meganuclease I-SceI. Our results demonstrate that targeted transgenesis in a reference inducible locus greatly facilitated the functional analysis of the selected recombinant cells. The efficient screening strategy we have designed makes possible automation of the transfection and selection steps. Furthermore, this strategy could be applied to a variety of highly differentiated cells.

Tracking Genomic Cancer Evolution for Precision Medicine: The Lung TRACERx Study

PubMed Central

Jamal-Hanjani, Mariam; Hackshaw, Alan; Ngai, Yenting; Shaw, Jacqueline; Dive, Caroline; Quezada, Sergio; Middleton, Gary; de Bruin, Elza; Le Quesne, John; Shafi, Seema; Falzon, Mary; Horswell, Stuart; Blackhall, Fiona; Khan, Iftekhar; Janes, Sam; Nicolson, Marianne; Lawrence, David; Forster, Martin; Fennell, Dean; Lee, Siow-Ming; Lester, Jason; Kerr, Keith; Muller, Salli; Iles, Natasha; Smith, Sean; Murugaesu, Nirupa; Mitter, Richard; Salm, Max; Stuart, Aengus; Matthews, Nik; Adams, Haydn; Ahmad, Tanya; Attanoos, Richard; Bennett, Jonathan; Birkbak, Nicolai Juul; Booton, Richard; Brady, Ged; Buchan, Keith; Capitano, Arrigo; Chetty, Mahendran; Cobbold, Mark; Crosbie, Philip; Davies, Helen; Denison, Alan; Djearman, Madhav; Goldman, Jacki; Haswell, Tom; Joseph, Leena; Kornaszewska, Malgorzata; Krebs, Matthew; Langman, Gerald; MacKenzie, Mairead; Millar, Joy; Morgan, Bruno; Naidu, Babu; Nonaka, Daisuke; Peggs, Karl; Pritchard, Catrin; Remmen, Hardy; Rowan, Andrew; Shah, Rajesh; Smith, Elaine; Summers, Yvonne; Taylor, Magali; Veeriah, Selvaraju; Waller, David; Wilcox, Ben; Wilcox, Maggie; Woolhouse, Ian; McGranahan, Nicholas; Swanton, Charles

2014-01-01

The importance of intratumour genetic and functional heterogeneity is increasingly recognised as a driver of cancer progression and survival outcome. Understanding how tumour clonal heterogeneity impacts upon therapeutic outcome, however, is still an area of unmet clinical and scientific need. TRACERx (TRAcking non-small cell lung Cancer Evolution through therapy [Rx]), a prospective study of patients with primary non-small cell lung cancer (NSCLC), aims to define the evolutionary trajectories of lung cancer in both space and time through multiregion and longitudinal tumour sampling and genetic analysis. By following cancers from diagnosis to relapse, tracking the evolutionary trajectories of tumours in relation to therapeutic interventions, and determining the impact of clonal heterogeneity on clinical outcomes, TRACERx may help to identify novel therapeutic targets for NSCLC and may also serve as a model applicable to other cancer types. PMID:25003521

Radiation-induced genomic instability and its implications for radiation carcinogenesis

NASA Technical Reports Server (NTRS)

Huang, Lei; Snyder, Andrew R.; Morgan, William F.

2003-01-01

Radiation-induced genomic instability is characterized by an increased rate of genetic alterations including cytogenetic rearrangements, mutations, gene amplifications, transformation and cell death in the progeny of irradiated cells multiple generations after the initial insult. Chromosomal rearrangements are the best-characterized end point of radiation-induced genomic instability, and many of the rearrangements described are similar to those found in human cancers. Chromosome breakage syndromes are defined by chromosome instability, and individuals with these diseases are cancer prone. Consequently, chromosomal instability as a phenotype may underlie some fraction of those changes leading to cancer. Here we attempt to relate current knowledge regarding radiation-induced chromosome instability with the emerging molecular information on the chromosome breakage syndromes. The goal is to understand how genetic and epigenetic factors might influence the onset of chromosome instability and the role of chromosomal instability in carcinogenesis.

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

PubMed

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.

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

PubMed Central

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

2011-01-01

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

Defining the Cellular Environment in the Organ of Corti following Extensive Hair Cell Loss: A Basis for Future Sensory Cell Replacement in the Cochlea

PubMed Central

Taylor, Ruth R.; Jagger, Daniel J.; Forge, Andrew

2012-01-01

Background Following the loss of hair cells from the mammalian cochlea, the sensory epithelium repairs to close the lesions but no new hair cells arise and hearing impairment ensues. For any cell replacement strategy to be successful, the cellular environment of the injured tissue has to be able to nurture new hair cells. This study defines characteristics of the auditory sensory epithelium after hair cell loss. Methodology/Principal Findings Studies were conducted in C57BL/6 and CBA/Ca mice. Treatment with an aminoglycoside-diuretic combination produced loss of all outer hair cells within 48 hours in both strains. The subsequent progressive tissue re-organisation was examined using immunohistochemistry and electron microscopy. There was no evidence of significant de-differentiation of the specialised columnar supporting cells. Kir4.1 was down regulated but KCC4, GLAST, microtubule bundles, connexin expression patterns and pathways of intercellular communication were retained. The columnar supporting cells became covered with non-specialised cells migrating from the outermost region of the organ of Corti. Eventually non-specialised, flat cells replaced the columnar epithelium. Flat epithelium developed in distributed patches interrupting regions of columnar epithelium formed of differentiated supporting cells. Formation of the flat epithelium was initiated within a few weeks post-treatment in C57BL/6 mice but not for several months in CBA/Ca's, suggesting genetic background influences the rate of re-organisation. Conclusions/Significance The lack of dedifferentiation amongst supporting cells and their replacement by cells from the outer side of the organ of Corti are factors that may need to be considered in any attempt to promote endogenous hair cell regeneration. The variability of the cellular environment along an individual cochlea arising from patch-like generation of flat epithelium, and the possible variability between individuals resulting from genetic influences on the rate at which remodelling occurs may pose challenges to devising the appropriate regenerative therapy for a deaf patient. PMID:22299045

Congenital Mirror Movements in Gorlin Syndrome: A Case Report With DTI and Functional MRI Features.

PubMed

Sag, Erdal; Gocmen, Rahsan; Yildiz, F Gokcem; Ozturk, Zeynelabidin; Temucin, Cagri; Teksam, Ozlem; Utine, Eda

2016-03-01

Congenital mirror movements are rare conditions that define the inability to perform unimanual movements. Gorlin syndrome, also known as nevoid basal cell carcinoma syndrome, is a genetic disorder with multiple nevi predisposing to basal cell carcinoma, odontogenic keratocysts, and skeletal malformations. Herein we report on an adolescent patient with Gorlin syndrome and coexisting congenital mirror movements. To our knowledge, this is the first patient in the literature who has both of these very rare conditions. Copyright © 2016 by the American Academy of Pediatrics.

Quantitative Imaging in Cancer Evolution and Ecology

PubMed Central

Grove, Olya; Gillies, Robert J.

2013-01-01

Cancer therapy, even when highly targeted, typically fails because of the remarkable capacity of malignant cells to evolve effective adaptations. These evolutionary dynamics are both a cause and a consequence of cancer system heterogeneity at many scales, ranging from genetic properties of individual cells to large-scale imaging features. Tumors of the same organ and cell type can have remarkably diverse appearances in different patients. Furthermore, even within a single tumor, marked variations in imaging features, such as necrosis or contrast enhancement, are common. Similar spatial variations recently have been reported in genetic profiles. Radiologic heterogeneity within tumors is usually governed by variations in blood flow, whereas genetic heterogeneity is typically ascribed to random mutations. However, evolution within tumors, as in all living systems, is subject to Darwinian principles; thus, it is governed by predictable and reproducible interactions between environmental selection forces and cell phenotype (not genotype). This link between regional variations in environmental properties and cellular adaptive strategies may permit clinical imaging to be used to assess and monitor intratumoral evolution in individual patients. This approach is enabled by new methods that extract, report, and analyze quantitative, reproducible, and mineable clinical imaging data. However, most current quantitative metrics lack spatialness, expressing quantitative radiologic features as a single value for a region of interest encompassing the whole tumor. In contrast, spatially explicit image analysis recognizes that tumors are heterogeneous but not well mixed and defines regionally distinct habitats, some of which appear to harbor tumor populations that are more aggressive and less treatable than others. By identifying regional variations in key environmental selection forces and evidence of cellular adaptation, clinical imaging can enable us to define intratumoral Darwinian dynamics before and during therapy. Advances in image analysis will place clinical imaging in an increasingly central role in the development of evolution-based patient-specific cancer therapy. © RSNA, 2013 PMID:24062559

A fungicide-responsive kinase as a tool for synthetic cell fate regulation.

PubMed

Furukawa, Kentaro; Hohmann, Stefan

2015-08-18

Engineered biological systems that precisely execute defined tasks have major potential for medicine and biotechnology. For instance, gene- or cell-based therapies targeting pathogenic cells may replace time- and resource-intensive drug development. Engineering signal transduction systems is a promising, yet presently underexplored approach. Here, we exploit a fungicide-responsive heterologous histidine kinase for pathway engineering and synthetic cell fate regulation in the budding yeast Saccharomyces cerevisiae. Rewiring the osmoregulatory Hog1 MAPK signalling system generates yeast cells programmed to execute three different tasks. First, a synthetic negative feedback loop implemented by employing the fungicide-responsive kinase and a fungicide-resistant derivative reshapes the Hog1 activation profile, demonstrating how signalling dynamics can be engineered. Second, combinatorial integration of different genetic parts including the histidine kinases, a pathway activator and chemically regulated promoters enables control of yeast growth and/or gene expression in a two-input Boolean logic manner. Finally, we implemented a genetic 'suicide attack' system, in which engineered cells eliminate target cells and themselves in a specific and controllable manner. Taken together, fungicide-responsive kinases can be applied in different constellations to engineer signalling behaviour. Sensitizing engineered cells to existing chemicals may be generally useful for future medical and biotechnological applications. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

A Parvovirus B19 synthetic genome: sequence features and functional competence.

PubMed

Manaresi, Elisabetta; Conti, Ilaria; Bua, Gloria; Bonvicini, Francesca; Gallinella, Giorgio

2017-08-01

Central to genetic studies for Parvovirus B19 (B19V) is the availability of genomic clones that may possess functional competence and ability to generate infectious virus. In our study, we established a new model genetic system for Parvovirus B19. A synthetic approach was followed, by design of a reference genome sequence, by generation of a corresponding artificial construct and its molecular cloning in a complete and functional form, and by setup of an efficient strategy to generate infectious virus, via transfection in UT7/EpoS1 cells and amplification in erythroid progenitor cells. The synthetic genome was able to generate virus with biological properties paralleling those of native virus, its infectious activity being dependent on the preservation of self-complementarity and sequence heterogeneity within the terminal regions. A virus of defined genome sequence, obtained from controlled cell culture conditions, can constitute a reference tool for investigation of the structural and functional characteristics of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

Establishing neural crest identity: a gene regulatory recipe

PubMed Central

Simões-Costa, Marcos; Bronner, Marianne E.

2015-01-01

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

A platform for rapid prototyping of synthetic gene networks in mammalian cells

PubMed Central

Duportet, Xavier; Wroblewska, Liliana; Guye, Patrick; Li, Yinqing; Eyquem, Justin; Rieders, Julianne; Rimchala, Tharathorn; Batt, Gregory; Weiss, Ron

2014-01-01

Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines. PMID:25378321

Norrin, frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization.

PubMed

Ye, Xin; Wang, Yanshu; Cahill, Hugh; Yu, Minzhong; Badea, Tudor C; Smallwood, Philip M; Peachey, Neal S; Nathans, Jeremy

2009-10-16

Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled-4 (Fz4) receptor, Lrp5 coreceptor, or Norrin ligand cause retinal hypovascularization, but the mechanisms by which Norrin/Fz4/Lrp signaling controls vascular development have not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, whereas excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is upregulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.

En1 directs superior olivary complex neuron positioning, survival, and expression of FoxP1.

PubMed

Altieri, Stefanie C; Jalabi, Walid; Zhao, Tianna; Romito-DiGiacomo, Rita R; Maricich, Stephen M

2015-12-01

Little is known about the genetic pathways and transcription factors that control development and maturation of central auditory neurons. En1, a gene expressed by a subset of developing and mature superior olivary complex (SOC) cells, encodes a homeodomain transcription factor important for neuronal development in the midbrain, cerebellum, hindbrain and spinal cord. Using genetic fate-mapping techniques, we show that all En1-lineal cells in the SOC are neurons and that these neurons are glycinergic, cholinergic and GABAergic in neurotransmitter phenotype. En1 deletion does not interfere with specification or neural fate of these cells, but does cause aberrant positioning and subsequent death of all En1-lineal SOC neurons by early postnatal ages. En1-null cells also fail to express the transcription factor FoxP1, suggesting that FoxP1 lies downstream of En1. Our data define important roles for En1 in the development and maturation of a diverse group of brainstem auditory neurons. Copyright © 2015 Elsevier Inc. All rights reserved.

A Multistate Toggle Switch Defines Fungal Cell Fates and Is Regulated by Synergistic Genetic Cues

PubMed Central

Anderson, Matthew Z.; Porman, Allison M.; Wang, Na; Mancera, Eugenio; Bennett, Richard J.

2016-01-01

Heritable epigenetic changes underlie the ability of cells to differentiate into distinct cell types. Here, we demonstrate that the fungal pathogen Candida tropicalis exhibits multipotency, undergoing stochastic and reversible switching between three cellular states. The three cell states exhibit unique cellular morphologies, growth rates, and global gene expression profiles. Genetic analysis identified six transcription factors that play key roles in regulating cell differentiation. In particular, we show that forced expression of Wor1 or Efg1 transcription factors can be used to manipulate transitions between all three cell states. A model for tristability is proposed in which Wor1 and Efg1 are self-activating but mutually antagonistic transcription factors, thereby forming a symmetrical self-activating toggle switch. We explicitly test this model and show that ectopic expression of WOR1 can induce white-to-hybrid-to-opaque switching, whereas ectopic expression of EFG1 drives switching in the opposite direction, from opaque-to-hybrid-to-white cell states. We also address the stability of induced cell states and demonstrate that stable differentiation events require ectopic gene expression in combination with chromatin-based cues. These studies therefore experimentally test a model of multistate stability and demonstrate that transcriptional circuits act synergistically with chromatin-based changes to drive cell state transitions. We also establish close mechanistic parallels between phenotypic switching in unicellular fungi and cell fate decisions during stem cell reprogramming. PMID:27711197

Flying podocytes.

PubMed

Simons, Matias; Huber, Tobias B

2009-03-01

Recent insights have defined the central role of podocytes both in rare genetic diseases and as a general determinant of the progression of human glomerular diseases. In a recent issue of Nature, Weavers et al. described a podocyte-like cell type in Drosophila, the nephrocyte, that closely resembles mammalian podocytes, including the nephrin-based slit diaphragm. This novel podocyte system might open new avenues toward the understanding of podocyte biology and pathophysiology.

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

Voigt, Christopher

SEED2014 focused on advances in the science and technology emerging from the field of synthetic biology. We broadly define this as technologies that accelerate the process of genetic engineering. It highlighted new tool development, as well as the application of these tools to diverse problems in biotechnology, including therapeutics, industrial chemicals and fuels, natural products, and agriculture. Systems spanned from in vitro experiments and viruses, through diverse bacteria, to eukaryotes (yeast, mammalian cells, plants).

Impaired intracellular trafficking defines early Parkinson's disease

PubMed Central

Hunn, Benjamin H.M.; Cragg, Stephanie J.; Bolam, J. Paul; Spillantini, Maria-Grazia; Wade-Martins, Richard

2015-01-01

Parkinson's disease (PD) is an insidious and incurable neurodegenerative disease, and represents a significant cost to individuals, carers, and ageing societies. It is defined at post-mortem by the loss of dopamine neurons in the substantia nigra together with the presence of Lewy bodies and Lewy neurites. We examine here the role of α-synuclein and other cellular transport proteins implicated in PD and how their aberrant activity may be compounded by the unique anatomy of the dopaminergic neuron. This review uses multiple lines of evidence from genetic studies, human tissue, induced pluripotent stem cells, and refined animal models to argue that prodromal PD can be defined as a disease of impaired intracellular trafficking. Dysfunction of the dopaminergic synapse heralds trafficking impairment. PMID:25639775

Factors influencing the efficiency of generating genetically engineered pigs by nuclear transfer: multi-factorial analysis of a large data set.

PubMed

Kurome, Mayuko; Geistlinger, Ludwig; Kessler, Barbara; Zakhartchenko, Valeri; Klymiuk, Nikolai; Wuensch, Annegret; Richter, Anne; Baehr, Andrea; Kraehe, Katrin; Burkhardt, Katinka; Flisikowski, Krzysztof; Flisikowska, Tatiana; Merkl, Claudia; Landmann, Martina; Durkovic, Marina; Tschukes, Alexander; Kraner, Simone; Schindelhauer, Dirk; Petri, Tobias; Kind, Alexander; Nagashima, Hiroshi; Schnieke, Angelika; Zimmer, Ralf; Wolf, Eckhard

2013-05-20

Somatic cell nuclear transfer (SCNT) using genetically engineered donor cells is currently the most widely used strategy to generate tailored pig models for biomedical research. Although this approach facilitates a similar spectrum of genetic modifications as in rodent models, the outcome in terms of live cloned piglets is quite variable. In this study, we aimed at a comprehensive analysis of environmental and experimental factors that are substantially influencing the efficiency of generating genetically engineered pigs. Based on a considerably large data set from 274 SCNT experiments (in total 18,649 reconstructed embryos transferred into 193 recipients), performed over a period of three years, we assessed the relative contribution of season, type of genetic modification, donor cell source, number of cloning rounds, and pre-selection of cloned embryos for early development to the cloning efficiency. 109 (56%) recipients became pregnant and 85 (78%) of them gave birth to offspring. Out of 318 cloned piglets, 243 (76%) were alive, but only 97 (40%) were clinically healthy and showed normal development. The proportion of stillborn piglets was 24% (75/318), and another 31% (100/318) of the cloned piglets died soon after birth. The overall cloning efficiency, defined as the number of offspring born per SCNT embryos transferred, including only recipients that delivered, was 3.95%. SCNT experiments performed during winter using fetal fibroblasts or kidney cells after additive gene transfer resulted in the highest number of live and healthy offspring, while two or more rounds of cloning and nuclear transfer experiments performed during summer decreased the number of healthy offspring. Although the effects of individual factors may be different between various laboratories, our results and analysis strategy will help to identify and optimize the factors, which are most critical to cloning success in programs aiming at the generation of genetically engineered pig models.

New Strategies in Engineering T-cell Receptor Gene-Modified T cells to More Effectively Target Malignancies.

PubMed

Schmitt, Thomas M; Stromnes, Ingunn M; Chapuis, Aude G; Greenberg, Philip D

2015-12-01

The immune system, T cells in particular, have the ability to target and destroy malignant cells. However, antitumor immune responses induced from the endogenous T-cell repertoire are often insufficient for the eradication of established tumors, as illustrated by the failure of cancer vaccination strategies or checkpoint blockade for most tumors. Genetic modification of T cells to express a defined T-cell receptor (TCR) can provide the means to rapidly generate large numbers of tumor-reactive T cells capable of targeting tumor cells in vivo. However, cell-intrinsic factors as well as immunosuppressive factors in the tumor microenvironment can limit the function of such gene-modified T cells. New strategies currently being developed are refining and enhancing this approach, resulting in cellular therapies that more effectively target tumors and that are less susceptible to tumor immune evasion. ©2015 American Association for Cancer Research.

Oligonucleotide arrays vs. metaphase-comparative genomic hybridisation and BAC arrays for single-cell analysis: first applications to preimplantation genetic diagnosis for Robertsonian translocation carriers.

PubMed

Ramos, Laia; del Rey, Javier; Daina, Gemma; García-Aragonés, Manel; Armengol, Lluís; Fernandez-Encinas, Alba; Parriego, Mònica; Boada, Montserrat; Martinez-Passarell, Olga; Martorell, Maria Rosa; Casagran, Oriol; Benet, Jordi; Navarro, Joaquima

2014-01-01

Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈ 20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers.

Oligonucleotide Arrays vs. Metaphase-Comparative Genomic Hybridisation and BAC Arrays for Single-Cell Analysis: First Applications to Preimplantation Genetic Diagnosis for Robertsonian Translocation Carriers

PubMed Central

Ramos, Laia; del Rey, Javier; Daina, Gemma; García-Aragonés, Manel; Armengol, Lluís; Fernandez-Encinas, Alba; Parriego, Mònica; Boada, Montserrat; Martinez-Passarell, Olga; Martorell, Maria Rosa; Casagran, Oriol; Benet, Jordi; Navarro, Joaquima

2014-01-01

Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers. PMID:25415307

Stem cells and bone diseases: new tools, new perspective

PubMed Central

Riminucci, Mara; Remoli, Cristina; Robey, Pamela G.; Bianco, Paolo

2017-01-01

Postnatal skeletal stem cells are a unique class of progenitors with biological properties that extend well beyond the limits of stemness as commonly defined. Skeletal stem cells sustain skeletal tissue homeostasis, organize and maintain the complex architectural structure of the bone marrow microenvironment and provide a niche for hematopoietic progenitor cells. The identification of stem cells in the human post-natal skeleton has profoundly changed our approach to the physiology and pathology of this system. Skeletal diseases have been long interpreted essentially in terms of defective function of differentiated cells and/or abnormal turnover of the matrix they produce. The notion of a skeletal stem cell has brought forth multiple, novel concepts in skeletal biology that provide potential alternative concepts. At the same time, the recognition of the complex functions played by skeletal progenitors, such as the structural and functional organization of the bone marrow, has provided an innovative, unifying perspective for understanding bone and bone marrow changes simultaneously occurring in many disorders. Finally, the possibility to isolate and highly enrich for skeletal progenitors, enables us to reproduce perfectly normal or pathological organ miniatures. These, in turn, provide suitable models to investigate and manipulate the pathogenetic mechanisms of many genetic and non-genetic skeletal diseases. PMID:25240458

Multiplexing clonality: combining RGB marking and genetic barcoding

PubMed Central

Cornils, Kerstin; Thielecke, Lars; Hüser, Svenja; Forgber, Michael; Thomaschewski, Michael; Kleist, Nadja; Hussein, Kais; Riecken, Kristoffer; Volz, Tassilo; Gerdes, Sebastian; Glauche, Ingmar; Dahl, Andreas; Dandri, Maura; Roeder, Ingo; Fehse, Boris

2014-01-01

RGB marking and DNA barcoding are two cutting-edge technologies in the field of clonal cell marking. To combine the virtues of both approaches, we equipped LeGO vectors encoding red, green or blue fluorescent proteins with complex DNA barcodes carrying color-specific signatures. For these vectors, we generated highly complex plasmid libraries that were used for the production of barcoded lentiviral vector particles. In proof-of-principle experiments, we used barcoded vectors for RGB marking of cell lines and primary murine hepatocytes. We applied single-cell polymerase chain reaction to decipher barcode signatures of individual RGB-marked cells expressing defined color hues. This enabled us to prove clonal identity of cells with one and the same RGB color. Also, we made use of barcoded vectors to investigate clonal development of leukemia induced by ectopic oncogene expression in murine hematopoietic cells. In conclusion, by combining RGB marking and DNA barcoding, we have established a novel technique for the unambiguous genetic marking of individual cells in the context of normal regeneration as well as malignant outgrowth. Moreover, the introduction of color-specific signatures in barcodes will facilitate studies on the impact of different variables (e.g. vector type, transgenes, culture conditions) in the context of competitive repopulation studies. PMID:24476916

Pioneer factors, genetic competence, and inductive signaling: programming liver and pancreas progenitors from the endoderm.

PubMed

Zaret, K S; Watts, J; Xu, J; Wandzioch, E; Smale, S T; Sekiya, T

2008-01-01

The endoderm is a multipotent progenitor cell population in the embryo that gives rise to the liver, pancreas, and other cell types and provides paradigms for understanding cell-type specification. Studies of isolated embryo tissue cells and genetic approaches in vivo have defined fibroblast growth factor/mitogen-activated protein kinase (FGF/MAPK) and bone morphogenetic protein (BMP) signaling pathways that induce liver and pancreatic fates in the endoderm. In undifferentiated endoderm cells, the FoxA and GATA transcription factors are among the first to engage silent genes, helping to endow competence for cell-type specification. FoxA proteins can bind their target sites in highly compacted chromatin and open up the local region for other factors to bind; hence, they have been termed "pioneer factors." We recently found that FoxA proteins remain bound to chromatin in mitosis, as an epigenetic mark. In embryonic stem cells, which lack FoxA, FoxA target sites can be occupied by FoxD3, which in turn helps to maintain a local demethylation of chromatin. By these means, a cascade of Fox factors helps to endow progenitor cells with the competence to activate genes in response to tissue-inductive signals. Understanding such epigenetic mechanisms for transcriptional competence coupled with knowledge of the relevant signals for cell-type specification should greatly facilitate efforts to predictably differentiate stem cells to liver and pancreatic fates.

Cross-talk between Rho and Rac GTPases drives deterministic exploration of cellular shape space and morphological heterogeneity.

PubMed

Sailem, Heba; Bousgouni, Vicky; Cooper, Sam; Bakal, Chris

2014-01-22

One goal of cell biology is to understand how cells adopt different shapes in response to varying environmental and cellular conditions. Achieving a comprehensive understanding of the relationship between cell shape and environment requires a systems-level understanding of the signalling networks that respond to external cues and regulate the cytoskeleton. Classical biochemical and genetic approaches have identified thousands of individual components that contribute to cell shape, but it remains difficult to predict how cell shape is generated by the activity of these components using bottom-up approaches because of the complex nature of their interactions in space and time. Here, we describe the regulation of cellular shape by signalling systems using a top-down approach. We first exploit the shape diversity generated by systematic RNAi screening and comprehensively define the shape space a migratory cell explores. We suggest a simple Boolean model involving the activation of Rac and Rho GTPases in two compartments to explain the basis for all cell shapes in the dataset. Critically, we also generate a probabilistic graphical model to show how cells explore this space in a deterministic, rather than a stochastic, fashion. We validate the predictions made by our model using live-cell imaging. Our work explains how cross-talk between Rho and Rac can generate different cell shapes, and thus morphological heterogeneity, in genetically identical populations.

Genetic framework for GATA factor function in vascular biology.

PubMed

Linnemann, Amelia K; O'Geen, Henriette; Keles, Sunduz; Farnham, Peggy J; Bresnick, Emery H

2011-08-16

Vascular endothelial dysfunction underlies the genesis and progression of numerous diseases. Although the GATA transcription factor GATA-2 is expressed in endothelial cells and is implicated in coronary heart disease, it has been studied predominantly as a master regulator of hematopoiesis. Because many questions regarding GATA-2 function in the vascular biology realm remain unanswered, we used ChIP sequencing and loss-of-function strategies to define the GATA-2-instigated genetic network in human endothelial cells. In contrast to erythroid cells, GATA-2 occupied a unique target gene ensemble consisting of genes encoding key determinants of endothelial cell identity and inflammation. GATA-2-occupied sites characteristically contained motifs that bind activator protein-1 (AP-1), a pivotal regulator of inflammatory genes. GATA-2 frequently occupied the same chromatin sites as c-JUN and c-FOS, heterodimeric components of AP-1. Although all three components were required for maximal AP-1 target gene expression, GATA-2 was not required for AP-1 chromatin occupancy. GATA-2 conferred maximal phosphorylation of chromatin-bound c-JUN at Ser-73, which stimulates AP-1-dependent transactivation, in a chromosomal context-dependent manner. This work establishes a link between a GATA factor and inflammatory genes, mechanistic insights underlying GATA-2-AP-1 cooperativity and a rigorous genetic framework for understanding GATA-2 function in normal and pathophysiological vascular states.

A drive through cellular therapy for CLL in 2015: allogeneic cell transplantation and CARs.

PubMed

Mato, Anthony; Porter, David L

2015-07-23

Over the past decade the development of safer reduced-intensity conditioning regimens, expanded donor pools, advances in supportive care, and prevention/management of graft-versus-host disease have expanded stem cell transplantation (SCT) availability for chronic lymphocytic leukemia (CLL) patients. However, there are now increasingly active treatment options available for CLL patients with favorable toxicity profiles and convenient administration schedules. This raises the critical issue of whether or not attainment of cure remains a necessary goal. It is now less clear that treatment with curative intention and with significant toxicity is required for long-term survival in CLL. In addition, the demonstrated safety and activity of genetically modified chimeric antigen receptor (CAR) T cells present the opportunity of harnessing the power of the immune system to kill CLL cells without the need for SCT. We attempt to define the role of SCT in the era of targeted therapies and discuss questions that remain to be answered. Furthermore, we highlight the potential for exciting new cellular therapy using genetically modified anti-CD19 CAR T cells and discuss its potential to alter treatment paradigms for CLL. © 2015 by The American Society of Hematology.

Gene therapy with autologous, interleukin 2-secreting tumor cells in patients with malignant melanoma.

PubMed

Palmer, K; Moore, J; Everard, M; Harris, J D; Rodgers, S; Rees, R C; Murray, A K; Mascari, R; Kirkwood, J; Riches, P G; Fisher, C; Thomas, J M; Harries, M; Johnston, S R; Collins, M K; Gore, M E

1999-05-20

We vaccinated metastatic melanoma patients with irradiated, autologous melanoma cells genetically engineered to secrete interleukin 2 (IL-2) to investigate whether an anti-tumor immune response would be induced. Melanoma cell cultures were established from surgical specimens and were engineered to secrete IL-2 by infection with recombinant retrovirus. Twelve patients were vaccinated subcutaneously one, two, or three times with approximately 10(7) irradiated, autologous, IL-2-secreting tumor cells. Treatment was well tolerated, with local reactions at 11 of 24 injection sites and minor systemic symptoms of fever and headache after 6 injections. One patient developed anti-tumor DTH after the first vaccination and showed an increased response after the second vaccination. Anti-autologous tumor CTLs could be detected prevaccination in the peripheral blood of seven patients and their activity increased after vaccination in four patients. No UICC-defined clinical responses were seen, but three patients had stable disease for 7-15 months, one of whom has not yet progressed (15+ months). Thus, patient vaccination with autologous, genetically engineered tumor cells is feasible and safe. Anti-tumor DTH and CTLs can be induced in some patients with such a vaccine.

Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers

PubMed Central

Blakely, Collin M.; Watkins, Thomas B.K.; Wu, Wei; Gini, Beatrice; Chabon, Jacob J.; McCoach, Caroline E.; McGranahan, Nicholas; Wilson, Gareth A.; Birkbak, Nicolai J.; Olivas, Victor R.; Rotow, Julia; Maynard, Ashley; Wang, Victoria; Gubens, Matthew A.; Banks, Kimberly C.; Lanman, Richard B.; Caulin, Aleah F.; John, John St.; Cordero, Anibal R.; Giannikopoulos, Petros; Simmons, Andrew D.; Mack, Philip C.; Gandara, David R.; Husain, Hatim; Doebele, Robert C.; Riess, Jonathan W.; Diehn, Maximilian; Swanton, Charles; Bivona, Trever G.

2017-01-01

A widespread approach to modern cancer therapy is to identify a single oncogenic driver gene and target its mutant protein product (e.g. EGFR inhibitor treatment in EGFR-mutant lung cancers). However, genetically-driven resistance to targeted therapy limits patient survival. Through genomic analysis of 1122 EGFR-mutant lung cancer cell-free DNA samples and whole exome analysis of seven longitudinally collected tumor samples from an EGFR-mutant lung cancer patient, we identify critical co-occurring oncogenic events present in most advanced-stage EGFR-mutant lung cancers. We define new pathways limiting EGFR inhibitor response, including WNT/β-catenin and cell cycle gene (e.g. CDK4, CDK6) alterations. Tumor genomic complexity increases with EGFR inhibitor treatment and co-occurring alterations in CTNNB1, and PIK3CA exhibit non-redundant functions that cooperatively promote tumor metastasis or limit EGFR inhibitor response. This study challenges the prevailing single-gene driver oncogene view and links clinical outcomes to co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancer patients. PMID:29106415

Advances in genetic circuit design: novel biochemistries, deep part mining, and precision gene expression.

PubMed

Nielsen, Alec A K; Segall-Shapiro, Thomas H; Voigt, Christopher A

2013-12-01

Cells use regulatory networks to perform computational operations to respond to their environment. Reliably manipulating such networks would be valuable for many applications in biotechnology; for example, in having genes turn on only under a defined set of conditions or implementing dynamic or temporal control of expression. Still, building such synthetic regulatory circuits remains one of the most difficult challenges in genetic engineering and as a result they have not found widespread application. Here, we review recent advances that address the key challenges in the forward design of genetic circuits. First, we look at new design concepts, including the construction of layered digital and analog circuits, and new approaches to control circuit response functions. Second, we review recent work to apply part mining and computational design to expand the number of regulators that can be used together within one cell. Finally, we describe new approaches to obtain precise gene expression and to reduce context dependence that will accelerate circuit design by more reliably balancing regulators while reducing toxicity. Copyright © 2013. Published by Elsevier Ltd.

The 4-Celled Tetrabaena socialis Nuclear Genome Reveals the Essential Components for Genetic Control of Cell Number at the Origin of Multicellularity in the Volvocine Lineage.

PubMed

Featherston, Jonathan; Arakaki, Yoko; Hanschen, Erik R; Ferris, Patrick J; Michod, Richard E; Olson, Bradley J S C; Nozaki, Hisayoshi; Durand, Pierre M

2018-04-01

Multicellularity is the premier example of a major evolutionary transition in individuality and was a foundational event in the evolution of macroscopic biodiversity. The volvocine chlorophyte lineage is well suited for studying this process. Extant members span unicellular, simple colonial, and obligate multicellular taxa with germ-soma differentiation. Here, we report the nuclear genome sequence of one of the most morphologically simple organisms in this lineage-the 4-celled colonial Tetrabaena socialis and compare this to the three other complete volvocine nuclear genomes. Using conservative estimates of gene family expansions a minimal set of expanded gene families was identified that associate with the origin of multicellularity. These families are rich in genes related to developmental processes. A subset of these families is lineage specific, which suggests that at a genomic level the evolution of multicellularity also includes lineage-specific molecular developments. Multiple points of evidence associate modifications to the ubiquitin proteasomal pathway (UPP) with the beginning of coloniality. Genes undergoing positive or accelerating selection in the multicellular volvocines were found to be enriched in components of the UPP and gene families gained at the origin of multicellularity include components of the UPP. A defining feature of colonial/multicellular life cycles is the genetic control of cell number. The genomic data presented here, which includes diversification of cell cycle genes and modifications to the UPP, align the genetic components with the evolution of this trait.

Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis

PubMed Central

Conklin, Patricia L.; Norris, Susan R.; Wheeler, Glen L.; Williams, Elizabeth H.; Smirnoff, Nicholas; Last, Robert L.

1999-01-01

Vitamin C (l-ascorbic acid; AsA) acts as a potent antioxidant and cellular reductant in plants and animals. AsA has long been known to have many critical physiological roles in plants, yet its biosynthesis is only currently being defined. A pathway for AsA biosynthesis that features GDP-mannose and l-galactose has recently been proposed for plants. We have isolated a collection of AsA-deficient mutants of Arabidopsis thaliana that are valuable tools for testing of an AsA biosynthetic pathway. The best-characterized of these mutants (vtc1) contains ≈25% of wild-type AsA and is defective in AsA biosynthesis. By using a combination of biochemical, molecular, and genetic techniques, we have demonstrated that the VTC1 locus encodes a GDP-mannose pyrophosphorylase (mannose-1-P guanyltransferase). This enzyme provides GDP-mannose, which is used for cell wall carbohydrate biosynthesis and protein glycosylation as well as for AsA biosynthesis. In addition to genetically defining the first locus involved in AsA biosynthesis, this work highlights the power of using traditional mutagenesis techniques coupled with the Arabidopsis Genome Initiative to rapidly clone physiologically important genes. PMID:10097187

Stem cells’ guided gene therapy of cancer: New frontier in personalized and targeted therapy

PubMed Central

Mavroudi, Maria; Zarogoulidis, Paul; Porpodis, Konstantinos; Kioumis, Ioannis; Lampaki, Sofia; Yarmus, Lonny; Malecki, Raf; Zarogoulidis, Konstantinos; Malecki, Marek

2014-01-01

Introduction Diagnosis and therapy of cancer remain to be the greatest challenges for all physicians working in clinical oncology and molecular medicine. The statistics speak for themselves with the grim reports of 1,638,910 men and women diagnosed with cancer and nearly 577,190 patients passed away due to cancer in the USA in 2012. For practicing clinicians, who treat patients suffering from advanced cancers with contemporary systemic therapies, the main challenge is to attain therapeutic efficacy, while minimizing side effects. Unfortunately, all contemporary systemic therapies cause side effects. In treated patients, these side effects may range from nausea to damaged tissues. In cancer survivors, the iatrogenic outcomes of systemic therapies may include genomic mutations and their consequences. Therefore, there is an urgent need for personalized and targeted therapies. Recently, we reviewed the current status of suicide gene therapy for cancer. Herein, we discuss the novel strategy: genetically engineered stem cells’ guided gene therapy. Review of therapeutic strategies in preclinical and clinical trials Stem cells have the unique potential for self renewal and differentiation. This potential is the primary reason for introducing them into medicine to regenerate injured or degenerated organs, as well as to rejuvenate aging tissues. Recent advances in genetic engineering and stem cell research have created the foundations for genetic engineering of stem cells as the vectors for delivery of therapeutic transgenes. Specifically in oncology, the stem cells are genetically engineered to deliver the cell suicide inducing genes selectively to the cancer cells only. Expression of the transgenes kills the cancer cells, while leaving healthy cells unaffected. Herein, we present various strategies to bioengineer suicide inducing genes and stem cell vectors. Moreover, we review results of the main preclinical studies and clinical trials. However, the main risk for therapeutic use of stem cells is their cancerous transformation. Therefore, we discuss various strategies to safeguard stem cell guided gene therapy against iatrogenic cancerogenesis. Perspectives Defining cancer biomarkers to facilitate early diagnosis, elucidating cancer genomics and proteomics with modern tools of next generation sequencing, and analyzing patients’ gene expression profiles provide essential data to elucidate molecular dynamics of cancer and to consider them for crafting pharmacogenomics-based personalized therapies. Streamlining of these data into genetic engineering of stem cells facilitates their use as the vectors delivering therapeutic genes into specific cancer cells. In this realm, stem cells guided gene therapy becomes a promising new frontier in personalized and targeted therapy of cancer. PMID:24860662

Differential chemosensitivity to antifolate drugs between RAS and BRAF melanoma cells

PubMed Central

2014-01-01

Background The importance of the genetic background of cancer cells for the individual susceptibility to cancer treatments is increasingly apparent. In melanoma, the existence of a BRAF mutation is a main predictor for successful BRAF-targeted therapy. However, despite initial successes with these therapies, patients relapse within a year and have to move on to other therapies. Moreover, patients harbouring a wild type BRAF gene (including 25% with NRAS mutations) still require alternative treatment such as chemotherapy. Multiple genetic parameters have been associated with response to chemotherapy, but despite their high frequency in melanoma nothing is known about the impact of BRAF or NRAS mutations on the response to chemotherapeutic agents. Methods Using cell proliferation and DNA methylation assays, FACS analysis and quantitative-RT-PCR we have characterised the response of a panel of NRAS and BRAF mutant melanoma cell lines to various chemotherapy drugs, amongst them dacarbazine (DTIC) and temozolomide (TMZ) and DNA synthesis inhibitors. Results Although both, DTIC and TMZ act as alkylating agents through the same intermediate, NRAS and BRAF mutant cells responded differentially only to DTIC. Further analysis revealed that the growth-inhibitory effects mediated by DTIC were rather due to interference with nucleotide salvaging, and that NRAS mutant melanoma cells exhibit higher activity of the nucleotide synthesis enzymes IMPDH and TK1. Importantly, the enhanced ability of RAS mutant cells to use nucleotide salvaging resulted in resistance to DHFR inhibitors. Conclusion In summary, our data suggest that the genetic background in melanoma cells influences the response to inhibitors blocking de novo DNA synthesis, and that defining the RAS mutation status could be used to stratify patients for the use of antifolate drugs. PMID:24941944

Optical dissection of odor information processing in vivo using GCaMPs expressed in specified cell types of the olfactory bulb

PubMed Central

Wachowiak, Matt; Economo, Michael N.; Díaz-Quesada, Marta; Brunert, Daniela; Wesson, Daniel W.; White, John. A.; Rothermel, Markus

2013-01-01

Understanding central processing requires precise monitoring of neural activity across populations of identified neurons in the intact brain. Here we used recently-optimized variants of the genetically-encoded calcium sensor GCaMP (GCaMP3 and GCaMPG5G) to image activity among genetically- and anatomically-defined neuronal populations in the olfactory bulb (OB), including two types of GABA-ergic interneurons (periglomerular (PG) and short axon (SA) cells) and OB output neurons (mitral/tufted (MT) cells) projecting to piriform cortex. We first established that changes in neuronal spiking can be accurately related to GCaMP fluorescence changes via a simple quantitative relationship over a large dynamic range. We next used in vivo two-photon imaging from individual neurons and epifluorescence signals reflecting population-level activity to investigate the spatiotemporal representation of odorants across these neuron types in anesthetized and awake mice. Under anesthesia, individual PG and SA cells showed temporally simple responses and little spontaneous activity, while MT cells were spontaneously active and showed diverse temporal responses. At the population level, response patterns of PG, SA and MT cells were surprisingly similar to those imaged from sensory inputs, with shared odorant-specific topography across the dorsal OB and inhalation-coupled temporal dynamics. During wakefulness, PG and SA cell responses increased in magnitude but remained temporally simple while those of MT cells changed to complex spatiotemporal patterns reflecting restricted excitation and widespread inhibition. These results point to multiple circuit elements with distinct roles in transforming odor representations in the OB and provide a framework for further dissecting early olfactory processing using optical and genetic tools. PMID:23516293

A novel approach for the generation of genetically modified mammary epithelial cell cultures yields new insights into TGFβ signaling in the mammary gland

PubMed Central

2010-01-01

Introduction Molecular dissection of the signaling pathways that underlie complex biological responses in the mammary epithelium is limited by the difficulty of propagating large numbers of mouse mammary epithelial cells, and by the inability of ribonucleic acid interference-based knockdown approaches to fully ablate gene function. Here we describe a method for the generation of conditionally immortalized mammary epithelial cells with defined genetic defects, and we show how such cells can be used to investigate complex signal transduction processes using the transforming growth factor beta (TGFβ)/Smad pathway as an example. Methods We intercrossed the previously described H-2Kb-tsA58 transgenic mouse (Immortomouse), which expresses a temperature-sensitive mutant of the simian virus-40 large T-antigen (tsTAg), with mice of differing Smad genotypes. Conditionally immortalized mammary epithelial cell cultures were derived from the virgin mammary glands of offspring of these crosses and were used to assess the Smad dependency of different biological responses to TGFβ. Results IMECs could be propagated indefinitely at permissive temperatures and had a stable epithelial phenotype, resembling primary mammary epithelial cells with respect to several criteria, including responsiveness to TGFβ. Using this panel of cells, we demonstrated that Smad3, but not Smad2, is necessary for TGFβ-induced apoptotic, growth inhibitory and epithelial-to-mesenchymal transition responses, whereas either Smad2 or Smad3 can support TGFβ-induced invasion as long as a threshold level of total Smad is exceeded. Conclusions The present work demonstrates the practicality and utility of generating conditionally immortalized mammary epithelial cell lines from genetically modified Immortomice for detailed investigation of complex signaling pathways in the mammary epithelium. PMID:20942910

Integrative analysis of 111 reference human epigenomes

PubMed Central

Kundaje, Anshul; Meuleman, Wouter; Ernst, Jason; Bilenky, Misha; Yen, Angela; Kheradpour, Pouya; Zhang, Zhizhuo; Heravi-Moussavi, Alireza; Liu, Yaping; Amin, Viren; Ziller, Michael J; Whitaker, John W; Schultz, Matthew D; Sandstrom, Richard S; Eaton, Matthew L; Wu, Yi-Chieh; Wang, Jianrong; Ward, Lucas D; Sarkar, Abhishek; Quon, Gerald; Pfenning, Andreas; Wang, Xinchen; Claussnitzer, Melina; Coarfa, Cristian; Harris, R Alan; Shoresh, Noam; Epstein, Charles B; Gjoneska, Elizabeta; Leung, Danny; Xie, Wei; Hawkins, R David; Lister, Ryan; Hong, Chibo; Gascard, Philippe; Mungall, Andrew J; Moore, Richard; Chuah, Eric; Tam, Angela; Canfield, Theresa K; Hansen, R Scott; Kaul, Rajinder; Sabo, Peter J; Bansal, Mukul S; Carles, Annaick; Dixon, Jesse R; Farh, Kai-How; Feizi, Soheil; Karlic, Rosa; Kim, Ah-Ram; Kulkarni, Ashwinikumar; Li, Daofeng; Lowdon, Rebecca; Mercer, Tim R; Neph, Shane J; Onuchic, Vitor; Polak, Paz; Rajagopal, Nisha; Ray, Pradipta; Sallari, Richard C; Siebenthall, Kyle T; Sinnott-Armstrong, Nicholas; Stevens, Michael; Thurman, Robert E; Wu, Jie; Zhang, Bo; Zhou, Xin; Beaudet, Arthur E; Boyer, Laurie A; De Jager, Philip; Farnham, Peggy J; Fisher, Susan J; Haussler, David; Jones, Steven; Li, Wei; Marra, Marco; McManus, Michael T; Sunyaev, Shamil; Thomson, James A; Tlsty, Thea D; Tsai, Li-Huei; Wang, Wei; Waterland, Robert A; Zhang, Michael; Chadwick, Lisa H; Bernstein, Bradley E; Costello, Joseph F; Ecker, Joseph R; Hirst, Martin; Meissner, Alexander; Milosavljevic, Aleksandar; Ren, Bing; Stamatoyannopoulos, John A; Wang, Ting; Kellis, Manolis

2015-01-01

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but a similar reference has lacked for epigenomic studies. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection to-date of human epigenomes for primary cells and tissues. Here, we describe the integrative analysis of 111 reference human epigenomes generated as part of the program, profiled for histone modification patterns, DNA accessibility, DNA methylation, and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically-relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation, and human disease. PMID:25693563

Acute lymphoblastic leukaemia

PubMed Central

Inaba, Hiroto; Greaves, Mel; Mullighan, Charles G.

2013-01-01

Summary Acute lymphoblastic leukaemia (ALL) is seen in both children and adults, but its incidence peaks between ages 2 and 5 years. The causation of ALL is considered to be multi-factorial, including exogenous or endogenous exposures, genetic susceptibility, and chance. The survival rate of paediatric ALL has improved to approximately 90% in recent trials with risk stratification by biologic features of leukaemic cells and response to therapy, therapy modification based on patient pharmacodynamics and pharmacogenomics, and improved supportive care. However, innovative approaches are needed to further improve survival while reducing adverse effects. While most children can be cured, the prognosis of infants and adults with ALL remains poor. Recent genome-wide profiling of germline and leukaemic cell DNA has identified novel submicroscopic structural genetic alterations and sequence mutations that contribute to leukaemogenesis, define new ALL subtypes, influence responsiveness to treatment, and may provide novel prognostic markers and therapeutic targets for personalized medicine. PMID:23523389

Interactions of Mitochondria/Metabolism and Calcium Regulation in Alzheimer’s Disease - A Calcinist Point of View

PubMed Central

Gibson, Gary E.; Thakkar, Ankita

2017-01-01

Decades of research suggest that alterations in calcium are central to the pathophysiology of Alzheimer’s Disease (AD). Highly reproducible changes in calcium dynamics occur in cells from patients with both genetic and non-genetic forms of AD relative to controls. The most robust change is an exaggerated release of calcium from internal stores. Detailed analysis of these changes in animal and cell models of the AD-causing presenilin mutations reveal robust changes in ryanodine receptors, inositol tris-phosphate receptors, calcium leak channels and store activated calcium entry. Similar anomalies in calcium result when AD-like changes in mitochondrial enzymes or oxidative stress are induced experimentally. The calcium abnormalities can be directly linked to the altered tau phosphorylation, amyloid precursor protein processing and synaptic dysfunction that are defining features of AD. A better understanding of these changes is required before using calcium abnormalities as therapeutic targets. PMID:28181072

Integrative analysis of 111 reference human epigenomes.

PubMed

Kundaje, Anshul; Meuleman, Wouter; Ernst, Jason; Bilenky, Misha; Yen, Angela; Heravi-Moussavi, Alireza; Kheradpour, Pouya; Zhang, Zhizhuo; Wang, Jianrong; Ziller, Michael J; Amin, Viren; Whitaker, John W; Schultz, Matthew D; Ward, Lucas D; Sarkar, Abhishek; Quon, Gerald; Sandstrom, Richard S; Eaton, Matthew L; Wu, Yi-Chieh; Pfenning, Andreas R; Wang, Xinchen; Claussnitzer, Melina; Liu, Yaping; Coarfa, Cristian; Harris, R Alan; Shoresh, Noam; Epstein, Charles B; Gjoneska, Elizabeta; Leung, Danny; Xie, Wei; Hawkins, R David; Lister, Ryan; Hong, Chibo; Gascard, Philippe; Mungall, Andrew J; Moore, Richard; Chuah, Eric; Tam, Angela; Canfield, Theresa K; Hansen, R Scott; Kaul, Rajinder; Sabo, Peter J; Bansal, Mukul S; Carles, Annaick; Dixon, Jesse R; Farh, Kai-How; Feizi, Soheil; Karlic, Rosa; Kim, Ah-Ram; Kulkarni, Ashwinikumar; Li, Daofeng; Lowdon, Rebecca; Elliott, GiNell; Mercer, Tim R; Neph, Shane J; Onuchic, Vitor; Polak, Paz; Rajagopal, Nisha; Ray, Pradipta; Sallari, Richard C; Siebenthall, Kyle T; Sinnott-Armstrong, Nicholas A; Stevens, Michael; Thurman, Robert E; Wu, Jie; Zhang, Bo; Zhou, Xin; Beaudet, Arthur E; Boyer, Laurie A; De Jager, Philip L; Farnham, Peggy J; Fisher, Susan J; Haussler, David; Jones, Steven J M; Li, Wei; Marra, Marco A; McManus, Michael T; Sunyaev, Shamil; Thomson, James A; Tlsty, Thea D; Tsai, Li-Huei; Wang, Wei; Waterland, Robert A; Zhang, Michael Q; Chadwick, Lisa H; Bernstein, Bradley E; Costello, Joseph F; Ecker, Joseph R; Hirst, Martin; Meissner, Alexander; Milosavljevic, Aleksandar; Ren, Bing; Stamatoyannopoulos, John A; Wang, Ting; Kellis, Manolis

2015-02-19

The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

A multiplexable TALE-based binary expression system for in vivo cellular interaction studies.

PubMed

Toegel, Markus; Azzam, Ghows; Lee, Eunice Y; Knapp, David J H F; Tan, Ying; Fa, Ming; Fulga, Tudor A

2017-11-21

Binary expression systems have revolutionised genetic research by enabling delivery of loss-of-function and gain-of-function transgenes with precise spatial-temporal resolution in vivo. However, at present, each existing platform relies on a defined exogenous transcription activator capable of binding a unique recognition sequence. Consequently, none of these technologies alone can be used to simultaneously target different tissues or cell types in the same organism. Here, we report a modular system based on programmable transcription activator-like effector (TALE) proteins, which enables parallel expression of multiple transgenes in spatially distinct tissues in vivo. Using endogenous enhancers coupled to TALE drivers, we demonstrate multiplexed orthogonal activation of several transgenes carrying cognate variable activating sequences (VAS) in distinct neighbouring cell types of the Drosophila central nervous system. Since the number of combinatorial TALE-VAS pairs is virtually unlimited, this platform provides an experimental framework for highly complex genetic manipulation studies in vivo.

Interactions of Mitochondria/Metabolism and Calcium Regulation in Alzheimer's Disease: A Calcinist Point of View.

PubMed

Gibson, Gary E; Thakkar, Ankita

2017-06-01

Decades of research suggest that alterations in calcium are central to the pathophysiology of Alzheimer's Disease (AD). Highly reproducible changes in calcium dynamics occur in cells from patients with both genetic and non-genetic forms of AD relative to controls. The most robust change is an exaggerated release of calcium from internal stores. Detailed analysis of these changes in animal and cell models of the AD-causing presenilin mutations reveal robust changes in ryanodine receptors, inositol tris-phosphate receptors, calcium leak channels and store activated calcium entry. Similar anomalies in calcium result when AD-like changes in mitochondrial enzymes or oxidative stress are induced experimentally. The calcium abnormalities can be directly linked to the altered tau phosphorylation, amyloid precursor protein processing and synaptic dysfunction that are defining features of AD. A better understanding of these changes is required before using calcium abnormalities as therapeutic targets.

Constitutively active transforming growth factor β receptor 1 in the mouse ovary promotes tumorigenesis

PubMed Central

Gao, Yang; Vincent, David F.; Davis, Anna Jane; Sansom, Owen J.; Bartholin, Laurent; Li, Qinglei

2016-01-01

Despite the well-established tumor suppressive role of TGFβ proteins, depletion of key TGFβ signaling components in the mouse ovary does not induce a growth advantage. To define the role of TGFβ signaling in ovarian tumorigenesis, we created a mouse model expressing a constitutively active TGFβ receptor 1 (TGFBR1) in ovarian somatic cells using conditional gain-of-function approach. Remarkably, these mice developed ovarian sex cord-stromal tumors with complete penetrance, leading to reproductive failure and mortality. The tumors expressed multiple granulosa cell markers and caused elevated serum inhibin and estradiol levels, reminiscent of granulosa cell tumors. Consistent with the tumorigenic effect, overactivation of TGFBR1 altered tumor microenvironment by promoting angiogenesis and enhanced ovarian cell proliferation, accompanied by impaired cell differentiation and dysregulated expression of critical genes in ovarian function. By further exploiting complementary genetic models, we substantiated our finding that constitutively active TGFBR1 is a potent oncogenic switch in mouse granulosa cells. In summary, overactivation of TGFBR1 drives gonadal tumor development. The TGFBR1 constitutively active mouse model phenocopies a number of morphological, hormonal, and molecular features of human granulosa cell tumors and are potentially valuable for preclinical testing of targeted therapies to treat granulosa cell tumors, a class of poorly defined ovarian malignancies. PMID:27344183

The role of the SCRAMBLED receptor-like kinase in patterning the Arabidopsis root epidermis.

PubMed

Kwak, Su-Hwan; Schiefelbein, John

2007-02-01

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

Forward genetics in Candida albicans that reveals the Arp2/3 complex is required for hyphal formation, but not endocytosis

PubMed Central

Epp, Elias; Walther, Andrea; Guylaine, Lépine; Leon, Zully; Mullick, Alaka; Raymond, Martine; Wendland, Jürgen; Whiteway, Malcolm

2014-01-01

Summary Candida albicans is a diploid fungal pathogen lacking a defined complete sexual cycle, and thus has been refractory to standard forward genetic analysis. Instead, transcription profiling and reverse genetic strategies based on Saccharomyces cerevisiae have typically been used to link genes to functions. To overcome restrictions inherent in such indirect approaches, we have investigated a forward genetic mutagenesis strategy based on the UAU1 technology. We screened 4700 random insertion mutants for defects in hyphal development and linked two new genes (ARP2 and VPS52) to hyphal growth. Deleting ARP2 abolished hyphal formation, generated round and swollen yeast phase cells, disrupted cortical actin patches and blocked virulence in mice. The mutants also showed a global lack of induction of hyphae-specific genes upon the yeast-to-hyphae switch. Surprisingly, both arp2Δ/Δ and arp2Δ/Δarp3Δ/Δ mutants were still able to endocytose FM4-64 and Lucifer Yellow, although as shown by time-lapse movies internalization of FM4-64 was somewhat delayed in mutant cells. Thus the non-essential role of the Arp2/3 complex discovered by forward genetic screening in C. albicans showed that uptake of membrane components from the plasma membrane to vacuolar structures is not dependent on this actin nucleating machinery. PMID:20141603

Quasi-programmed aging of budding yeast: a trade-off between programmed processes of cell proliferation, differentiation, stress response, survival and death defines yeast lifespan

PubMed Central

Arlia-Ciommo, Anthony; Piano, Amanda; Leonov, Anna; Svistkova, Veronika; Titorenko, Vladimir I

2014-01-01

Recent findings suggest that evolutionarily distant organisms share the key features of the aging process and exhibit similar mechanisms of its modulation by certain genetic, dietary and pharmacological interventions. The scope of this review is to analyze mechanisms that in the yeast Saccharomyces cerevisiae underlie: (1) the replicative and chronological modes of aging; (2) the convergence of these 2 modes of aging into a single aging process; (3) a programmed differentiation of aging cell communities in liquid media and on solid surfaces; and (4) longevity-defining responses of cells to some chemical compounds released to an ecosystem by other organisms populating it. Based on such analysis, we conclude that all these mechanisms are programs for upholding the long-term survival of the entire yeast population inhabiting an ecological niche; however, none of these mechanisms is a ʺprogram of agingʺ - i.e., a program for progressing through consecutive steps of the aging process. PMID:25485579

Programming Enzyme-Initiated Autonomous DNAzyme Nanodevices in Living Cells.

PubMed

Chen, Feng; Bai, Min; Cao, Ke; Zhao, Yue; Cao, Xiaowen; Wei, Jing; Wu, Na; Li, Jiang; Wang, Lihua; Fan, Chunhai; Zhao, Yongxi

2017-12-26

Molecular nanodevices are computational assemblers that switch defined states upon external stimulation. However, interfacing artificial nanodevices with natural molecular machineries in living cells remains a great challenge. Here, we delineate a generic method for programming assembly of enzyme-initiated DNAzyme nanodevices (DzNanos). Two programs including split assembly of two partzymes and toehold exchange displacement assembly of one intact DNAzyme initiated by telomerase are computed. The intact one obtains higher assembly yield and catalytic performance ascribed to proper conformation folding and active misplaced assembly. By employing MnO 2 nanosheets as both DNA carriers and source of Mn 2+ as DNAzyme cofactor, we find that this DzNano is well assembled via a series of conformational states in living cells and operates autonomously with sustained cleavage activity. Other enzymes can also induce corresponding DzNano assembly with defined programming modules. These DzNanos not only can monitor enzyme catalysis in situ but also will enable the implementation of cellular stages, behaviors, and pathways for basic science, diagnostic, and therapeutic applications as genetic circuits.

Ethnicity-Dependent and -Independent Heterogeneity in Healthy Normal Breast Hierarchy Impacts Tumor Characterization

PubMed Central

Nakshatri, Harikrishna; Anjanappa, Manjushree; Bhat-Nakshatri, Poornima

2015-01-01

Recent reports of widespread genetic variation affecting regulation of gene expression raise the possibility of significant inter-individual differences in stem-progenitor-mature cell hierarchy in adult organs. This has not been explored because of paucity of methods to quantitatively assess subpopulation of normal epithelial cells on individual basis. We report the remarkable inter-individual differences in differentiation capabilities as documented by phenotypic heterogeneity in stem-progenitor-mature cell hierarchy of the normal breast. Ethnicity and genetic predisposition are partly responsible for this heterogeneity, evidenced by the finding that CD44+/CD24- and PROCR+/EpCAM- multi-potent stem cells were elevated significantly in African American women compared with Caucasians. ALDEFLUOR+ luminal stem/progenitor cells were lower in BRCA1-mutation carriers compared with cells from healthy donors (p = 0.0014). Moreover, tumor and adjoining-normal breast cells of the same patients showed distinct CD49f+/EpCAM+ progenitor, CD271+/EpCAM- basal, and ALDEFLUOR+ cell profiles. These inter-individual differences in the rate of differentiation in the normal breast may contribute to a substantial proportion of transcriptome, epigenome, and signaling pathway alterations and consequently has the potential to spuriously magnify the extent of documented tumor-specific gene expression. Therefore, comparative analysis of phenotypically defined subpopulations of normal and tumor cells on an individual basis may be required to identify cancer-specific aberrations. PMID:26311223

Ethnicity-Dependent and -Independent Heterogeneity in Healthy Normal Breast Hierarchy Impacts Tumor Characterization.

PubMed

Nakshatri, Harikrishna; Anjanappa, Manjushree; Bhat-Nakshatri, Poornima

2015-08-27

Recent reports of widespread genetic variation affecting regulation of gene expression raise the possibility of significant inter-individual differences in stem-progenitor-mature cell hierarchy in adult organs. This has not been explored because of paucity of methods to quantitatively assess subpopulation of normal epithelial cells on individual basis. We report the remarkable inter-individual differences in differentiation capabilities as documented by phenotypic heterogeneity in stem-progenitor-mature cell hierarchy of the normal breast. Ethnicity and genetic predisposition are partly responsible for this heterogeneity, evidenced by the finding that CD44+/CD24- and PROCR+/EpCAM- multi-potent stem cells were elevated significantly in African American women compared with Caucasians. ALDEFLUOR+ luminal stem/progenitor cells were lower in BRCA1-mutation carriers compared with cells from healthy donors (p = 0.0014). Moreover, tumor and adjoining-normal breast cells of the same patients showed distinct CD49f+/EpCAM+ progenitor, CD271+/EpCAM- basal, and ALDEFLUOR+ cell profiles. These inter-individual differences in the rate of differentiation in the normal breast may contribute to a substantial proportion of transcriptome, epigenome, and signaling pathway alterations and consequently has the potential to spuriously magnify the extent of documented tumor-specific gene expression. Therefore, comparative analysis of phenotypically defined subpopulations of normal and tumor cells on an individual basis may be required to identify cancer-specific aberrations.

Diversity amongst trigeminal neurons revealed by high throughput single cell sequencing

PubMed Central

Nguyen, Minh Q.; Wu, Youmei; Bonilla, Lauren S.; von Buchholtz, Lars J.

2017-01-01

The trigeminal ganglion contains somatosensory neurons that detect a range of thermal, mechanical and chemical cues and innervate unique sensory compartments in the head and neck including the eyes, nose, mouth, meninges and vibrissae. We used single-cell sequencing and in situ hybridization to examine the cellular diversity of the trigeminal ganglion in mice, defining thirteen clusters of neurons. We show that clusters are well conserved in dorsal root ganglia suggesting they represent distinct functional classes of somatosensory neurons and not specialization associated with their sensory targets. Notably, functionally important genes (e.g. the mechanosensory channel Piezo2 and the capsaicin gated ion channel Trpv1) segregate into multiple clusters and often are expressed in subsets of cells within a cluster. Therefore, the 13 genetically-defined classes are likely to be physiologically heterogeneous rather than highly parallel (i.e., redundant) lines of sensory input. Our analysis harnesses the power of single-cell sequencing to provide a unique platform for in silico expression profiling that complements other approaches linking gene-expression with function and exposes unexpected diversity in the somatosensory system. PMID:28957441

GoIFISH: a system for the quantification of single cell heterogeneity from IFISH images.

PubMed

Trinh, Anne; Rye, Inga H; Almendro, Vanessa; Helland, Aslaug; Russnes, Hege G; Markowetz, Florian

2014-08-26

Molecular analysis has revealed extensive intra-tumor heterogeneity in human cancer samples, but cannot identify cell-to-cell variations within the tissue microenvironment. In contrast, in situ analysis can identify genetic aberrations in phenotypically defined cell subpopulations while preserving tissue-context specificity. GoIFISHGoIFISH is a widely applicable, user-friendly system tailored for the objective and semi-automated visualization, detection and quantification of genomic alterations and protein expression obtained from fluorescence in situ analysis. In a sample set of HER2-positive breast cancers GoIFISHGoIFISH is highly robust in visual analysis and its accuracy compares favorably to other leading image analysis methods. GoIFISHGoIFISH is freely available at www.sourceforge.net/projects/goifish/.

M13 Bacteriophage-Polymer Nanoassemblies as Drug Delivery Vehicles

DTIC Science & Technology

2011-01-01

the M13 bacteriophage is well-defined and can be genetically engineered to produce conductive fibers [35] or display peptides or proteins in...36], and fluorescent dyes [42, 43], can be chemically anchored on the surface of M13 bacterio- phage . Our group has systematically investigated the...bioconjugation chemistry of M13 , and used such modified M13 phages to produce conductive nanofibers [35], direct cell growth [36], and target

Genetic and Phenotypic Characterization of Manufacturing Seeds for a Tetravalent Dengue Vaccine (DENVax)

PubMed Central

Huang, Claire Y.-H.; Kinney, Richard M.; Livengood, Jill A.; Bolling, Bethany; Arguello, John J.; Luy, Betty E.; Silengo, Shawn J.; Boroughs, Karen L.; Stovall, Janae L.; Kalanidhi, Akundi P.; Brault, Aaron C.; Osorio, Jorge E.; Stinchcomb, Dan T.

2013-01-01

Background We have developed a manufacturing strategy that can improve the safety and genetic stability of recombinant live-attenuated chimeric dengue vaccine (DENVax) viruses. These viruses, containing the pre-membrane (prM) and envelope (E) genes of dengue serotypes 1–4 in the replicative background of the attenuated dengue-2 PDK-53 vaccine virus candidate, were manufactured under cGMP. Methodology/Principal Findings After deriving vaccine viruses from RNA-transfected Vero cells, six plaque-purified viruses for each serotype were produced. The plaque-purified strains were then analyzed to select one stock for generation of the master seed. Full genetic and phenotypic characterizations of the master virus seeds were conducted to ensure these viruses retained the previously identified attenuating determinants and phenotypes of the vaccine viruses. We also assessed vector competence of the vaccine viruses in sympatric (Thai) Aedes aegypti mosquito vectors. Conclusion/Significance All four serotypes of master vaccine seeds retained the previously defined safety features, including all three major genetic loci of attenuation, small plaques, temperature sensitivity in mammalian cells, reduced replication in mosquito cell cultures, and reduced neurovirulence in new-born mice. In addition, the candidate vaccine viruses demonstrated greatly reduced infection and dissemination in Aedes aegypti mosquitoes, and are not likely to be transmissible by these mosquitoes. This manufacturing strategy has successfully been used to produce the candidate tetravalent vaccine, which is currently being tested in human clinical trials in the United States, Central and South America, and Asia. PMID:23738026

Transsynaptic Mapping of Second-Order Taste Neurons in Flies by trans-Tango.

PubMed

Talay, Mustafa; Richman, Ethan B; Snell, Nathaniel J; Hartmann, Griffin G; Fisher, John D; Sorkaç, Altar; Santoyo, Juan F; Chou-Freed, Cambria; Nair, Nived; Johnson, Mark; Szymanski, John R; Barnea, Gilad

2017-11-15

Mapping neural circuits across defined synapses is essential for understanding brain function. Here we describe trans-Tango, a technique for anterograde transsynaptic circuit tracing and manipulation. At the core of trans-Tango is a synthetic signaling pathway that is introduced into all neurons in the animal. This pathway converts receptor activation at the cell surface into reporter expression through site-specific proteolysis. Specific labeling is achieved by presenting a tethered ligand at the synapses of genetically defined neurons, thereby activating the pathway in their postsynaptic partners and providing genetic access to these neurons. We first validated trans-Tango in the Drosophila olfactory system and then implemented it in the gustatory system, where projections beyond the first-order receptor neurons are not fully characterized. We identified putative second-order neurons within the sweet circuit that include projection neurons targeting known neuromodulation centers in the brain. These experiments establish trans-Tango as a flexible platform for transsynaptic circuit analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Evidence of intermetastatic heterogeneity for pathological response and genetic mutations within colorectal liver metastases following preoperative chemotherapy.

PubMed

Sebagh, Mylène; Allard, Marc-Antoine; Bosselut, Nelly; Dao, Myriam; Vibert, Eric; Lewin, Maïté; Lemoine, Antoinette; Cherqui, Daniel; Adam, René; Sa Cunha, Antonio

2016-04-19

In patients receiving preoperative chemotherapy, colorectal liver metastases (CLM) are expected to demonstrate a similar behaviour because of similar organ microenvironment and tumour cell chemosensitivity. We focused on the occurrence of pathological and genetic heterogeneity within CLM. Patients resected for multiple CLM between 2004 and 2011 after > three cycles of chemotherapy were included. Pathological heterogeneity was arbitrarily defined as a > 50% difference in the percentage of remaining tumour cells between individual CLM. In patients with pathological heterogeneity, the mutational genotyping (KRAS, NRAS, BRAF and PIK3CA) was determined from the most heterogeneous CLM. Pathological heterogeneity was observed in 31 of 157 patients with multiple CLM (median = 4, range, 2-32) (19.7%). In 72.4% of them, we found a concordance of the mutation status between the paired CLM: both wild-type in 55%, and both mutated in 17.2%. We observed a discordance of the mutation status of 27.6% between CLM: one mutated and the other wild-type. The mutated CLM was the less florid one in 75% of patients with genetic heterogeneity. Pathological heterogeneity is present in 19.7% of patients with multiple CLM. Genetic heterogeneity is present in 27.6% of patients with pathological heterogeneity. Heterogeneity could refine guide management for tissue sampling.

University of Texas Southwestern Medical Center: High-Throughput siRNA Screening of a Non-Small Cell Lung Cancer (NSCLC) Cell Line Panel | Office of Cancer Genomics

Cancer.gov

The goal of this project is to use siRNA screens to identify NSCLC-selective siRNAs from two genome-wide libraries that will allow us to functionally define genetic dependencies of subtypes of NSCLC. Using bioinformatics tools, the CTD2 center at the University of Texas Southwestern Medical Center are discovering associations between this functional data (siRNAs) and NSCLC mutational status, methylation arrays, gene expression arrays, and copy number variation data that will help us identify new targets and enrollment biomarkers. 

University of Texas Southwestern Medical Center (UTSW): High-Throughput siRNA Screening of a Non-Small Cell Lung Cancer (NSCLC) Cell Line Panel | Office of Cancer Genomics

Cancer.gov

The goal of this project is to use siRNA screens to identify NSCLC-selective siRNAs from two genome-wide libraries that will allow us to functionally define genetic dependencies of subtypes of NSCLC. Using bioinformatics tools, the CTD2 center at the University of Texas Southwestern Medical Center are discovering associations between this functional data (siRNAs) and NSCLC mutational status, methylation arrays, gene expression arrays, and copy number variation data that will help us identify new targets and enrollment biomarkers. 

Conceptual Challenges of the Systemic Approach in Understanding Cell Differentiation.

PubMed

Paldi, Andras

2018-01-01

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

Genetic and Pharmacological Inhibition of PDK1 in Cancer Cells: Characterization of a Selective Allosteric Kinase Inhibitor

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

Nagashima, Kumiko; Shumway, Stuart D.; Sathyanarayanan, Sriram

2013-11-20

Phosphoinositide-dependent kinase 1 (PDK1) is a critical activator of multiple prosurvival and oncogenic protein kinases and has garnered considerable interest as an oncology drug target. Despite progress characterizing PDK1 as a therapeutic target, pharmacological support is lacking due to the prevalence of nonspecific inhibitors. Here, we benchmark literature and newly developed inhibitors and conduct parallel genetic and pharmacological queries into PDK1 function in cancer cells. Through kinase selectivity profiling and x-ray crystallographic studies, we identify an exquisitely selective PDK1 inhibitor (compound 7) that uniquely binds to the inactive kinase conformation (DFG-out). In contrast to compounds 1-5, which are classical ATP-competitivemore » kinase inhibitors (DFG-in), compound 7 specifically inhibits cellular PDK1 T-loop phosphorylation (Ser-241), supporting its unique binding mode. Interfering with PDK1 activity has minimal antiproliferative effect on cells growing as plastic-attached monolayer cultures (i.e. standard tissue culture conditions) despite reduced phosphorylation of AKT, RSK, and S6RP. However, selective PDK1 inhibition impairs anchorage-independent growth, invasion, and cancer cell migration. Compound 7 inhibits colony formation in a subset of cancer cell lines (four of 10) and primary xenograft tumor lines (nine of 57). RNAi-mediated knockdown corroborates the PDK1 dependence in cell lines and identifies candidate biomarkers of drug response. In summary, our profiling studies define a uniquely selective and cell-potent PDK1 inhibitor, and the convergence of genetic and pharmacological phenotypes supports a role of PDK1 in tumorigenesis in the context of three-dimensional in vitro culture systems.« less

Diffuse large B-cell lymphoma patient-derived xenograft models capture the molecular and biological heterogeneity of the disease.

PubMed

Chapuy, Bjoern; Cheng, Hongwei; Watahiki, Akira; Ducar, Matthew D; Tan, Yuxiang; Chen, Linfeng; Roemer, Margaretha G M; Ouyang, Jing; Christie, Amanda L; Zhang, Liye; Gusenleitner, Daniel; Abo, Ryan P; Farinha, Pedro; von Bonin, Frederike; Thorner, Aaron R; Sun, Heather H; Gascoyne, Randy D; Pinkus, Geraldine S; van Hummelen, Paul; Wulf, Gerald G; Aster, Jon C; Weinstock, David M; Monti, Stefano; Rodig, Scott J; Wang, Yuzhuo; Shipp, Margaret A

2016-05-05

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease defined by transcriptional classifications, specific signaling and survival pathways, and multiple low-frequency genetic alterations. Preclinical model systems that capture the genetic and functional heterogeneity of DLBCL are urgently needed. Here, we generated and characterized a panel of large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) models, including 8 that reflect the immunophenotypic, transcriptional, genetic, and functional heterogeneity of primary DLBCL and 1 that is a plasmablastic lymphoma. All LBCL PDX models were subjected to whole-transcriptome sequencing to classify cell of origin and consensus clustering classification (CCC) subtypes. Mutations and chromosomal rearrangements were evaluated by whole-exome sequencing with an extended bait set. Six of the 8 DLBCL models were activated B-cell (ABC)-type tumors that exhibited ABC-associated mutations such as MYD88, CD79B, CARD11, and PIM1. The remaining 2 DLBCL models were germinal B-cell type, with characteristic alterations of GNA13, CREBBP, and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC Only 25% of the DLBCL PDX models harbored inactivating TP53 mutations, whereas 75% exhibited copy number alterations of TP53 or its upstream modifier, CDKN2A, consistent with the reported incidence and type of p53 pathway alterations in primary DLBCL. By CCC criteria, 6 of 8 DLBCL PDX models were B-cell receptor (BCR)-type tumors that exhibited selective surface immunoglobulin expression and sensitivity to entospletinib, a recently developed spleen tyrosine kinase inhibitor. In summary, we have established and characterized faithful PDX models of DLBCL and demonstrated their usefulness in functional analyses of proximal BCR pathway inhibition. © 2016 by The American Society of Hematology.

A Newly Defined and Xeno-Free Culture Medium Supports Every-Other-Day Medium Replacement in the Generation and Long-Term Cultivation of Human Pluripotent Stem Cells.

PubMed

Ahmadian Baghbaderani, Behnam; Tian, Xinghui; Scotty Cadet, Jean; Shah, Kevan; Walde, Amy; Tran, Huan; Kovarcik, Don Paul; Clarke, Diana; Fellner, Thomas

2016-01-01

Human pluripotent stem cells (hPSCs) present an unprecedented opportunity to advance human health by offering an alternative and renewable cell resource for cellular therapeutics and regenerative medicine. The present demand for high quality hPSCs for use in both research and clinical studies underscores the need to develop technologies that will simplify the cultivation process and control variability. Here we describe the development of a robust, defined and xeno-free hPSC medium that supports reliable propagation of hPSCs and generation of human induced pluripotent stem cells (hiPSCs) from multiple somatic cell types; long-term serial subculturing of hPSCs with every-other-day (EOD) medium replacement; and banking fully characterized hPSCs. The hPSCs cultured in this medium for over 40 passages are genetically stable, retain high expression levels of the pluripotency markers TRA-1-60, TRA-1-81, Oct-3/4 and SSEA-4, and readily differentiate into ectoderm, mesoderm and endoderm. Importantly, the medium plays an integral role in establishing a cGMP-compliant process for the manufacturing of hiPSCs that can be used for generation of clinically relevant cell types for cell replacement therapy applications.

In vitro models.

PubMed

Mather, Jennie Powell

2012-02-01

The current resurgence of interest in the cancer stem cell (CSC) hypothesis as possibly providing a unifying theory of cancer biology is fueled by the growing body of work on normal adult tissue stem cells and the promise that CSC may hold the key to one of the central problems of clinical oncology: tumor recurrence. Many studies suggest that the microenvironment plays a role, perhaps a seminal one, in cancer development and progression. In addition, the possibility that the stem cell-like component of tumors is capable of rapid and reversible changes of phenotype raises questions concerning studies with these populations and the application of what we learn to the clinical situation. These types of questions are extremely difficult to study using in vivo models or freshly isolated cells. Established cell lines grown in defined conditions provide important model systems for these studies. There are three types of in vitro models for CSCs: (a) selected subpopulations of existing tumor lines (derived from serum-containing medium; (b) creation of lines from tumor or normal cells by genetic manipulation; or (c) direct in vitro selection of CSC from tumors or sorted tumor cells using defined serum-free conditions. We review the problems associated with creating and maintaining in vitro cultures of CSCs and the progress to date on the establishment of these important models. Copyright © 2011 AlphaMed Press.

Stem cells and bone diseases: new tools, new perspective.

PubMed

Riminucci, Mara; Remoli, Cristina; Robey, Pamela G; Bianco, Paolo

2015-01-01

Postnatal skeletal stem cells are a unique class of progenitors with biological properties that extend well beyond the limits of stemness as commonly defined. Skeletal stem cells sustain skeletal tissue homeostasis, organize and maintain the complex architectural structure of the bone marrow microenvironment and provide a niche for hematopoietic progenitor cells. The identification of stem cells in the human post-natal skeleton has profoundly changed our approach to the physiology and pathology of this system. Skeletal diseases have been long interpreted essentially in terms of defective function of differentiated cells and/or abnormal turnover of the matrix that they produce. The notion of a skeletal stem cell has brought forth multiple, novel concepts in skeletal biology that provide potential alternative concepts. At the same time, the recognition of the complex functions played by skeletal progenitors, such as the structural and functional organization of the bone marrow, has provided an innovative, unifying perspective for understanding bone and bone marrow changes simultaneously occurring in many disorders. Finally, the possibility to isolate and highly enrich for skeletal progenitors, enables us to reproduce perfectly normal or pathological organ miniatures. These, in turn, provide suitable models to investigate and manipulate the pathogenetic mechanisms of many genetic and non-genetic skeletal diseases. This article is part of a Special Issue entitled Stem cells and Bone. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

How and where to build a root hair.

PubMed

Dolan, L

2001-12-01

The root hair of Arabidopsis has become a model system for investigations of the patterning and morphogenesis of cells in plants. A cascade of transcriptional regulators controls the pattern of cellular differentiation. Recently, one of the genes that plays a specific role in cellular differentiation in roots, WEREWOLF, has been shown to be functionally equivalent to GLABRA1, which functions only in the shoot. The cloning of genes defined by mutants with defective root-hair growth has provided insights into the roles of the cell wall, ion transport and the cytoskeleton during hair growth. Genetic analyses continue to identify mutants that will be instructive in furthering our understanding of the growth and development of root-hair cells.

Single-Cell Whole-Genome Amplification and Sequencing: Methodology and Applications.

PubMed

Huang, Lei; Ma, Fei; Chapman, Alec; Lu, Sijia; Xie, Xiaoliang Sunney

2015-01-01

We present a survey of single-cell whole-genome amplification (WGA) methods, including degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), multiple displacement amplification (MDA), and multiple annealing and looping-based amplification cycles (MALBAC). The key parameters to characterize the performance of these methods are defined, including genome coverage, uniformity, reproducibility, unmappable rates, chimera rates, allele dropout rates, false positive rates for calling single-nucleotide variations, and ability to call copy-number variations. Using these parameters, we compare five commercial WGA kits by performing deep sequencing of multiple single cells. We also discuss several major applications of single-cell genomics, including studies of whole-genome de novo mutation rates, the early evolution of cancer genomes, circulating tumor cells (CTCs), meiotic recombination of germ cells, preimplantation genetic diagnosis (PGD), and preimplantation genomic screening (PGS) for in vitro-fertilized embryos.

Proceedings from the National Cancer Institute's Second International Workshop on the Biology, Prevention, and Treatment of Relapse after Hematopoietic Stem Cell Transplantation: Part I. Biology of relapse after transplantation.

PubMed

Gress, Ronald E; Miller, Jeffrey S; Battiwalla, Minoo; Bishop, Michael R; Giralt, Sergio A; Hardy, Nancy M; Kröger, Nicolaus; Wayne, Alan S; Landau, Dan A; Wu, Catherine J

2013-11-01

In the National Cancer Institute's Second Workshop on the Biology, Prevention, and Treatment of Relapse after Hematopoietic Stem Cell Transplantation, the Scientific/Educational Session on the Biology of Relapse discussed recent advances in understanding some of the host-, disease-, and transplantation-related contributions to relapse, emphasizing concepts with potential therapeutic implications. Relapse after hematopoietic stem cell transplantation (HSCT) represents tumor escape, from the cytotoxic effects of the conditioning regimen and from immunologic control mediated by reconstituted lymphocyte populations. Factors influencing the biology of the therapeutic graft-versus-malignancy (GVM) effect-and relapse-include conditioning regimen effects on lymphocyte populations and homeostasis, immunologic niches, and the tumor microenvironment; reconstitution of lymphocyte populations and establishment of functional immune competence; and genetic heterogeneity within the malignancy defining potential for clonal escape. Recent developments in T cell and natural killer cell homeostasis and reconstitution are reviewed, with implications for prevention and treatment of relapse, as is the application of modern genome sequencing to defining the biologic basis of GVM, clonal escape, and relapse after HSCT. Published by Elsevier Inc.

"Ant" and "grasshopper" life-history strategies in Saccharomyces cerevisiae.

PubMed

Spor, Aymé; Wang, Shaoxiao; Dillmann, Christine; de Vienne, Dominique; Sicard, Delphine

2008-02-13

From the evolutionary and ecological points of view, it is essential to distinguish between the genetic and environmental components of the variability of life-history traits and of their trade-offs. Among the factors affecting this variability, the resource uptake rate deserves particular attention, because it depends on both the environment and the genetic background of the individuals. In order to unravel the bases of the life-history strategies in yeast, we grew a collection of twelve strains of Saccharomyces cerevisiae from different industrial and geographical origins in three culture media differing for their glucose content. Using a population dynamics model to fit the change of population size over time, we estimated the intrinsic growth rate (r), the carrying capacity (K), the mean cell size and the glucose consumption rate per cell. The life-history traits, as well as the glucose consumption rate, displayed large genetic and plastic variability and genetic-by-environment interactions. Within each medium, growth rate and carrying capacity were not correlated, but a marked trade-off between these traits was observed over the media, with high K and low r in the glucose rich medium and low K and high r in the other media. The cell size was tightly negatively correlated to carrying capacity in all conditions. The resource consumption rate appeared to be a clear-cut determinant of both the carrying capacity and the cell size in all media, since it accounted for 37% to 84% of the variation of those traits. In a given medium, the strains that consume glucose at high rate have large cell size and low carrying capacity, while the strains that consume glucose at low rate have small cell size but high carrying capacity. These two contrasted behaviors may be metaphorically defined as "ant" and "grasshopper" strategies of resource utilization. Interestingly, a strain may be "ant" in one medium and "grasshopper" in another. These life-history strategies are discussed with regards to yeast physiology, and in an evolutionary perspective.

Inner ear symptoms and disease: Pathophysiological understanding and therapeutic options

PubMed Central

Ciuman, Raphael R.

2013-01-01

In recent years, huge advances have taken place in understanding of inner ear pathophysiology causing sensorineural hearing loss, tinnitus, and vertigo. Advances in understanding comprise biochemical and physiological research of stimulus perception and conduction, inner ear homeostasis, and hereditary diseases with underlying genetics. This review describes and tabulates the various causes of inner ear disease and defines inner ear and non-inner ear causes of hearing loss, tinnitus, and vertigo. The aim of this review was to comprehensively breakdown this field of otorhinolaryngology for specialists and non-specialists and to discuss current therapeutic options in distinct diseases and promising research for future therapies, especially pharmaceutic, genetic, or stem cell therapy. PMID:24362017

Cancer: Bad Luck or Punishment?

PubMed

Lichtenstein, A V

2017-01-01

Contrasting opinions on the role of extrinsic and intrinsic factors in cancer etiology (Tomasetti, C., and Vogelstein, B. (2015) Science, 347, 78-81; Wu, S., et al. (2016) Nature, 529, 43-47) variously define priorities in the war on cancer. The correlation between the lifetime risk of several types of cancer and the total number of divisions of normal self-renewing cells revealed by the authors has given them grounds to put forward the "bad luck" hypothesis. It assumes that ~70% of cancer variability is attributed to random errors arising during DNA replication in normal, noncancerous stem cells, i.e. to internal factors, which is impossible either to expect or to prevent. This assumption caused many critical responses that emphasize, on the contrary, the defining role of extrinsic factors in cancer etiology. The analysis of epidemiological and genetic data presented in this work testifies in favor of the "bad luck" hypothesis.

BCR-ABL1-like acute lymphoblastic leukaemia: From bench to bedside.

PubMed

Boer, Judith M; den Boer, Monique L

2017-09-01

Acute lymphoblastic leukaemia (ALL) occurs in approximately 1:1500 children and is less frequently found in adults. The most common immunophenotype of ALL is the B cell lineage and within B cell precursor ALL, specific genetic aberrations define subtypes with distinct biological and clinical characteristics. With more advanced genetic analysis methods such as whole genome and transcriptome sequencing, novel genetic subtypes have recently been discovered. One novel class of genetic aberrations comprises tyrosine kinase-activating lesions, including translocations and rearrangements of tyrosine kinase and cytokine receptor genes. These newly discovered genetic aberrations are harder to detect by standard diagnostic methods such as karyotyping, fluorescent in situ hybridisation (FISH) or polymerase chain reaction (PCR) because they are diverse and often cryptic. These lesions involve one of several tyrosine kinase genes (among others, v-abl Abelson murine leukaemia viral oncogene homologue 1 (ABL1), v-abl Abelson murine leukaemia viral oncogene homologue 2 (ABL2), platelet-derived growth factor receptor beta polypeptide (PDGFRB)), each of which can be fused to up to 15 partner genes. Together, they compose 2-3% of B cell precursor ALL (BCP-ALL), which is similar in size to the well-known fusion gene BCR-ABL1 subtype. These so-called BCR-ABL1-like fusions are mutually exclusive with the sentinel translocations in BCP-ALL (BCR-ABL1, ETV6-RUNX1, TCF3-PBX1, and KMT2A (MLL) rearrangements) and have the promising prospect to be sensitive to tyrosine kinase inhibitors similar to BCR-ABL1. In this review, we discuss the types of tyrosine kinase-activating lesions discovered, and the preclinical and clinical evidence for the use of tyrosine kinase inhibitors in the treatment of this novel subtype of ALL. Copyright © 2017 Elsevier Ltd. All rights reserved.

Improving cell therapy – experiments using transplanted telomerase-immortalized cells in immunodeficient mice

PubMed Central

Huang, Qin; Chen, Meizhen; Liang, Sitai; Acha, Victor; Liu, Dan; Yuan, Furong; Hawks, Christina L.; Hornsby, Peter J.

2007-01-01

Cell therapy is the use of stem cells and other types of cells in various therapies for age-related diseases. Two issues that must be addressed before cell therapy could be used routinely in medicine are improved efficacy of the transplanted cells and demonstrated long-term safety. Desirable genetic modifications that could be made to cells to be used for cell therapy include immortalization with hTERT (human telomerase reverse transcriptase). We have used a model for cell therapy in which transplantation of adrenocortical cells restores glucocorticoid and mineralocorticoid hormone levels in adrenalectomized immunodeficient mice. In this model, clones of cells that had been immortalized with hTERT were shown to be able to replace the function of the animals'adrenal glands by forming vascularized tissue structures when cells were transplanted beneath the capsule of the kidney. hTERT-modified cells showed no tendency for neoplastic changes. Moreover, a series of experiments showed that hTERT does not cooperate with known oncoproteins in tumorigenesis either in adrenocortical cells or in human fibroblasts. Nevertheless, hTERT was required for tumorigenesis when cells were implanted subcutaneously rather than in the subrenal capsule space. Changes in gene expression make hTERT-modified cells more robust. Understanding these changes is important so as to be able to separately control immortalization and other desirable properties of cells that could be used in cell therapy. Alternatively, desirable properties of transplants might be provided by co-transplanted mesenchymal cells: mesenchymal cell-assisted cell therapy. For both hTERT modification and mesenchymal cell-assisted cell therapy, genomics approaches will be needed to define what genetic modifications are desirable and safe in cells used in cell therapy. PMID:17123586

Humanization of the mouse mammary gland by replacement of the luminal layer with genetically engineered preneoplastic human cells.

PubMed

Verbeke, Stephanie; Richard, Elodie; Monceau, Elodie; Schmidt, Xenia; Rousseau, Benoit; Velasco, Valerie; Bernard, David; Bonnefoi, Herve; MacGrogan, Gaetan; Iggo, Richard D

2014-12-20

The cell of origin for estrogen receptor α-positive (ERα+) breast cancer is probably a luminal stem cell in the terminal duct lobular units. To model these cells, we have used the murine myoepithelial layer in the mouse mammary ducts as a scaffold upon which to build a human luminal layer. To prevent squamous metaplasia, a common artifact in genetically-engineered breast cancer models, we sought to limit activation of the epidermal growth factor receptor (EGFR) during in vitro cell culture before grafting the cells. Human reduction mammoplasty cells were grown in vitro in WIT medium. Epidermal growth factor in the medium was replaced with amphiregulin and neuregulin to decrease activation of EGFR and increase activation of EGFR homologs 3 and 4 (ERBB3 and ERBB4). Lentiviral vectors were used to express oncogenic transgenes and fluorescent proteins. Human mammary epithelial cells were mixed with irradiated mouse fibroblasts and Matrigel, then injected through the nipple into the mammary ducts of immunodeficient mice. Engrafted cells were visualized by stereomicroscopy for fluorescent proteins and characterized by histology and immunohistochemistry. Growth of normal mammary epithelial cells in conditions favoring ERBB3/4 signaling prevented squamous metaplasia in vitro. Normal human cells were quickly lost after intraductal injection, but cells infected with lentiviruses expressing CCND1, MYC, TERT, BMI1 and a short-hairpin RNA targeting TP53 were able to engraft and progressively replace the luminal layer in the mouse mammary ducts, resulting in the formation of an extensive network of humanized ducts. Despite expressing multiple oncogenes, the human cells formed a morphologically normal luminal layer. Expression of a single additional oncogene, PIK3CA-H1047R, converted the cells into invasive cancer cells. The resulting tumors were ERα+, Ki67+ luminal B adenocarcinomas that were resistant to treatment with fulvestrant. Injection of preneoplastic human mammary epithelial cells into the mammary ducts of immunodeficient mice leads to replacement of the murine luminal layer with morphologically normal human cells. Genetic manipulation of the injected cells makes it possible to study defined steps in the transformation of human mammary epithelial cells in a more physiological environment than has hitherto been possible.

Regional control of Drosophila gut stem cell proliferation: EGF establishes GSSC proliferative set point & controls emergence from quiescence.

PubMed

Strand, Marie; Micchelli, Craig A

2013-01-01

Adult stem cells vary widely in their rates of proliferation. Some stem cells are constitutively active, while others divide only in response to injury. The mechanism controlling this differential proliferative set point is not well understood. The anterior-posterior (A/P) axis of the adult Drosophila midgut has a segmental organization, displaying physiological compartmentalization and region-specific epithelia. These distinct midgut regions are maintained by defined stem cell populations with unique division schedules, providing an excellent experimental model with which to investigate this question. Here, we focus on the quiescent gastric stem cells (GSSCs) of the acidic copper cell region (CCR), which exhibit the greatest period of latency between divisions of all characterized gut stem cells, to define the molecular basis of differential stem cell activity. Our molecular genetic analysis demonstrates that the mitogenic EGF signaling pathway is a limiting factor controlling GSSC proliferation. We find that under baseline conditions, when GSSCs are largely quiescent, the lowest levels of EGF ligands in the midgut are found in the CCR. However, acute epithelial injury by enteric pathogens leads to an increase in EGF ligand expression in the CCR and rapid expansion of the GSSC lineage. Thus, the unique proliferative set points for gut stem cells residing in physiologically distinct compartments are governed by regional control of niche signals along the A/P axis.

A deterministic method for estimating free energy genetic network landscapes with applications to cell commitment and reprogramming paths.

PubMed

Olariu, Victor; Manesso, Erica; Peterson, Carsten

2017-06-01

Depicting developmental processes as movements in free energy genetic landscapes is an illustrative tool. However, exploring such landscapes to obtain quantitative or even qualitative predictions is hampered by the lack of free energy functions corresponding to the biochemical Michaelis-Menten or Hill rate equations for the dynamics. Being armed with energy landscapes defined by a network and its interactions would open up the possibility of swiftly identifying cell states and computing optimal paths, including those of cell reprogramming, thereby avoiding exhaustive trial-and-error simulations with rate equations for different parameter sets. It turns out that sigmoidal rate equations do have approximate free energy associations. With this replacement of rate equations, we develop a deterministic method for estimating the free energy surfaces of systems of interacting genes at different noise levels or temperatures. Once such free energy landscape estimates have been established, we adapt a shortest path algorithm to determine optimal routes in the landscapes. We explore the method on three circuits for haematopoiesis and embryonic stem cell development for commitment and reprogramming scenarios and illustrate how the method can be used to determine sequential steps for onsets of external factors, essential for efficient reprogramming.

Leber hereditary optic neuropathy: current perspectives

PubMed Central

Meyerson, Cherise; Van Stavern, Greg; McClelland, Collin

2015-01-01

Leber hereditary optic neuropathy (LHON) is one of the most common inherited optic neuropathies causing bilateral central vision loss. The disorder results from point mutations in mitochondrial DNA and subsequent mitochondrial dysfunction. The primary cell type that is lost in LHON is the retinal ganglion cell, which is highly susceptible to disrupted ATP production and oxidative stress. Inheritance of LHON follows that of mitochondrial genetics, and it has a highly variable clinical phenotype, as other genetic and environmental factors also play a role. Although LHON usually presents with isolated vision loss, some patients suffer other neurological sequelae. For ill-defined reasons, male LHON mutation carriers are more affected than females. Most LHON patients remain legally blind, but a small proportion can experience spontaneous partial recovery, often within the first year of symptom onset. Unfortunately, at this time there are no established curative interventions and treatment is largely supportive. Patients should be offered low vision services and counseled on mitigating risk factors for additional vision loss, such as smoking and consuming alcohol. Encouraging treatments currently undergoing investigation includes ubiquinone analogs, such as idebenone, as well as gene therapy and stem cells to restore ATP synthesis and provide neuroprotection to surviving retinal ganglion cells. PMID:26170609

Bacterial interactions in dental biofilm development.

PubMed

Hojo, K; Nagaoka, S; Ohshima, T; Maeda, N

2009-11-01

Recent analyses with ribosomal RNA-based technologies have revealed the diversity of bacterial populations within dental biofilms, and have highlighted their important contributions to oral health and disease. Dental biofilms are exceedingly complex and multispecies ecosystems, where oral bacteria interact cooperatively or competitively with other members. Bacterial interactions that influence dental biofilm communities include various different mechanisms. During the early stage of biofilm formation, it is known that planktonic bacterial cells directly attach to surfaces of the oral cavity or indirectly bind to other bacterial cells that have already colonized. Adherence through co-aggregation may be critical for the temporary retention of bacteria on dental surfaces, and may facilitate eventual bacterial colonization. It is likely that metabolic communication, genetic exchange, production of inhibitory factors (e.g., bacteriocins, hydrogen peroxide, etc.), and quorum-sensing are pivotal regulatory factors that determine the bacterial composition and/or metabolism. Since each bacterium can easily access a neighboring bacterial cell and its metabolites, genetic exchanges and metabolic communication may occur frequently in dental biofilms. Quorum-sensing is defined as gene regulation in response to cell density, which influences various functions, e.g., virulence and bacteriocin production. In this review, we discuss these important interactions among oral bacteria within the dental biofilm communities.

Biotechnological applications in in vitro plant regeneration studies of broccoli (Brassica oleracea L. var. italica), an important vegetable crop.

PubMed

Kumar, Pankaj; Srivastava, Dinesh Kumar

2016-04-01

Biotechnology holds promise for genetic improvement of important vegetable crops. Broccoli (Brassica oleracea L. var. italica) is an important vegetable crop of the family Brassicaceae. However, various biotic and abiotic stresses cause enormous crop yield losses during commercial cultivation of broccoli. Establishment of a reliable, reproducible and efficient in vitro plant regeneration system with cell and tissue culture is a vital prerequisite for biotechnological application of crop improvement programme. An in vitro plant regeneration technique refers to culturing, cell division, cell multiplication, de-differentiation and differentiation of cells, protoplasts, tissues and organs on defined liquid/solid medium under aseptic and controlled environment. Recent progress in the field of plant tissue culture has made this area one of the most dynamic and promising in experimental biology. There are many published reports on in vitro plant regeneration studies in broccoli including direct organogenesis, indirect organogenesis and somatic embryogenesis. This review summarizes those plant regeneration studies in broccoli that could be helpful in drawing the attention of the researchers and scientists to work on it to produce healthy, biotic and abiotic stress resistant plant material and to carry out genetic transformation studies for the production of transgenic plants.

A deterministic method for estimating free energy genetic network landscapes with applications to cell commitment and reprogramming paths

PubMed Central

Olariu, Victor; Manesso, Erica

2017-01-01

Depicting developmental processes as movements in free energy genetic landscapes is an illustrative tool. However, exploring such landscapes to obtain quantitative or even qualitative predictions is hampered by the lack of free energy functions corresponding to the biochemical Michaelis–Menten or Hill rate equations for the dynamics. Being armed with energy landscapes defined by a network and its interactions would open up the possibility of swiftly identifying cell states and computing optimal paths, including those of cell reprogramming, thereby avoiding exhaustive trial-and-error simulations with rate equations for different parameter sets. It turns out that sigmoidal rate equations do have approximate free energy associations. With this replacement of rate equations, we develop a deterministic method for estimating the free energy surfaces of systems of interacting genes at different noise levels or temperatures. Once such free energy landscape estimates have been established, we adapt a shortest path algorithm to determine optimal routes in the landscapes. We explore the method on three circuits for haematopoiesis and embryonic stem cell development for commitment and reprogramming scenarios and illustrate how the method can be used to determine sequential steps for onsets of external factors, essential for efficient reprogramming. PMID:28680655

Isolation and characterization of chromosome-gain and increase-in-ploidy mutants in yeast.

PubMed

Chan, C S; Botstein, D

1993-11-01

We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37 degrees. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37 degrees. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth.

Two Membrane-Associated Tyrosine Phosphatase Homologs Potentiate C. elegans AKT-1/PKB Signaling

PubMed Central

Hu, Patrick J; Xu, Jinling; Ruvkun, Gary

2006-01-01

Akt/protein kinase B (PKB) functions in conserved signaling cascades that regulate growth and metabolism. In humans, Akt/PKB is dysregulated in diabetes and cancer; in Caenorhabditis elegans, Akt/PKB functions in an insulin-like signaling pathway to regulate larval development. To identify molecules that modulate C. elegans Akt/PKB signaling, we performed a genetic screen for enhancers of the akt-1 mutant phenotype (eak). We report the analysis of three eak genes. eak-6 and eak-5/sdf-9 encode protein tyrosine phosphatase homologs; eak-4 encodes a novel protein with an N-myristoylation signal. All three genes are expressed primarily in the two endocrine XXX cells, and their predicted gene products localize to the plasma membrane. Genetic evidence indicates that these proteins function in parallel to AKT-1 to inhibit the FoxO transcription factor DAF-16. These results define two membrane-associated protein tyrosine phosphatase homologs that may potentiate C. elegans Akt/PKB signaling by cell autonomous and cell nonautonomous mechanisms. Similar molecules may modulate Akt/PKB signaling in human endocrine tissues. PMID:16839187

Synthetic virus seeds for improved vaccine safety: Genetic reconstruction of poliovirus seeds for a PER.C6 cell based inactivated poliovirus vaccine.

PubMed

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

2015-10-13

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

Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP

PubMed Central

Hafner, Markus; Landthaler, Markus; Burger, Lukas; Khorshid, Mohsen; Hausser, Jean; Berninger, Philipp; Rothballer, Andrea; Ascano, Manuel; Jungkamp, Anna-Carina; Munschauer, Mathias; Ulrich, Alexander; Wardle, Greg S.; Dewell, Scott; Zavolan, Mihaela; Tuschl, Thomas

2010-01-01

Summary RNA transcripts are subject to post-transcriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases. PMID:20371350

Lmo2 expression defines tumor cell identity during T-cell leukemogenesis.

PubMed

García-Ramírez, Idoia; Bhatia, Sanil; Rodríguez-Hernández, Guillermo; González-Herrero, Inés; Walter, Carolin; González de Tena-Dávila, Sara; Parvin, Salma; Haas, Oskar; Woessmann, Wilhelm; Stanulla, Martin; Schrappe, Martin; Dugas, Martin; Natkunam, Yasodha; Orfao, Alberto; Domínguez, Verónica; Pintado, Belén; Blanco, Oscar; Alonso-López, Diego; De Las Rivas, Javier; Martín-Lorenzo, Alberto; Jiménez, Rafael; García Criado, Francisco Javier; García Cenador, María Begoña; Lossos, Izidore S; Vicente-Dueñas, Carolina; Borkhardt, Arndt; Hauer, Julia; Sánchez-García, Isidro

2018-06-07

The impact of LMO2 expression on cell lineage decisions during T-cell leukemogenesis remains largely elusive. Using genetic lineage tracing, we have explored the potential of LMO2 in dictating a T-cell malignant phenotype. We first initiated LMO2 expression in hematopoietic stem/progenitor cells and maintained its expression in all hematopoietic cells. These mice develop exclusively aggressive human-like T-ALL In order to uncover a potential exclusive reprogramming effect of LMO2 in murine hematopoietic stem/progenitor cells, we next showed that transient LMO2 expression is sufficient for oncogenic function and induction of T-ALL The resulting T-ALLs lacked LMO2 and its target-gene expression, and histologically, transcriptionally, and genetically similar to human LMO2-driven T-ALL We next found that during T-ALL development, secondary genomic alterations take place within the thymus. However, the permissiveness for development of T-ALL seems to be associated with wider windows of differentiation than previously appreciated. Restricted Cre-mediated activation of Lmo2 at different stages of B-cell development induces systematically and unexpectedly T-ALL that closely resembled those of their natural counterparts. Together, these results provide a novel paradigm for the generation of tumor T cells through reprogramming in vivo and could be relevant to improve the response of T-ALL to current therapies. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Single-cell protein secretomic signatures as potential correlates to tumor cell lineage evolution and cell–cell interaction

PubMed Central

Kwak, Minsuk; Mu, Luye; Lu, Yao; Chen, Jonathan J.; Brower, Kara; Fan, Rong

2013-01-01

Secreted proteins including cytokines, chemokines, and growth factors represent important functional regulators mediating a range of cellular behavior and cell–cell paracrine/autocrine signaling, e.g., in the immunological system (Rothenberg, 2007), tumor microenvironment (Hanahan and Weinberg, 2011), or stem cell niche (Gnecchi etal., 2008). Detection of these proteins is of great value not only in basic cell biology but also for diagnosis and therapeutic monitoring of human diseases such as cancer. However, due to co-production of multiple effector proteins from a single cell, referred to as polyfunctionality, it is biologically informative to measure a panel of secreted proteins, or secretomic signature, at the level of single cells. Recent evidence further indicates that a genetically identical cell population can give rise to diverse phenotypic differences (Niepel etal., 2009). Non-genetic heterogeneity is also emerging as a potential barrier to accurate monitoring of cellular immunity and effective pharmacological therapies (Cohen etal., 2008; Gascoigne and Taylor, 2008), but can hardly assessed using conventional approaches that do not examine cellular phenotype at the functional level. It is known that cytokines, for example, in the immune system define the effector functions and lineage differentiation of immune cells. In this article, we hypothesize that protein secretion profile may represent a universal measure to identify the definitive correlate in the larger context of cellular functions to dissect cellular heterogeneity and evolutionary lineage relationship in human cancer. PMID:23390614

Product quality considerations for mammalian cell culture process development and manufacturing.

PubMed

Gramer, Michael J

2014-01-01

The manufacturing of a biologic drug from mammalian cells results in not a single substance, but an array of product isoforms, also known as variants. These isoforms arise due to intracellular or extracellular events as a result of biological or chemical modification. The most common examples related to biomanufacturing include amino acid modifications (glycosylation, isomerization, oxidation, adduct formation, pyroglutamate formation, phosphorylation, sulfation, amidation), amino acid sequence variants (genetic mutations, amino acid misincorporation, N- and C-terminal heterogeneity, clipping), and higher-order structure modifications (misfolding, aggregation, disulfide pairing). Process-related impurities (HCP, DNA, media components, viral particles) are also important quality attributes related to product safety. The observed ranges associated with each quality attribute define the product quality profile. A biologic drug must have a correct and consistent quality profile throughout clinical development and scale-up to commercial production to ensure product safety and efficacy. In general, the upstream process (cell culture) defines the quality of product-related substances, whereas the downstream process (purification) defines the residual level of process- and product-related impurities. The purpose of this chapter is to review the impact of the cell culture process on product quality. Emphasis is placed on studies with industrial significance and where the direct mechanism of product quality impact was determined. Where possible, recommendations for maintaining consistent or improved quality are provided.

Senescence-Derived Extracellular Molecules as Modulators of Oral Cancer Development: A Mini-Review.

PubMed

Parkinson, Eric Kenneth; James, Emma L; Prime, Stephen S

2016-01-01

Oral cancers are predominantly oral squamous cell carcinomas (OSCCs) derived from keratinocytes, and there is now very detailed knowledge of the genetics and molecular biology of the epithelial tumourigenic component of these cancers, including the identification of cancer stem or tumour-initiating cells. Several key genetic alterations have been identified including the near ubiquitous loss of the CDKN2A/p16INK4A and p53 pathways and telomerase activation, together with frequent inactivation of the NOTCH1 canonical pathway either by somatic genetic alterations or by the presence of human papilloma virus. There is also evidence that OSCCs arise from a 'field' of altered cells and that malignant conversion takes place pre-dominantly at the microscopic level. However, in the last decade, it has been realised that tumour development and progression are influenced by the cells of the microenvironment with cross-talk between the epithelial (tumour) and mesenchymal components. OSCCs, especially those that have bypassed cellular senescence, produce an array of proteins and metabolites that induce cellular senescence in the normal surrounding cells; indeed, senescence is a common property of cancer-associated fibroblasts (CAFs). Cellular senescence is defined as an irreversible cell cycle arrest and is associated with the release of molecules known as the senescence-associated secretory phenotype that can selectively promote the growth of pre-neoplastic keratinocytes (osteopontin) and cancer invasion (transforming growth factor β, matrix metalloproteinases, interleukin 6 and lactate). In addition, both old and new work has shown that keratinocytes harbouring NOTCH loss-of-function mutations that lead to defective keratinocyte differentiation and loss of squamous epithelial barrier function may act as a tumour-promoting stimulus for initiated cells harbouring RAS pathway mutations by activating a wound response in the tumour mesenchyme. Thus, not all keratinocytes in the tumour tissue may be tumourigenic and may instead act as promoters of tumour growth and progression analogous to the much-studied CAFs which co-evolve with the genetically altered tumourigenic cells. This new data is discussed in relation to attempts to develop novel non-invasive diagnostics and therapeutics for oral cancer. © 2015 S. Karger AG, Basel.

Genetic Factors That Might Lead to Different Responses in Individuals Exposed to Perchlorate

PubMed Central

Scinicariello, Franco; Murray, H. Edward; Smith, Lester; Wilbur, Sharon; Fowler, Bruce A.

2005-01-01

Perchlorate has been detected in groundwater in many parts of the United States, and recent detection in vegetable and dairy food products indicates that contamination by perchlorate is more widespread than previously thought. Perchlorate is a competitive inhibitor of the sodium iodide symporter, the thyroid cell–surface protein responsible for transporting iodide from the plasma into the thyroid. An estimated 4.3% of the U.S. population is subclinically hypothyroid, and 6.9% of pregnant women may have low iodine intake. Congenital hypothyroidism affects 1 in 3,000 to 1 in 4,000 infants, and 15% of these cases have been attributed to genetic defects. Our objective in this review is to identify genetic biomarkers that would help define subpopulations sensitive to environmental perchlorate exposure. We review the literature to identify genetic defects involved in the iodination process of the thyroid hormone synthesis, particularly defects in iodide transport from circulation into the thyroid cell, defects in iodide transport from the thyroid cell to the follicular lumen (Pendred syndrome), and defects of iodide organification. Furthermore, we summarize relevant studies of perchlorate in humans. Because of perchlorate inhibition of iodide uptake, it is biologically plausible that chronic ingestion of perchlorate through contaminated sources may cause some degree of iodine discharge in populations that are genetically susceptible to defects in the iodination process of the thyroid hormone synthesis, thus deteriorating their conditions. We conclude that future studies linking human disease and environmental perchlorate exposure should consider the genetic makeup of the participants, actual perchlorate exposure levels, and individual iodine intake/excretion levels. PMID:16263499

Genetic susceptibility to Chagas disease cardiomyopathy: involvement of several genes of the innate immunity and chemokine-dependent migration pathways

PubMed Central

2013-01-01

Background Chagas disease, caused by the protozoan Trypanosoma cruzi is endemic in Latin America. Thirty percent of infected individuals develop chronic Chagas cardiomyopathy (CCC), an inflammatory dilated cardiomyopathy that is, by far, the most important clinical consequence of T. cruzi infection. The others remain asymptomatic (ASY). A possible genetic component to disease progression was suggested by familial aggregation of cases and the association of markers of innate and adaptive immunity genes with CCC development. Migration of Th1-type T cells play a major role in myocardial damage. Methods Our genetic analysis focused on CCR5, CCL2 and MAL/TIRAP genes. We used the Tag SNPs based approach, defined to catch all the genetic information from each gene. The study was conducted on a large Brazilian population including 315 CCC cases and 118 ASY subjects. Results The CCL2rs2530797A/A and TIRAPrs8177376A/A were associated to an increase susceptibility whereas the CCR5rs3176763C/C genotype is associated to protection to CCC. These associations were confirmed when we restricted the analysis to severe CCC, characterized by a left ventricular ejection fraction under 40%. Conclusions Our data show that polymorphisms affecting key molecules involved in several immune parameters (innate immunity signal transduction and T cell/monocyte migration) play a role in genetic susceptibility to CCC development. This also points out to the multigenic character of CCC, each polymorphism imparting a small contribution. The identification of genetic markers for CCC will provide information for pathogenesis as well as therapeutic targets. PMID:24330528

Effects of different extracts of curcumin on TPC1 papillary thyroid cancer cell line.

PubMed

Perna, Angelica; De Luca, Antonio; Adelfi, Laura; Pasquale, Tammaro; Varriale, Bruno; Esposito, Teresa

2018-02-15

The thyroid gland is one of the largest endocrine glands in the body. The vast majority of TCs (> 90%) originate from follicular cells and are defined as differentiated thyroid cancers (DTC) and the two histological subtypes are the papillary TC with its variants and the follicular TC. Curcumin possesses a wide variety of biological functions, and thanks to its properties, it has gained considerable attention due to its profound medicinal values (Prasad, Gupta, Tyagi, and Aggarwal, Biotechnol Adv 32:1053-1064, 2014). We have undertaken the present work in order to define the possible role of curcumin in modulating the genetic expression of cell markers and to understand the effectiveness of this nutraceutical in modulating the regression of cancer phenotype. As a template we used the TPC-1 cells treated with the different extracts of turmeric, and examined the levels of expression of different markers (proliferative, inflammatory, antioxidant, apoptotic). Treatment with the three different curcumin extracts displays anti-inflammatory, antioxidant properties and it is able to influence cell cycle with slightly different effects upon the extracts. Furthermore curcumin is able to influence cell metabolic activity vitality. In conclusion curcumin has the potential to be developed as a safe therapeutic but further studies are needed to verify its antitumor ability in vivo.

Stem Cells and the Pathogenesis of Endometriosis

PubMed Central

Sasson, Isaac E.; Taylor, Hugh S.

2011-01-01

Endometriosis is a common gynecological disorder that is defined by the presence of endometrial tissue outside the uterine cavity. This disease often results in extensive morbidity, including chronic pelvic pain and infertility. The pathogenesis of endometriosis is likely multifactorial, and extensive investigation has explored the role of genetics, environmental factors, and the immune system in predisposing patients to developing endometriosis. A series of recent publications have described the identification of endometrial stem/progenitor cells. Such cells have long been speculated to function in the cyclic regeneration of the endometrium during the menstrual cycle and in the pathogenesis of several gynecological disorders. This narrative review will (i) examine the evidence for endometrial stem cells, (ii) examine their potential role in the pathogenesis of endometriosis, and (iii) identify important unanswered questions with suggestions for future investigation. PMID:18443337

Arraying proteins by cell-free synthesis.

PubMed

He, Mingyue; Wang, Ming-Wei

2007-10-01

Recent advances in life science have led to great motivation for the development of protein arrays to study functions of genome-encoded proteins. While traditional cell-based methods have been commonly used for generating protein arrays, they are usually a time-consuming process with a number of technical challenges. Cell-free protein synthesis offers an attractive system for making protein arrays, not only does it rapidly converts the genetic information into functional proteins without the need for DNA cloning, but also presents a flexible environment amenable to production of folded proteins or proteins with defined modifications. Recent advancements have made it possible to rapidly generate protein arrays from PCR DNA templates through parallel on-chip protein synthesis. This article reviews current cell-free protein array technologies and their proteomic applications.

Locally disordered methylation forms the basis of intratumor methylome variation in chronic lymphocytic leukemia.

PubMed

Landau, Dan A; Clement, Kendell; Ziller, Michael J; Boyle, Patrick; Fan, Jean; Gu, Hongcang; Stevenson, Kristen; Sougnez, Carrie; Wang, Lili; Li, Shuqiang; Kotliar, Dylan; Zhang, Wandi; Ghandi, Mahmoud; Garraway, Levi; Fernandes, Stacey M; Livak, Kenneth J; Gabriel, Stacey; Gnirke, Andreas; Lander, Eric S; Brown, Jennifer R; Neuberg, Donna; Kharchenko, Peter V; Hacohen, Nir; Getz, Gad; Meissner, Alexander; Wu, Catherine J

2014-12-08

Intratumoral heterogeneity plays a critical role in tumor evolution. To define the contribution of DNA methylation to heterogeneity within tumors, we performed genome-scale bisulfite sequencing of 104 primary chronic lymphocytic leukemias (CLLs). Compared with 26 normal B cell samples, CLLs consistently displayed higher intrasample variability of DNA methylation patterns across the genome, which appears to arise from stochastically disordered methylation in malignant cells. Transcriptome analysis of bulk and single CLL cells revealed that methylation disorder was linked to low-level expression. Disordered methylation was further associated with adverse clinical outcome. We therefore propose that disordered methylation plays a similar role to that of genetic instability, enhancing the ability of cancer cells to search for superior evolutionary trajectories. Copyright © 2014 Elsevier Inc. All rights reserved.

Locally disordered methylation forms the basis of intra-tumor methylome variation in chronic lymphocytic leukemia

PubMed Central

Landau, Dan A.; Clement, Kendell; Ziller, Michael J.; Boyle, Patrick; Fan, Jean; Gu, Hongcang; Stevenson, Kristen; Sougnez, Carrie; Wang, Lili; Li, Shuqiang; Kotliar, Dylan; Zhang, Wandi; Ghandi, Mahmoud; Garraway, Levi; Fernandes, Stacey M.; Livak, Kenneth J.; Gabriel, Stacey; Gnirke, Andreas; Lander, Eric S.; Brown, Jennifer R.; Neuberg, Donna; Kharchenko, Peter V.; Hacohen, Nir; Getz, Gad; Meissner, Alexander; Wu, Catherine J.

2014-01-01

SUMMARY Intra-tumoral heterogeneity plays a critical role in tumor evolution. To define the contribution of DNA methylation to heterogeneity within tumors, we performed genome-scale bisulfite sequencing of 104 primary chronic lymphocytic leukemias (CLL). Compared to 26 normal B cell samples, CLLs consistently displayed higher intra-sample variability of DNA methylation patterns across the genome, which appears to arise from stochastically disordered methylation in malignant cells. Transcriptome analysis of bulk and single CLL cells revealed that methylation disorder was linked to low-level expression. Disordered methylation was further associated with adverse clinical outcome. We therefore propose that disordered methylation plays a similar role to genetic instability, enhancing the ability of cancer cells to search for superior evolutionary trajectories. PMID:25490447

The genomic landscape of acute lymphoblastic leukemia in children and young adults.

PubMed

Mullighan, Charles G

2014-12-05

Our understanding of the genetic basis of childhood acute lymphoblastic leukemia (ALL) has been greatly advanced by genomic profiling and sequencing studies. These efforts have characterized the genetic basis of recently described and poorly understood subtypes of ALL, including early T-cell precursor ALL, Philadelphia chromosome-like (Ph-like) ALL, and ALL with intrachromosomal amplification of chromosome 21, and have identified several rational therapeutic targets in high-risk ALL, notably ABL1-class and JAK-STAT inhibitors in Ph-like ALL. Deep sequencing studies are also refining our understanding of the genetic basis of clonal heterogeneity and relapse. These studies have elucidated the nature of clonal evolution during disease progression and identified genetic changes that confer resistance to specific therapeutic agents, including CREBBP and NT5C2. Genomic profiling has also identified common and rare inherited genetic variants that influence the risk of developing leukemia. These efforts are now being extended to ALL in adolescents and adults with the goal of fully defining the genetic landscape of ALL to further improve treatment outcomes in high-risk populations. © 2014 by The American Society of Hematology. All rights reserved.

Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines.

PubMed

Rahman, Maryam; Reyner, Karina; Deleyrolle, Loic; Millette, Sebastien; Azari, Hassan; Day, Bryan W; Stringer, Brett W; Boyd, Andrew W; Johns, Terrance G; Blot, Vincent; Duggal, Rohit; Reynolds, Brent A

2015-03-01

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture.

Neurosphere and adherent culture conditions are equivalent for malignant glioma stem cell lines

PubMed Central

Reyner, Karina; Deleyrolle, Loic; Millette, Sebastien; Azari, Hassan; Day, Bryan W.; Stringer, Brett W.; Boyd, Andrew W.; Johns, Terrance G.; Blot, Vincent; Duggal, Rohit; Reynolds, Brent A.

2015-01-01

Certain limitations of the neurosphere assay (NSA) have resulted in a search for alternative culture techniques for brain tumor-initiating cells (TICs). Recently, reports have described growing glioblastoma (GBM) TICs as a monolayer using laminin. We performed a side-by-side analysis of the NSA and laminin (adherent) culture conditions to compare the growth and expansion of GBM TICs. GBM cells were grown using the NSA and adherent culture conditions. Comparisons were made using growth in culture, apoptosis assays, protein expression, limiting dilution clonal frequency assay, genetic affymetrix analysis, and tumorigenicity in vivo. In vitro expansion curves for the NSA and adherent culture conditions were virtually identical (P=0.24) and the clonogenic frequencies (5.2% for NSA vs. 5.0% for laminin, P=0.9) were similar as well. Likewise, markers of differentiation (glial fibrillary acidic protein and beta tubulin III) and proliferation (Ki67 and MCM2) revealed no statistical difference between the sphere and attachment methods. Several different methods were used to determine the numbers of dead or dying cells (trypan blue, DiIC, caspase-3, and annexin V) with none of the assays noting a meaningful variance between the two methods. In addition, genetic expression analysis with microarrays revealed no significant differences between the two groups. Finally, glioma cells derived from both methods of expansion formed large invasive tumors exhibiting GBM features when implanted in immune-compromised animals. A detailed functional, protein and genetic characterization of human GBM cells cultured in serum-free defined conditions demonstrated no statistically meaningful differences when grown using sphere (NSA) or adherent conditions. Hence, both methods are functionally equivalent and remain suitable options for expanding primary high-grade gliomas in tissue culture. PMID:25806119

Mapping genetic vulnerabilities reveals BTK as a novel therapeutic target in oesophageal cancer.

PubMed

Chong, Irene Yushing; Aronson, Lauren; Bryant, Hanna; Gulati, Aditi; Campbell, James; Elliott, Richard; Pettitt, Stephen; Wilkerson, Paul; Lambros, Maryou B; Reis-Filho, Jorge S; Ramessur, Anisha; Davidson, Michael; Chau, Ian; Cunningham, David; Ashworth, Alan; Lord, Christopher J

2017-08-22

Oesophageal cancer is the seventh most common cause of cancer-related death worldwide. Disease relapse is frequent and treatment options are limited. To identify new biomarker-defined therapeutic approaches for patients with oesophageal cancer, we integrated the genomic profiles of 17 oesophageal tumour-derived cell lines with drug sensitivity data from small molecule inhibitor profiling, identifying drug sensitivity effects associated with cancer driver gene alterations. We also interrogated recently described RNA interference screen data for these tumour cell lines to identify candidate genetic dependencies or vulnerabilities that could be exploited as therapeutic targets. By integrating the genomic features of oesophageal tumour cell lines with siRNA and drug screening data, we identified a series of candidate targets in oesophageal cancer, including a sensitivity to inhibition of the kinase BTK in MYC amplified oesophageal tumour cell lines. We found that this genetic dependency could be elicited with the clinical BTK/ERBB2 kinase inhibitor, ibrutinib. In both MYC and ERBB2 amplified tumour cells, ibrutinib downregulated ERK-mediated signal transduction, cMYC Ser-62 phosphorylation and levels of MYC protein, and elicited G 1 cell cycle arrest and apoptosis, suggesting that this drug could be used to treat biomarker-selected groups of patients with oesophageal cancer. BTK represents a novel candidate therapeutic target in oesophageal cancer that can be targeted with ibrutinib. On the basis of this work, a proof-of-concept phase II clinical trial evaluating the efficacy of ibrutinib in patients with MYC and/or ERBB2 amplified advanced oesophageal cancer is currently underway (NCT02884453). NCT02884453; Pre-results. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

A genetic screen in Myxococcus xanthus identifies mutants that uncouple outer membrane exchange from a downstream cellular response.

PubMed

Dey, Arup; Wall, Daniel

2014-12-01

Upon physical contact with sibling cells, myxobacteria transiently fuse their outer membranes (OMs) and exchange OM proteins and lipids. From previous work, TraA and TraB were identified to be essential factors for OM exchange (OME) in donor and recipient cells. To define the genetic complexity of OME, we carried out a comprehensive forward genetic screen. The screen was based on the observation that Myxococcus xanthus nonmotile cells, by a Tra-dependent mechanism, block swarm expansion of motile cells when mixed. Thus, mutants defective in OME or a downstream responsive pathway were readily identified as escape flares from mixed inocula seeded on agar. This screen was surprisingly powerful, as we found >50 mutants defective in OME. Importantly, all of the mutations mapped to the traAB operon, suggesting that there may be few, if any, proteins besides TraA and TraB directly required for OME. We also found a second and phenotypically different class of mutants that exhibited wild-type OME but were defective in a responsive pathway. This pathway is postulated to control inner membrane homeostasis by covalently attaching amino acids to phospholipids. The identified proteins are homologous to the Staphylococcus aureus MprF protein, which is involved in membrane adaptation and antibiotic resistance. Interestingly, we also found that a small number of nonmotile cells were sufficient to block the swarming behavior of a large gliding-proficient population. This result suggests that an OME-derived signal could be amplified from a few nonmotile producers to act on many responder cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Auxin minimum triggers the developmental switch from cell division to cell differentiation in the Arabidopsis root

PubMed Central

De Ruvo, Micol; Pacifici, Elena; Salvi, Elena; Sozzani, Rosangela; Benfey, Philip N.; Di Paola, Luisa; Marée, Athanasius F. M.; Costantino, Paolo; Grieneisen, Verônica A.; Sabatini, Sabrina

2017-01-01

In multicellular organisms, a stringent control of the transition between cell division and differentiation is crucial for correct tissue and organ development. In the Arabidopsis root, the boundary between dividing and differentiating cells is positioned by the antagonistic interaction of the hormones auxin and cytokinin. Cytokinin affects polar auxin transport, but how this impacts the positional information required to establish this tissue boundary, is still unknown. By combining computational modeling with molecular genetics, we show that boundary formation is dependent on cytokinin’s control on auxin polar transport and degradation. The regulation of both processes shapes the auxin profile in a well-defined auxin minimum. This auxin minimum positions the boundary between dividing and differentiating cells, acting as a trigger for this developmental transition, thus controlling meristem size. PMID:28831001

Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis.

PubMed

Byrne, M E; Barley, R; Curtis, M; Arroyo, J M; Dunham, M; Hudson, A; Martienssen, R A

Meristem function in plants requires both the maintenance of stem cells and the specification of founder cells from which lateral organs arise. Lateral organs are patterned along proximodistal, dorsoventral and mediolateral axes. Here we show that the Arabidopsis mutant asymmetric leaves1 (as1) disrupts this process. AS1 encodes a myb domain protein, closely related to PHANTASTICA in Antirrhinum and ROUGH SHEATH2 in maize, both of which negatively regulate knotted-class homeobox genes. AS1 negatively regulates the homeobox genes KNAT1 and KNAT2 and is, in turn, negatively regulated by the meristematic homeobox gene SHOOT MERISTEMLESS. This genetic pathway defines a mechanism for differentiating between stem cells and organ founder cells within the shoot apical meristem and demonstrates that genes expressed in organ primordia interact with meristematic genes to regulate shoot morphogenesis.

Cells of origin in the embryonic nerve roots for NF1-associated plexiform neurofibroma

PubMed Central

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

2014-01-01

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

Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells

PubMed Central

Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

2013-01-01

Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001 PMID:24252877

Expansion and conversion of human pancreatic ductal cells into insulin-secreting endocrine cells.

PubMed

Lee, Jonghyeob; Sugiyama, Takuya; Liu, Yinghua; Wang, Jing; Gu, Xueying; Lei, Ji; Markmann, James F; Miyazaki, Satsuki; Miyazaki, Jun-Ichi; Szot, Gregory L; Bottino, Rita; Kim, Seung K

2013-11-19

Pancreatic islet β-cell insufficiency underlies pathogenesis of diabetes mellitus; thus, functional β-cell replacement from renewable sources is the focus of intensive worldwide effort. However, in vitro production of progeny that secrete insulin in response to physiological cues from primary human cells has proven elusive. Here we describe fractionation, expansion and conversion of primary adult human pancreatic ductal cells into progeny resembling native β-cells. FACS-sorted adult human ductal cells clonally expanded as spheres in culture, while retaining ductal characteristics. Expression of the cardinal islet developmental regulators Neurog3, MafA, Pdx1 and Pax6 converted exocrine duct cells into endocrine progeny with hallmark β-cell properties, including the ability to synthesize, process and store insulin, and secrete it in response to glucose or other depolarizing stimuli. These studies provide evidence that genetic reprogramming of expandable human pancreatic cells with defined factors may serve as a general strategy for islet replacement in diabetes. DOI: http://dx.doi.org/10.7554/eLife.00940.001.

Prolyl isomerase Pin1 acts downstream of miR-200 to promote cancer stem-like cell traits in breast cancer

PubMed Central

Luo, Man-Li; Gong, Chang; Chen, Chun-Hau; Lee, Daniel Y.; Hu, Hai; Huang, Pengyu; Yao, Yandan; Guo, Wenjun; Reinhardt, Ferenc; Wulf, Gerburg; Lieberman, Judy; Zhou, Xiao Zhen; Song, Erwei; Lu, Kun Ping

2014-01-01

Breast cancer stem-like cells (BCSC) have been implicated in tumor growth, metastasis, drug resistance and relapse but druggable targets in appropriate subsets of this cell population have yet to be identified. Here we identify a fundamental role for the prolyl isomerase Pin1 in driving BCSC expansion, invasiveness and tumorigenicity, defining it as a key target of miR-200c which is known to be a critical regluator in BSCS. Pin1 overexpression expanded the growth and tumorigenicity of BCSC and triggered epithelial-mesenchymal transition (EMT). Conversely, genetic or pharmaacological inhibition of Pin1 reduced the abundance and self-renewal activity of BCSC. Moreover, moderate overexpression of miR-200c-resistant Pin1 rescued the BCSC defect in miR-200c-expressing cells. Genetic deletion of Pin1 also decreased the abundance and repopulating capability of normal mouse mammary stem cells. In human cells freshly isolated from reduction mammoplasty tissues, Pin1 overexpression endowed BCSC traits to normal breast epithelial cells, expanding both luminal and basal/myoepithelial lineages in these cells. In contrast, Pin1 silencing in primary breast cancer cells isolated from clinical samples inhibited the expansion, self-renewal activity and tumorigenesis of BCSC in vitro and in vivo. Overall, our work demonstrated that Pin1 is a pivotal regulator acting downstream of miR-200c to drive BCSC and breast tumorigenicity, highlighting a new therapeutic target to eradicate BCSC. PMID:24786790

Physical and functional mapping of a tumor suppressor locus for renal cell carcinoma within chromosome 3p12.

PubMed

Lott, S T; Lovell, M; Naylor, S L; Killary, A M

1998-08-15

Using a functional genetic approach, we previously identified a novel genetic locus, NRC-1 (Nonpapillary Renal Cell Carcinoma 1), that mediated tumor suppression and rapid cell death of renal cell carcinoma (RCC) cells in vivo. For these experiments, a defined subchromosomal fragment of human chromosome 3p was transferred into a sporadic RCC cell line via microcell fusion, and microcell hybrid clones were tested for tumorigenicity in vivo. The results indicated functional evidence for a novel tumor suppressor locus within the 3p14-p12 interval known to contain the most common fragile site of the human genome (FRA3B), the FHIT gene, and the breakpoint region associated with the familial form of RCC. We now report the physical mapping of the NRC-1 critical region by detailed microsatellite analyses of novel microcell hybrid clones containing transferred fragments of chromosome 3p in the RCC cell background that were phenotypically suppressed or unsuppressed for tumorigenicity in vivo. The results limit the region containing the tumor suppressor locus within chromosome 3p12. The FHIT gene, FRA3B, and the familial RCC breakpoint region were excluded from the NRC-1 critical region. Furthermore, the NRC-1 locus falls within a well-characterized homozygous deletion region of 5-7 Mb associated with a small cell lung carcinoma cell line, U2020, suggesting that a more general tumor suppressor gene may reside in this region.

Recent biologic and genetic advances in neuroblastoma: Implications for diagnostic, risk stratification, and treatment strategies.

PubMed

Newman, Erika A; Nuchtern, Jed G

2016-10-01

Neuroblastoma is an embryonic cancer of neural crest cell lineage, accounting for up to 10% of all pediatric cancer. The clinical course is heterogeneous ranging from spontaneous regression in neonates to life-threatening metastatic disease in older children. Much of this clinical variance is thought to result from distinct pathologic characteristics that predict patient outcomes. Consequently, many research efforts have been focused on identifying the underlying biologic and genetic features of neuroblastoma tumors in order to more clearly define prognostic subgroups for treatment stratification. Recent technological advances have placed emphasis on the integration of genetic alterations and predictive biologic variables into targeted treatment approaches to improve patient survival outcomes. This review will focus on these recent advances and the implications they have on the diagnostic, staging, and treatment approaches in modern neuroblastoma clinical management. Copyright © 2016 Elsevier Inc. All rights reserved.

Decoding the genome beyond sequencing: the new phase of genomic research.

PubMed

Heng, Henry H Q; Liu, Guo; Stevens, Joshua B; Bremer, Steven W; Ye, Karen J; Abdallah, Batoul Y; Horne, Steven D; Ye, Christine J

2011-10-01

While our understanding of gene-based biology has greatly improved, it is clear that the function of the genome and most diseases cannot be fully explained by genes and other regulatory elements. Genes and the genome represent distinct levels of genetic organization with their own coding systems; Genes code parts like protein and RNA, but the genome codes the structure of genetic networks, which are defined by the whole set of genes, chromosomes and their topological interactions within a cell. Accordingly, the genetic code of DNA offers limited understanding of genome functions. In this perspective, we introduce the genome theory which calls for the departure of gene-centric genomic research. To make this transition for the next phase of genomic research, it is essential to acknowledge the importance of new genome-based biological concepts and to establish new technology platforms to decode the genome beyond sequencing. Copyright © 2011 Elsevier Inc. All rights reserved.

The Chern-Simons Current in Systems of DNA-RNA Transcriptions

NASA Astrophysics Data System (ADS)

Capozziello, Salvatore; Pincak, Richard; Kanjamapornkul, Kabin; Saridakis, Emmanuel N.

2018-04-01

A Chern-Simons current, coming from ghost and anti-ghost fields of supersymmetry theory, can be used to define a spectrum of gene expression in new time series data where a spinor field, as alternative representation of a gene, is adopted instead of using the standard alphabet sequence of bases $A, T, C, G, U$. After a general discussion on the use of supersymmetry in biological systems, we give examples of the use of supersymmetry for living organism, discuss the codon and anti-codon ghost fields and develop an algebraic construction for the trash DNA, the DNA area which does not seem active in biological systems. As a general result, all hidden states of codon can be computed by Chern-Simons 3 forms. Finally, we plot a time series of genetic variations of viral glycoprotein gene and host T-cell receptor gene by using a gene tensor correlation network related to the Chern-Simons current. An empirical analysis of genetic shift, in host cell receptor genes with separated cluster of gene and genetic drift in viral gene, is obtained by using a tensor correlation plot over time series data derived as the empirical mode decomposition of Chern-Simons current.

Novel Multiplex Fluorescent PCR-Based Method for HLA Typing and Preimplantational Genetic Diagnosis of β-Thalassemia.

PubMed

Khosravi, Sharifeh; Salehi, Mansour; Ramezanzadeh, Mahboobeh; Mirzaei, Hamed; Salehi, Rasoul

2016-05-01

Thalassemia is curable by bone marrow transplantation; however, finding suitable donors with defined HLA combination remains a major challenge. Cord blood stem cells with preselected HLA system through preimplantation genetic diagnosis (PGD) proved very useful for resolving scarce HLA-matched bone marrow donors. A thalassemia trait couple with an affected child was included in this study. We used informative STR markers at the HLA and beta globin loci to develop a single cell multiplex fluorescent PCR protocol. The protocol was extensively optimized on single lymphocytes isolated from the couple's peripheral blood. The optimized protocol was applied on single blastomeres biopsied from day 3 cleavage stage IVF embryos of the couple. Four IVF embryos biopsied on day 3 and a single blastomere of each were provided for genetic diagnosis of combined β-thalassemia mutations and HLA typing. Of these, one embryo was diagnosed as homozygous normal for the thalassemia mutation and HLA matched with the existing affected sibling. The optimized protocol worked well in PGD clinical cycle for selection of thalassemia-unaffected embryos with the desired HLA system. Copyright © 2016 IMSS. Published by Elsevier Inc. All rights reserved.

Rare cancers: a sea of opportunity.

PubMed

Boyd, Niki; Dancey, Janet E; Gilks, C Blake; Huntsman, David G

2016-02-01

Rare cancers, as a collective, account for around a quarter of all cancer diagnoses and deaths. Historically, they have been divided into two groups: cancers defined by their unusual histogenesis (cell of origin or differentiation state)--including chordomas or adult granulosa cell tumours--and histologically defined subtypes of common cancers. Most tumour types in the first group are still clinically and biologically relevant, and have been disproportionately important as sources of insight into cancer biology. By contrast, most of those in the second group have been shown to have neither defining molecular features nor clinical utility. Omics-based analyses have splintered common cancers into a myriad of molecularly, rather than histologically, defined subsets of common cancers, many of which have immediate clinical relevance. Now, almost all rare cancers are either histomolecular entities, which often have pathognomonic mutations, or molecularly defined subsets of more common cancers. The presence of specific genetic variants provides rationale for the testing of targeted drugs in rare cancers. However, in addition to molecular alterations, it is crucial to consider the contributions of both mutation and cell context in the development, biology, and behaviour of these cancers. Patients with rare cancers are disadvantaged because of the challenge of leading clinical trials in this setting due to poor accrual. However, the number of patients with rare cancers will only increase as more molecular subsets of common cancers are identified, necessitating a shift in the focus of clinical trials and research into these cancer types, which, by epidemiological definitions, will become rare tumours. Copyright © 2016 Elsevier Ltd. All rights reserved.

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy

PubMed Central

Etheridge, Thomas J.; Boulineau, Rémi L.; Herbert, Alex; Watson, Adam T.; Daigaku, Yasukazu; Tucker, Jem; George, Sophie; Jönsson, Peter; Palayret, Matthieu; Lando, David; Laue, Ernest; Osborne, Mark A.; Klenerman, David; Lee, Steven F.; Carr, Antony M.

2014-01-01

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds. PMID:25106872

Notch1 acts via Foxc2 to promote definitive hematopoiesis via effects on hemogenic endothelium

PubMed Central

Jang, Il Ho; Lu, Yi-Fen; Zhao, Long; Wenzel, Pamela L.; Kume, Tsutomu; Datta, Sumon M.; Arora, Natasha; Guiu, Jordi; Lagha, Mounia; Kim, Peter G.; Do, Eun Kyoung; Kim, Jae Ho; Schlaeger, Thorsten M.; Zon, Leonard I.; Bigas, Anna; Burns, Caroline E.

2015-01-01

Hematopoietic and vascular development share many common features, including cell surface markers and sites of origin. Recent lineage-tracing studies have established that definitive hematopoietic stem and progenitor cells arise from vascular endothelial–cadherin+ hemogenic endothelial cells of the aorta-gonad-mesonephros region, but the genetic programs underlying the specification of hemogenic endothelial cells remain poorly defined. Here, we discovered that Notch induction enhances hematopoietic potential and promotes the specification of hemogenic endothelium in differentiating cultures of mouse embryonic stem cells, and we identified Foxc2 as a highly upregulated transcript in the hemogenic endothelial population. Studies in zebrafish and mouse embryos revealed that Foxc2 and its orthologs are required for the proper development of definitive hematopoiesis and function downstream of Notch signaling in the hemogenic endothelium. These data establish a pathway linking Notch signaling to Foxc2 in hemogenic endothelial cells to promote definitive hematopoiesis. PMID:25587036

Definition of MYC genetic heteroclonality in diffuse large B-cell lymphoma with 8q24 rearrangement and its impact on protein expression.

PubMed

Valera, Alexandra; Epistolio, Samantha; Colomo, Lluis; Riva, Alice; Balagué, Olga; Dlouhy, Ivan; Tzankov, Alexandar; Bühler, Marco; Haralambieva, Eugenia; Campo, Elias; Soldini, Davide; Mazzucchelli, Luca; Martin, Vittoria

2016-08-01

MYC rearrangement can be detected in a subgroup of diffuse large B-cell lymphoma characterized by unfavorable prognosis. In contrast to Burkitt lymphoma, the correlation between MYC rearrangement and MYC protein expression in diffuse large B-cell lymphoma is less clear, as approximately one-third of rearranged cases show negative or low expression by immunohistochemistry. To better understand whether specific characteristics of the MYC rearrangement may influence its protein expression, we investigated 43 de novo diffuse large B-cell lymphoma positive for 8q24 rearrangement by FISH, using 14 Burkitt lymphoma for comparison. Different cell populations (clones), breakpoints (classical vs non-classical FISH patterns), partner genes (IGH vs non-IGH) and immunostaining were detected and analyzed using computerized image systems. In a subgroup of diffuse large B-cell lymphoma, we observed different clones within the same tumor distinguishing the founder clone with MYC rearrangement alone from other subclones, carrying MYC rearrangement coupled with loss/extra copies of derivatives/normal alleles. This picture, which we defined MYC genetic heteroclonality, was found in 42% of cases and correlated to negative MYC expression (P=0.026). Non-classical FISH breakpoints were detected in 16% of diffuse large B-cell lymphoma without affecting expression (P=0.040). Non-IGH gene was the preferential partner of rearrangement in those diffuse large B-cell lymphoma showing MYC heteroclonality (P=0.016) and/or non-classical FISH breakpoints (P=0.058). MYC heteroclonality was not observed in Burkitt lymphoma and all cases had positive MYC expression. Non-classical FISH MYC breakpoint and non-IGH partner were found in 29 and 20% of Burkitt lymphoma, respectively. In conclusion, MYC genetic heteroclonality is a frequent event in diffuse large B-cell lymphoma and may have a relevant role in modulating MYC expression.

Integument pattern formation involves genetic and epigenetic controls: feather arrays simulated by digital hormone models

PubMed Central

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

2015-01-01

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

The 19q12 bladder cancer GWAS signal: association with cyclin E function and aggressive disease

PubMed Central

Fu, Yi-Ping; Kohaar, Indu; Moore, Lee E.; Lenz, Petra; Figueroa, Jonine D.; Tang, Wei; Porter-Gill, Patricia; Chatterjee, Nilanjan; Scott-Johnson, Alexandra; Garcia-Closas, Montserrat; Muchmore, Brian; Baris, Dalsu; Paquin, Ashley; Ylaya, Kris; Schwenn, Molly; Apolo, Andrea B.; Karagas, Margaret R.; Tarway, McAnthony; Johnson, Alison; Mumy, Adam; Schned, Alan; Guedez, Liliana; Jones, Michael A.; Kida, Masatoshi; Monawar Hosain, GM; Malats, Nuria; Kogevinas, Manolis; Tardon, Adonina; Serra, Consol; Carrato, Alfredo; Garcia-Closas, Reina; Lloreta, Josep; Wu, Xifeng; Purdue, Mark; Andriole, Gerald L.; Grubb, Robert L.; Black, Amanda; Landi, Maria T.; Caporaso, Neil E.; Vineis, Paolo; Siddiq, Afshan; Bueno-de-Mesquita, H. Bas; Trichopoulos, Dimitrios; Ljungberg, Börje; Severi, Gianluca; Weiderpass, Elisabete; Krogh, Vittorio; Dorronsoro, Miren; Travis, Ruth C.; Tjønneland, Anne; Brennan, Paul; Chang-Claude, Jenny; Riboli, Elio; Prescott, Jennifer; Chen, Constance; De Vivo, Immaculata; Govannucci, Edward; Hunter, David; Kraft, Peter; Lindstrom, Sara; Gapstur, Susan M.; Jacobs, Eric J.; Diver, W. Ryan; Albanes, Demetrius; Weinstein, Stephanie J.; Virtamo, Jarmo; Kooperberg, Charles; Hohensee, Chancellor; Rodabough, Rebecca J.; Cortessis, Victoria K.; Conti, David V.; Gago-Dominguez, Manuela; Stern, Mariana C.; Pike, Malcolm C.; Van Den Berg, David; Yuan, Jian-Min; Haiman, Christopher A.; Cussenot, Olivier; Cancel-Tassin, Geraldine; Roupret, Morgan; Comperat, Eva; Porru, Stefano; Carta, Angela; Pavanello, Sofia; Arici, Cecilia; Mastrangelo, Giuseppe; Grossman, H. Barton; Wang, Zhaoming; Deng, Xiang; Chung, Charles C.; Hutchinson, Amy; Burdette, Laurie; Wheeler, William; Fraumeni, Joseph; Chanock, Stephen J.; Hewitt, Stephen M.; Silverman, Debra T.; Rothman, Nathaniel; Prokunina-Olsson, Ludmila

2014-01-01

A genome-wide association study (GWAS) of bladder cancer identified a genetic marker rs8102137 within the 19q12 region as a novel susceptibility variant. This marker is located upstream of the CCNE1 gene, which encodes cyclin E, a cell cycle protein. We performed genetic fine mapping analysis of the CCNE1 region using data from two bladder cancer GWAS (5,942 cases and 10,857 controls). We found that the original GWAS marker rs8102137 represents a group of 47 linked SNPs (with r2≥0.7) associated with increased bladder cancer risk. From this group we selected a functional promoter variant rs7257330, which showed strong allele-specific binding of nuclear proteins in several cell lines. In both GWAS, rs7257330 was associated only with aggressive bladder cancer, with a combined per-allele odds ratio (OR) =1.18 (95%CI=1.09-1.27, p=4.67×10−5 vs. OR =1.01 (95%CI=0.93-1.10, p=0.79) for non-aggressive disease, with p=0.0015 for case-only analysis. Cyclin E protein expression analyzed in 265 bladder tumors was increased in aggressive tumors (p=0.013) and, independently, with each rs7257330-A risk allele (ptrend=0.024). Over-expression of recombinant cyclin E in cell lines caused significant acceleration of cell cycle. In conclusion, we defined the 19q12 signal as the first GWAS signal specific for aggressive bladder cancer. Molecular mechanisms of this genetic association may be related to cyclin E over-expression and alteration of cell cycle in carriers of CCNE1 risk variants. In combination with established bladder cancer risk factors and other somatic and germline genetic markers, the CCNE1 variants could be useful for inclusion into bladder cancer risk prediction models. PMID:25320178

Gene expression signature of tolerance and lymphocyte subsets in stable renal transplants: results of a cross-sectional study.

PubMed

Moreso, Francesc; Torres, Irina B; Martínez-Gallo, Monica; Benlloch, Susana; Cantarell, Carme; Perelló, Manel; Jimeno, José; Pujol-Borrell, Ricardo; Seron, Daniel

2014-06-01

In kidney transplants operational tolerance has been associated with up-regulation of B cell differentiation genes and an increased number of total, naive and transitional peripheral B cells. The aim is to evaluate tolerance biomarkers in different cohorts of stable renal transplants under immunosuppression. This is a cross-sectional study conducted in renal transplants. We evaluate genetic tolerance signature and lymphocyte subsets in stable transplants treated with calcineurin inhibitors (CNI) at 1 (n=15), 5 (n=14) and 10 (n=16) years, and azathioprine-treated transplants followed 30 years (n=8). Healthy volunteers (n=10) and patients with chronic rejection (n=15) served as controls. We confirm that peripheral expression of IGKV1D-13 and IGKV4-1 genes by RT-PCR distinguish tolerant (n=10) from stable transplants (n=10) provided by the International Tolerance Network. Tolerance signature was defined as the lowest expression for both genes in tolerant patients. In CNI-treated patients, genetic signature of tolerance and B cells showed a time-dependent increase not observed in azathioprine-treated patients (p<0.01). Genetic tolerance signature was observed in 0% at 1, 7% at 5 and 25% at 10-years while it was not observed in azathioprine-treated and chronic rejection patients. Fifteen out of 16 CNI-treated transplants at 10 years were revaluated 3 months apart. Nine did not show the tolerance signature in any determination, 4 in one and 2 in both determinations. Genetic signature of tolerance was associated with an increase of total, naive and transitional B cells (p<0.05). IGKV1D-13 and IGKV4-1 gene expression and its linked B cell populations increase during follow up in CNI-treated patients. At 10 years, 2 out of 15 CNI treated patients consistently express biomarkers associated with true tolerance. In azathioprine-treated patients these biomarkers were down-regulated. Copyright © 2014 Elsevier B.V. All rights reserved.

CLO: The cell line ontology

PubMed Central

2014-01-01

Background Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. Construction and content Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. Utility and discussion The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development. PMID:25852852

B cells in operational tolerance.

PubMed

Chesneau, M; Danger, R; Soulillou, J-P; Brouard, S

2018-05-01

Transplantation is currently the therapy of choice for endstage organ failure even though it requires long-term immunosuppresive therapy, with its numerous side effects, for acceptance of the transplanted organ. In rare cases however, patients develop operational tolerance, that is, graft survival without immunosuppression. Studies conducted on these patients reveal genetic, phenotypic, and functional signatures. They provide a better understanding of the immunological mechanisms involved in operational tolerance and define biomarkers that could be used to adapt immunosuppressive treatment to the individual, safely reduce immunosuppression doses, and ideally and safely guide immunosuppression withdrawal. This review summarizes studies that suggest a role for B cells as biomarkers of operational tolerance and discusses the use of B cells as a predictive tool for immunologic risk. Copyright © 2018. Published by Elsevier Inc.

Three-Dimensional Transgenic Cell Models to Quantify Space Genotoxic Effects

NASA Technical Reports Server (NTRS)

Gonda, S.; Wu, H.; Pingerelli, P.; Glickman, B.

2000-01-01

In this paper we describe a three-dimensional, multicellular tissue-equivalent model, produced in NASA-designed, rotating wall bioreactors using mammalian cells engineered for genomic containment of mUltiple copies of defined target genes for genotoxic assessment. The Rat 2(lambda) fibroblasts (Stratagene, Inc.) were genetically engineered to contain high-density target genes for mutagenesis. Stable three-dimensional, multicellular spheroids were formed when human mammary epithelial cells and Rat 2(lambda) fibroblasts were cocultured on Cytodex 3 Beads in a rotating wall bioreactor. The utility of this spheroidal model for genotoxic assessment was indicated by a linear dose response curve and by results of gene sequence analysis of mutant clones from 400micron diameter spheroids following low-dose, high-energy, neon radiation exposure

Satellite DNA-based artificial chromosomes for use in gene therapy.

PubMed

Hadlaczky, G

2001-04-01

Satellite DNA-based artificial chromosomes (SATACs) can be made by induced de novo chromosome formation in cells of different mammalian species. These artificially generated accessory chromosomes are composed of predictable DNA sequences and they contain defined genetic information. Prototype human SATACs have been successfully constructed in different cell types from 'neutral' endogenous DNA sequences from the short arm of the human chromosome 15. SATACs have already passed a number of hurdles crucial to their further development as gene therapy vectors, including: large-scale purification; transfer of purified artificial chromosomes into different cells and embryos; generation of transgenic animals and germline transmission with purified SATACs; and the tissue-specific expression of a therapeutic gene from an artificial chromosome in the milk of transgenic animals.

Genomic architecture and evolution of clear cell renal cell carcinomas defined by multiregion sequencing

PubMed Central

Varela, Ignacio; Fisher, Rosalie; McGranahan, Nicholas; Matthews, Nicholas; Santos, Claudio R; Martinez, Pierre; Phillimore, Benjamin; Begum, Sharmin; Rabinowitz, Adam; Spencer-Dene, Bradley; Gulati, Sakshi; Bates, Paul A; Stamp, Gordon; Pickering, Lisa; Gore, Martin; Nicol, David L; Hazell, Steven; Futreal, P Andrew; Stewart, Aengus; Swanton, Charles

2015-01-01

Clear cell renal carcinomas (ccRCCs) can display intratumor heterogeneity (ITH). We applied multiregion exome sequencing (M-seq) to resolve the genetic architecture and evolutionary histories of ten ccRCCs. Ultra-deep sequencing identified ITH in all cases. We found that 73–75% of identified ccRCC driver aberrations were subclonal, confounding estimates of driver mutation prevalence. ITH increased with the number of biopsies analyzed, without evidence of saturation in most tumors. Chromosome 3p loss and VHL aberrations were the only ubiquitous events. The proportion of C>T transitions at CpG sites increased during tumor progression. M-seq permits the temporal resolution of ccRCC evolution and refines mutational signatures occurring during tumor development. PMID:24487277

Computational power and generative capacity of genetic systems.

PubMed

Igamberdiev, Abir U; Shklovskiy-Kordi, Nikita E

2016-01-01

Semiotic characteristics of genetic sequences are based on the general principles of linguistics formulated by Ferdinand de Saussure, such as the arbitrariness of sign and the linear nature of the signifier. Besides these semiotic features that are attributable to the basic structure of the genetic code, the principle of generativity of genetic language is important for understanding biological transformations. The problem of generativity in genetic systems arises to a possibility of different interpretations of genetic texts, and corresponds to what Alexander von Humboldt called "the infinite use of finite means". These interpretations appear in the individual development as the spatiotemporal sequences of realizations of different textual meanings, as well as the emergence of hyper-textual statements about the text itself, which underlies the process of biological evolution. These interpretations are accomplished at the level of the readout of genetic texts by the structures defined by Efim Liberman as "the molecular computer of cell", which includes DNA, RNA and the corresponding enzymes operating with molecular addresses. The molecular computer performs physically manifested mathematical operations and possesses both reading and writing capacities. Generativity paradoxically resides in the biological computational system as a possibility to incorporate meta-statements about the system, and thus establishes the internal capacity for its evolution. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A method of evolving novel feature extraction algorithms for detecting buried objects in FLIR imagery using genetic programming

NASA Astrophysics Data System (ADS)

Paino, A.; Keller, J.; Popescu, M.; Stone, K.

2014-06-01

In this paper we present an approach that uses Genetic Programming (GP) to evolve novel feature extraction algorithms for greyscale images. Our motivation is to create an automated method of building new feature extraction algorithms for images that are competitive with commonly used human-engineered features, such as Local Binary Pattern (LBP) and Histogram of Oriented Gradients (HOG). The evolved feature extraction algorithms are functions defined over the image space, and each produces a real-valued feature vector of variable length. Each evolved feature extractor breaks up the given image into a set of cells centered on every pixel, performs evolved operations on each cell, and then combines the results of those operations for every cell using an evolved operator. Using this method, the algorithm is flexible enough to reproduce both LBP and HOG features. The dataset we use to train and test our approach consists of a large number of pre-segmented image "chips" taken from a Forward Looking Infrared Imagery (FLIR) camera mounted on the hood of a moving vehicle. The goal is to classify each image chip as either containing or not containing a buried object. To this end, we define the fitness of a candidate solution as the cross-fold validation accuracy of the features generated by said candidate solution when used in conjunction with a Support Vector Machine (SVM) classifier. In order to validate our approach, we compare the classification accuracy of an SVM trained using our evolved features with the accuracy of an SVM trained using mainstream feature extraction algorithms, including LBP and HOG.

DNA Ligase C1 Mediates the LigD-Independent Nonhomologous End-Joining Pathway of Mycobacterium smegmatis

PubMed Central

Bhattarai, Hitesh; Gupta, Richa

2014-01-01

Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3′ phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo. PMID:24957619

Molecular characterization of circulating colorectal tumor cells defines genetic signatures for individualized cancer care.

PubMed

Kong, Say Li; Liu, Xingliang; Suhaimi, Nur-Afidah Mohamed; Koh, Kenneth Jia Hao; Hu, Min; Lee, Daniel Yoke San; Cima, Igor; Phyo, Wai Min; Lee, Esther Xing Wei; Tai, Joyce A; Foong, Yu Miin; Vo, Jess Honganh; Koh, Poh Koon; Zhang, Tong; Ying, Jackie Y; Lim, Bing; Tan, Min-Han; Hillmer, Axel M

2017-09-15

Studies on circulating tumor cells (CTCs) have largely focused on platform development and CTC enumeration rather than on the genomic characterization of CTCs. To address this, we performed targeted sequencing of CTCs of colorectal cancer patients and compared the mutations with the matched primary tumors. We collected preoperative blood and matched primary tumor samples from 48 colorectal cancer patients. CTCs were isolated using a label-free microfiltration device on a silicon microsieve. Upon whole genome amplification, we performed amplicon-based targeted sequencing on a panel of 39 druggable and frequently mutated genes on both CTCs and fresh-frozen tumor samples. We developed an analysis pipeline to minimize false-positive detection of somatic mutations in amplified DNA. In 60% of the CTC-enriched blood samples, we detected primary tumor matching mutations. We found a significant positive correlation between the allele frequencies of somatic mutations detected in CTCs and abnormal CEA serum level. Strikingly, we found driver mutations and amplifications in cancer and druggable genes such as APC, KRAS, TP53, ERBB3 , FBXW7 and ERBB2 . In addition, we found that CTCs carried mutation signatures that resembled the signatures of their primary tumors. Cumulatively, our study defined genetic signatures and somatic mutation frequency of colorectal CTCs. The identification of druggable mutations in CTCs of preoperative colorectal cancer patients could lead to more timely and focused therapeutic interventions.

Molecular characterization of circulating colorectal tumor cells defines genetic signatures for individualized cancer care

PubMed Central

Kong, Say Li; Liu, Xingliang; Suhaimi, Nur-Afidah Mohamed; Koh, Kenneth Jia Hao; Hu, Min; Lee, Daniel Yoke San; Cima, Igor; Phyo, Wai Min; Lee, Esther Xing Wei; Tai, Joyce A.; Foong, Yu Miin; Vo, Jess Honganh; Koh, Poh Koon; Zhang, Tong; Ying, Jackie Y.; Lim, Bing; Tan, Min-Han; Hillmer, Axel M.

2017-01-01

Studies on circulating tumor cells (CTCs) have largely focused on platform development and CTC enumeration rather than on the genomic characterization of CTCs. To address this, we performed targeted sequencing of CTCs of colorectal cancer patients and compared the mutations with the matched primary tumors. We collected preoperative blood and matched primary tumor samples from 48 colorectal cancer patients. CTCs were isolated using a label-free microfiltration device on a silicon microsieve. Upon whole genome amplification, we performed amplicon-based targeted sequencing on a panel of 39 druggable and frequently mutated genes on both CTCs and fresh-frozen tumor samples. We developed an analysis pipeline to minimize false-positive detection of somatic mutations in amplified DNA. In 60% of the CTC-enriched blood samples, we detected primary tumor matching mutations. We found a significant positive correlation between the allele frequencies of somatic mutations detected in CTCs and abnormal CEA serum level. Strikingly, we found driver mutations and amplifications in cancer and druggable genes such as APC, KRAS, TP53, ERBB3, FBXW7 and ERBB2. In addition, we found that CTCs carried mutation signatures that resembled the signatures of their primary tumors. Cumulatively, our study defined genetic signatures and somatic mutation frequency of colorectal CTCs. The identification of druggable mutations in CTCs of preoperative colorectal cancer patients could lead to more timely and focused therapeutic interventions. PMID:28978093

Activation of antioxidant pathways in ras-mediated oncogenic transformation of human surface ovarian epithelial cells revealed by functional proteomics and mass spectrometry.

PubMed

Young, Travis W; Mei, Fang C; Yang, Gong; Thompson-Lanza, Jennifer A; Liu, Jinsong; Cheng, Xiaodong

2004-07-01

Cellular transformation is a complex process involving genetic alterations associated with multiple signaling pathways. Development of a transformation model using defined genetic elements has provided an opportunity to elucidate the role of oncogenes and tumor suppressor genes in the initiation and development of ovarian cancer. To study the cellular and molecular mechanisms of Ras-mediated oncogenic transformation of ovarian epithelial cells, we used a proteomic approach involving two-dimensional electrophoresis and mass spectrometry to profile two ovarian epithelial cell lines, one immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase and the other transformed with an additional oncogenic ras(V12) allele. Of approximately 2200 observed protein spots, we have identified >30 protein targets that showed significant changes between the immortalized and transformed cell lines using peptide mass fingerprinting. Among these identified targets, one most notable group of proteins altered significantly consists of enzymes involved in cellular redox balance. Detailed analysis of these protein targets suggests that activation of Ras-signaling pathways increases the threshold of reactive oxidative species (ROS) tolerance by up-regulating the overall antioxidant capacity of cells, especially in mitochondria. This enhanced antioxidant capacity protects the transformed cells from high levels of ROS associated with the uncontrolled growth potential of tumor cells. It is conceivable that an enhanced antioxidation capability may constitute a common mechanism for tumor cells to evade apoptosis induced by oxidative stresses at high ROS levels.

Leveraging premalignant biology for immune-based cancer prevention.

PubMed

Spira, Avrum; Disis, Mary L; Schiller, John T; Vilar, Eduardo; Rebbeck, Timothy R; Bejar, Rafael; Ideker, Trey; Arts, Janine; Yurgelun, Matthew B; Mesirov, Jill P; Rao, Anjana; Garber, Judy; Jaffee, Elizabeth M; Lippman, Scott M

2016-09-27

Prevention is an essential component of cancer eradication. Next-generation sequencing of cancer genomes and epigenomes has defined large numbers of driver mutations and molecular subgroups, leading to therapeutic advances. By comparison, there is a relative paucity of such knowledge in premalignant neoplasia, which inherently limits the potential to develop precision prevention strategies. Studies on the interplay between germ-line and somatic events have elucidated genetic processes underlying premalignant progression and preventive targets. Emerging data hint at the immune system's ability to intercept premalignancy and prevent cancer. Genetically engineered mouse models have identified mechanisms by which genetic drivers and other somatic alterations recruit inflammatory cells and induce changes in normal cells to create and interact with the premalignant tumor microenvironment to promote oncogenesis and immune evasion. These studies are currently limited to only a few lesion types and patients. In this Perspective, we advocate a large-scale collaborative effort to systematically map the biology of premalignancy and the surrounding cellular response. By bringing together scientists from diverse disciplines (e.g., biochemistry, omics, and computational biology; microbiology, immunology, and medical genetics; engineering, imaging, and synthetic chemistry; and implementation science), we can drive a concerted effort focused on cancer vaccines to reprogram the immune response to prevent, detect, and reject premalignancy. Lynch syndrome, clonal hematopoiesis, and cervical intraepithelial neoplasia which also serve as models for inherited syndromes, blood, and viral premalignancies, are ideal scenarios in which to launch this initiative.

Efficient generation of induced pluripotent stem cells from human bone marrow mesenchymal stem cells.

PubMed

Yulin, X; Lizhen, L; Lifei, Z; Shan, F; Ru, L; Kaimin, H; Huang, H

2012-01-01

Ectopic expression of defined sets of genetic factors can reprogramme somatic cells to induced pluripotent stem cells (iPSCs) that closely resemble embryonic stem cells. However, the low reprogramming efficiency is a significant handicap for mechanistic studies and potential clinical application. In this study, we used human bone marrow-derived mesenchymal stem cells (hBMMSCs) as target cells for reprogramming and investigated efficient iPSC generation from hBMMSCs using the compounds of p53 siRNA, valproic acid (VPA) and vitamin C (Vc) with four transcription factors OCT4, SOX2, KLF4, and c-MYC (compound induction system). The synergetic mechanism of the compounds was studied. Our results showed that the compound induction system could efficiently reprogramme hBMMSCs to iPSCs. hBMMSC-derived iPSC populations expressed pluripotent markers and had multi-potential to differentiate into three germ layer-derived cells. p53 siRNA, VPA and Vc had a synergetic effect on cell reprogramming and the combinatorial use of these substances greatly improved the efficiency of iPSC generation by suppressing the expression of p53, decreasing cell apoptosis, up-regulating the expression of the pluripotent gene OCT4 and modifying the cell cycle. Therefore, our study highlights a straightforward method for improving the speed and efficiency of iPSC generation and provides versatile tools for investigating early developmental processes such as haemopoiesis and relevant diseases. In addition, this study provides a paradigm for the combinatorial use of genetic factors and molecules to improve the efficiency of iPSC generation.

Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function.

PubMed

Pattaro, Cristian; Teumer, Alexander; Gorski, Mathias; Chu, Audrey Y; Li, Man; Mijatovic, Vladan; Garnaas, Maija; Tin, Adrienne; Sorice, Rossella; Li, Yong; Taliun, Daniel; Olden, Matthias; Foster, Meredith; Yang, Qiong; Chen, Ming-Huei; Pers, Tune H; Johnson, Andrew D; Ko, Yi-An; Fuchsberger, Christian; Tayo, Bamidele; Nalls, Michael; Feitosa, Mary F; Isaacs, Aaron; Dehghan, Abbas; d'Adamo, Pio; Adeyemo, Adebowale; Dieffenbach, Aida Karina; Zonderman, Alan B; Nolte, Ilja M; van der Most, Peter J; Wright, Alan F; Shuldiner, Alan R; Morrison, Alanna C; Hofman, Albert; Smith, Albert V; Dreisbach, Albert W; Franke, Andre; Uitterlinden, Andre G; Metspalu, Andres; Tonjes, Anke; Lupo, Antonio; Robino, Antonietta; Johansson, Åsa; Demirkan, Ayse; Kollerits, Barbara; Freedman, Barry I; Ponte, Belen; Oostra, Ben A; Paulweber, Bernhard; Krämer, Bernhard K; Mitchell, Braxton D; Buckley, Brendan M; Peralta, Carmen A; Hayward, Caroline; Helmer, Catherine; Rotimi, Charles N; Shaffer, Christian M; Müller, Christian; Sala, Cinzia; van Duijn, Cornelia M; Saint-Pierre, Aude; Ackermann, Daniel; Shriner, Daniel; Ruggiero, Daniela; Toniolo, Daniela; Lu, Yingchang; Cusi, Daniele; Czamara, Darina; Ellinghaus, David; Siscovick, David S; Ruderfer, Douglas; Gieger, Christian; Grallert, Harald; Rochtchina, Elena; Atkinson, Elizabeth J; Holliday, Elizabeth G; Boerwinkle, Eric; Salvi, Erika; Bottinger, Erwin P; Murgia, Federico; Rivadeneira, Fernando; Ernst, Florian; Kronenberg, Florian; Hu, Frank B; Navis, Gerjan J; Curhan, Gary C; Ehret, George B; Homuth, Georg; Coassin, Stefan; Thun, Gian-Andri; Pistis, Giorgio; Gambaro, Giovanni; Malerba, Giovanni; Montgomery, Grant W; Eiriksdottir, Gudny; Jacobs, Gunnar; Li, Guo; Wichmann, H-Erich; Campbell, Harry; Schmidt, Helena; Wallaschofski, Henri; Völzke, Henry; Brenner, Hermann; Kroemer, Heyo K; Kramer, Holly; Lin, Honghuang; Leach, I Mateo; Ford, Ian; Guessous, Idris; Rudan, Igor; Prokopenko, Inga; Borecki, Ingrid; Heid, Iris M; Kolcic, Ivana; Persico, Ivana; Jukema, J Wouter; Wilson, James F; Felix, Janine F; Divers, Jasmin; Lambert, Jean-Charles; Stafford, Jeanette M; Gaspoz, Jean-Michel; Smith, Jennifer A; Faul, Jessica D; Wang, Jie Jin; Ding, Jingzhong; Hirschhorn, Joel N; Attia, John; Whitfield, John B; Chalmers, John; Viikari, Jorma; Coresh, Josef; Denny, Joshua C; Karjalainen, Juha; Fernandes, Jyotika K; Endlich, Karlhans; Butterbach, Katja; Keene, Keith L; Lohman, Kurt; Portas, Laura; Launer, Lenore J; Lyytikäinen, Leo-Pekka; Yengo, Loic; Franke, Lude; Ferrucci, Luigi; Rose, Lynda M; Kedenko, Lyudmyla; Rao, Madhumathi; Struchalin, Maksim; Kleber, Marcus E; Cavalieri, Margherita; Haun, Margot; Cornelis, Marilyn C; Ciullo, Marina; Pirastu, Mario; de Andrade, Mariza; McEvoy, Mark A; Woodward, Mark; Adam, Martin; Cocca, Massimiliano; Nauck, Matthias; Imboden, Medea; Waldenberger, Melanie; Pruijm, Menno; Metzger, Marie; Stumvoll, Michael; Evans, Michele K; Sale, Michele M; Kähönen, Mika; Boban, Mladen; Bochud, Murielle; Rheinberger, Myriam; Verweij, Niek; Bouatia-Naji, Nabila; Martin, Nicholas G; Hastie, Nick; Probst-Hensch, Nicole; Soranzo, Nicole; Devuyst, Olivier; Raitakari, Olli; Gottesman, Omri; Franco, Oscar H; Polasek, Ozren; Gasparini, Paolo; Munroe, Patricia B; Ridker, Paul M; Mitchell, Paul; Muntner, Paul; Meisinger, Christa; Smit, Johannes H; Kovacs, Peter; Wild, Philipp S; Froguel, Philippe; Rettig, Rainer; Mägi, Reedik; Biffar, Reiner; Schmidt, Reinhold; Middelberg, Rita P S; Carroll, Robert J; Penninx, Brenda W; Scott, Rodney J; Katz, Ronit; Sedaghat, Sanaz; Wild, Sarah H; Kardia, Sharon L R; Ulivi, Sheila; Hwang, Shih-Jen; Enroth, Stefan; Kloiber, Stefan; Trompet, Stella; Stengel, Benedicte; Hancock, Stephen J; Turner, Stephen T; Rosas, Sylvia E; Stracke, Sylvia; Harris, Tamara B; Zeller, Tanja; Zemunik, Tatijana; Lehtimäki, Terho; Illig, Thomas; Aspelund, Thor; Nikopensius, Tiit; Esko, Tonu; Tanaka, Toshiko; Gyllensten, Ulf; Völker, Uwe; Emilsson, Valur; Vitart, Veronique; Aalto, Ville; Gudnason, Vilmundur; Chouraki, Vincent; Chen, Wei-Min; Igl, Wilmar; März, Winfried; Koenig, Wolfgang; Lieb, Wolfgang; Loos, Ruth J F; Liu, Yongmei; Snieder, Harold; Pramstaller, Peter P; Parsa, Afshin; O'Connell, Jeffrey R; Susztak, Katalin; Hamet, Pavel; Tremblay, Johanne; de Boer, Ian H; Böger, Carsten A; Goessling, Wolfram; Chasman, Daniel I; Köttgen, Anna; Kao, W H Linda; Fox, Caroline S

2016-01-21

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways.

A common bacterial metabolite elicits prion-based bypass of glucose repression

PubMed Central

Garcia, David M; Dietrich, David; Clardy, Jon; Jarosz, Daniel F

2016-01-01

Robust preference for fermentative glucose metabolism has motivated domestication of the budding yeast Saccharomyces cerevisiae. This program can be circumvented by a protein-based genetic element, the [GAR+] prion, permitting simultaneous metabolism of glucose and other carbon sources. Diverse bacteria can elicit yeast cells to acquire [GAR+], although the molecular details of this interaction remain unknown. Here we identify the common bacterial metabolite lactic acid as a strong [GAR+] inducer. Transient exposure to lactic acid caused yeast cells to heritably circumvent glucose repression. This trait had the defining genetic properties of [GAR+], and did not require utilization of lactic acid as a carbon source. Lactic acid also induced [GAR+]-like epigenetic states in fungi that diverged from S. cerevisiae ~200 million years ago, and in which glucose repression evolved independently. To our knowledge, this is the first study to uncover a bacterial metabolite with the capacity to potently induce a prion. DOI: http://dx.doi.org/10.7554/eLife.17978.001 PMID:27906649

Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function

PubMed Central

Pattaro, Cristian; Teumer, Alexander; Gorski, Mathias; Chu, Audrey Y.; Li, Man; Mijatovic, Vladan; Garnaas, Maija; Tin, Adrienne; Sorice, Rossella; Li, Yong; Taliun, Daniel; Olden, Matthias; Foster, Meredith; Yang, Qiong; Chen, Ming-Huei; Pers, Tune H.; Johnson, Andrew D.; Ko, Yi-An; Fuchsberger, Christian; Tayo, Bamidele; Nalls, Michael; Feitosa, Mary F.; Isaacs, Aaron; Dehghan, Abbas; d'Adamo, Pio; Adeyemo, Adebowale; Dieffenbach, Aida Karina; Zonderman, Alan B.; Nolte, Ilja M.; van der Most, Peter J.; Wright, Alan F.; Shuldiner, Alan R.; Morrison, Alanna C.; Hofman, Albert; Smith, Albert V.; Dreisbach, Albert W.; Franke, Andre; Uitterlinden, Andre G.; Metspalu, Andres; Tonjes, Anke; Lupo, Antonio; Robino, Antonietta; Johansson, Åsa; Demirkan, Ayse; Kollerits, Barbara; Freedman, Barry I.; Ponte, Belen; Oostra, Ben A.; Paulweber, Bernhard; Krämer, Bernhard K.; Mitchell, Braxton D.; Buckley, Brendan M.; Peralta, Carmen A.; Hayward, Caroline; Helmer, Catherine; Rotimi, Charles N.; Shaffer, Christian M.; Müller, Christian; Sala, Cinzia; van Duijn, Cornelia M.; Saint-Pierre, Aude; Ackermann, Daniel; Shriner, Daniel; Ruggiero, Daniela; Toniolo, Daniela; Lu, Yingchang; Cusi, Daniele; Czamara, Darina; Ellinghaus, David; Siscovick, David S.; Ruderfer, Douglas; Gieger, Christian; Grallert, Harald; Rochtchina, Elena; Atkinson, Elizabeth J.; Holliday, Elizabeth G.; Boerwinkle, Eric; Salvi, Erika; Bottinger, Erwin P.; Murgia, Federico; Rivadeneira, Fernando; Ernst, Florian; Kronenberg, Florian; Hu, Frank B.; Navis, Gerjan J.; Curhan, Gary C.; Ehret, George B.; Homuth, Georg; Coassin, Stefan; Thun, Gian-Andri; Pistis, Giorgio; Gambaro, Giovanni; Malerba, Giovanni; Montgomery, Grant W.; Eiriksdottir, Gudny; Jacobs, Gunnar; Li, Guo; Wichmann, H-Erich; Campbell, Harry; Schmidt, Helena; Wallaschofski, Henri; Völzke, Henry; Brenner, Hermann; Kroemer, Heyo K.; Kramer, Holly; Lin, Honghuang; Leach, I. Mateo; Ford, Ian; Guessous, Idris; Rudan, Igor; Prokopenko, Inga; Borecki, Ingrid; Heid, Iris M.; Kolcic, Ivana; Persico, Ivana; Jukema, J. Wouter; Wilson, James F.; Felix, Janine F.; Divers, Jasmin; Lambert, Jean-Charles; Stafford, Jeanette M.; Gaspoz, Jean-Michel; Smith, Jennifer A.; Faul, Jessica D.; Wang, Jie Jin; Ding, Jingzhong; Hirschhorn, Joel N.; Attia, John; Whitfield, John B.; Chalmers, John; Viikari, Jorma; Coresh, Josef; Denny, Joshua C.; Karjalainen, Juha; Fernandes, Jyotika K.; Endlich, Karlhans; Butterbach, Katja; Keene, Keith L.; Lohman, Kurt; Portas, Laura; Launer, Lenore J.; Lyytikäinen, Leo-Pekka; Yengo, Loic; Franke, Lude; Ferrucci, Luigi; Rose, Lynda M.; Kedenko, Lyudmyla; Rao, Madhumathi; Struchalin, Maksim; Kleber, Marcus E.; Cavalieri, Margherita; Haun, Margot; Cornelis, Marilyn C.; Ciullo, Marina; Pirastu, Mario; de Andrade, Mariza; McEvoy, Mark A.; Woodward, Mark; Adam, Martin; Cocca, Massimiliano; Nauck, Matthias; Imboden, Medea; Waldenberger, Melanie; Pruijm, Menno; Metzger, Marie; Stumvoll, Michael; Evans, Michele K.; Sale, Michele M.; Kähönen, Mika; Boban, Mladen; Bochud, Murielle; Rheinberger, Myriam; Verweij, Niek; Bouatia-Naji, Nabila; Martin, Nicholas G.; Hastie, Nick; Probst-Hensch, Nicole; Soranzo, Nicole; Devuyst, Olivier; Raitakari, Olli; Gottesman, Omri; Franco, Oscar H.; Polasek, Ozren; Gasparini, Paolo; Munroe, Patricia B.; Ridker, Paul M.; Mitchell, Paul; Muntner, Paul; Meisinger, Christa; Smit, Johannes H.; Abecasis, Goncalo R.; Adair, Linda S.; Alexander, Myriam; Altshuler, David; Amin, Najaf; Arking, Dan E.; Arora, Pankaj; Aulchenko, Yurii; Bakker, Stephan J. L.; Bandinelli, Stefania; Barroso, Ines; Beckmann, Jacques S.; Beilby, John P.; Bergman, Richard N.; Bergmann, Sven; Bis, Joshua C.; Boehnke, Michael; Bonnycastle, Lori L.; Bornstein, Stefan R.; Bots, Michiel L.; Bragg-Gresham, Jennifer L.; Brand, Stefan-Martin; Brand, Eva; Braund, Peter S.; Brown, Morris J.; Burton, Paul R.; Casas, Juan P.; Caulfield, Mark J.; Chakravarti, Aravinda; Chambers, John C.; Chandak, Giriraj R.; Chang, Yen-Pei C.; Charchar, Fadi J.; Chaturvedi, Nish; Shin Cho, Yoon; Clarke, Robert; Collins, Francis S.; Collins, Rory; Connell, John M.; Cooper, Jackie A.; Cooper, Matthew N.; Cooper, Richard S.; Corsi, Anna Maria; Dörr, Marcus; Dahgam, Santosh; Danesh, John; Smith, George Davey; Day, Ian N. M.; Deloukas, Panos; Denniff, Matthew; Dominiczak, Anna F.; Dong, Yanbin; Doumatey, Ayo; Elliott, Paul; Elosua, Roberto; Erdmann, Jeanette; Eyheramendy, Susana; Farrall, Martin; Fava, Cristiano; Forrester, Terrence; Fowkes, F. Gerald R.; Fox, Ervin R.; Frayling, Timothy M.; Galan, Pilar; Ganesh, Santhi K.; Garcia, Melissa; Gaunt, Tom R.; Glazer, Nicole L.; Go, Min Jin; Goel, Anuj; Grässler, Jürgen; Grobbee, Diederick E.; Groop, Leif; Guarrera, Simonetta; Guo, Xiuqing; Hadley, David; Hamsten, Anders; Han, Bok-Ghee; Hardy, Rebecca; Hartikainen, Anna-Liisa; Heath, Simon; Heckbert, Susan R.; Hedblad, Bo; Hercberg, Serge; Hernandez, Dena; Hicks, Andrew A.; Hilton, Gina; Hingorani, Aroon D.; Bolton, Judith A Hoffman; Hopewell, Jemma C.; Howard, Philip; Humphries, Steve E.; Hunt, Steven C.; Hveem, Kristian; Ikram, M. Arfan; Islam, Muhammad; Iwai, Naoharu; Jarvelin, Marjo-Riitta; Jackson, Anne U.; Jafar, Tazeen H.; Janipalli, Charles S.; Johnson, Toby; Kathiresan, Sekar; Khaw, Kay-Tee; Kim, Hyung-Lae; Kinra, Sanjay; Kita, Yoshikuni; Kivimaki, Mika; Kooner, Jaspal S.; Kumar, M. J. Kranthi; Kuh, Diana; Kulkarni, Smita R.; Kumari, Meena; Kuusisto, Johanna; Kuznetsova, Tatiana; Laakso, Markku; Laan, Maris; Laitinen, Jaana; Lakatta, Edward G.; Langefeld, Carl D.; Larson, Martin G.; Lathrop, Mark; Lawlor, Debbie A.; Lawrence, Robert W.; Lee, Jong-Young; Lee, Nanette R.; Levy, Daniel; Li, Yali; Longstreth, Will T.; Luan, Jian'an; Lucas, Gavin; Ludwig, Barbara; Mangino, Massimo; Mani, K. Radha; Marmot, Michael G.; Mattace-Raso, Francesco U. S.; Matullo, Giuseppe; McArdle, Wendy L.; McKenzie, Colin A.; Meitinger, Thomas; Melander, Olle; Meneton, Pierre; Meschia, James F.; Miki, Tetsuro; Milaneschi, Yuri; Mohlke, Karen L.; Mooser, Vincent; Morken, Mario A.; Morris, Richard W.; Mosley, Thomas H.; Najjar, Samer; Narisu, Narisu; Newton-Cheh, Christopher; Nguyen, Khanh-Dung Hoang; Nilsson, Peter; Nyberg, Fredrik; O'Donnell, Christopher J.; Ogihara, Toshio; Ohkubo, Takayoshi; Okamura, Tomonori; Ong, RickTwee-Hee; Ongen, Halit; Onland-Moret, N. Charlotte; O'Reilly, Paul F.; Org, Elin; Orru, Marco; Palmas, Walter; Palmen, Jutta; Palmer, Lyle J.; Palmer, Nicholette D.; Parker, Alex N.; Peden, John F.; Peltonen, Leena; Perola, Markus; Pihur, Vasyl; Platou, Carl G. P.; Plump, Andrew; Prabhakaran, Dorairajan; Psaty, Bruce M.; Raffel, Leslie J.; Rao, Dabeeru C.; Rasheed, Asif; Ricceri, Fulvio; Rice, Kenneth M.; Rosengren, Annika; Rotter, Jerome I.; Rudock, Megan E.; Sõber, Siim; Salako, Tunde; Saleheen, Danish; Salomaa, Veikko; Samani, Nilesh J.; Schwartz, Steven M.; Schwarz, Peter E. H.; Scott, Laura J.; Scott, James; Scuteri, Angelo; Sehmi, Joban S.; Seielstad, Mark; Seshadri, Sudha; Sharma, Pankaj; Shaw-Hawkins, Sue; Shi, Gang; Shrine, Nick R. G.; Sijbrands, Eric J. G.; Sim, Xueling; Singleton, Andrew; Sjögren, Marketa; Smith, Nicholas L.; Artigas, Maria Soler; Spector, Tim D.; Staessen, Jan A.; Stancakova, Alena; Steinle, Nanette I.; Strachan, David P.; Stringham, Heather M.; Sun, Yan V.; Swift, Amy J.; Tabara, Yasuharu; Tai, E-Shyong; Talmud, Philippa J.; Taylor, Andrew; Terzic, Janos; Thelle, Dag S.; Tobin, Martin D.; Tomaszewski, Maciej; Tripathy, Vikal; Tuomilehto, Jaakko; Tzoulaki, Ioanna; Uda, Manuela; Ueshima, Hirotsugu; Uiterwaal, Cuno S. P. M.; Umemura, Satoshi; van der Harst, Pim; van der Schouw, Yvonne T.; van Gilst, Wiek H.; Vartiainen, Erkki; Vasan, Ramachandran S.; Veldre, Gudrun; Verwoert, Germaine C.; Viigimaa, Margus; Vinay, D. G.; Vineis, Paolo; Voight, Benjamin F.; Vollenweider, Peter; Wagenknecht, Lynne E.; Wain, Louise V.; Wang, Xiaoling; Wang, Thomas J.; Wareham, Nicholas J.; Watkins, Hugh; Weder, Alan B.; Whincup, Peter H.; Wiggins, Kerri L.; Witteman, Jacqueline C. M.; Wong, Andrew; Wu, Ying; Yajnik, Chittaranjan S.; Yao, Jie; Young, J. H.; Zelenika, Diana; Zhai, Guangju; Zhang, Weihua; Zhang, Feng; Zhao, Jing Hua; Zhu, Haidong; Zhu, Xiaofeng; Zitting, Paavo; Zukowska-Szczechowska, Ewa; Okada, Yukinori; Wu, Jer-Yuarn; Gu, Dongfeng; Takeuchi, Fumihiko; Takahashi, Atsushi; Maeda, Shiro; Tsunoda, Tatsuhiko; Chen, Peng; Lim, Su-Chi; Wong, Tien-Yin; Liu, Jianjun; Young, Terri L.; Aung, Tin; Teo, Yik-Ying; Kim, Young Jin; Kang, Daehee; Chen, Chien-Hsiun; Tsai, Fuu-Jen; Chang, Li-Ching; Fann, S. -J. Cathy; Mei, Hao; Hixson, James E.; Chen, Shufeng; Katsuya, Tomohiro; Isono, Masato; Albrecht, Eva; Yamamoto, Kazuhiko; Kubo, Michiaki; Nakamura, Yusuke; Kamatani, Naoyuki; Kato, Norihiro; He, Jiang; Chen, Yuan-Tsong; Tanaka, Toshihiro; Reilly, Muredach P; Schunkert, Heribert; Assimes, Themistocles L.; Hall, Alistair; Hengstenberg, Christian; König, Inke R.; Laaksonen, Reijo; McPherson, Ruth; Thompson, John R.; Thorsteinsdottir, Unnur; Ziegler, Andreas; Absher, Devin; Chen, Li; Cupples13, L. Adrienne; Halperin, Eran; Li, Mingyao; Musunuru, Kiran; Preuss, Michael; Schillert, Arne; Thorleifsson, Gudmar; Wells, George A.; Holm, Hilma; Roberts, Robert; Stewart, Alexandre F. R.; Fortmann, Stephen; Go, Alan; Hlatky, Mark; Iribarren, Carlos; Knowles, Joshua; Myers, Richard; Quertermous, Thomas; Sidney, Steven; Risch, Neil; Tang, Hua; Blankenberg, Stefan; Schnabel, Renate; Sinning, Christoph; Lackner, Karl J.; Tiret, Laurence; Nicaud, Viviane; Cambien, Francois; Bickel, Christoph; Rupprecht, Hans J.; Perret, Claire; Proust, Carole; Münzel, Thomas F.; Barbalic, Maja; Chen, Ida Yii-Der; Demissie-Banjaw, Serkalem; Folsom, Aaron; Lumley, Thomas; Marciante, Kristin; Taylor, Kent D.; Volcik, Kelly; Gretarsdottir, Solveig; Gulcher, Jeffrey R.; Kong, Augustine; Stefansson, Kari; Thorgeirsson, Gudmundur; Andersen, Karl; Fischer, Marcus; Grosshennig, Anika; Linsel-Nitschke, Patrick; Stark, Klaus; Schreiber, Stefan; Aherrahrou, Zouhair; Bruse, Petra; Doering, Angela; Klopp, Norman; Diemert, Patrick; Loley, Christina; Medack, Anja; Nahrstedt, Janja; Peters, Annette; Wagner, Arnika K.; Willenborg, Christina; Böhm, Bernhard O.; Dobnig, Harald; Grammer, Tanja B.; Hoffmann, Michael M.; Meinitzer, Andreas; Winkelmann, Bernhard R.; Pilz, Stefan; Renner, Wilfried; Scharnagl, Hubert; Stojakovic, Tatjana; Tomaschitz, Andreas; Winkler, Karl; Guiducci, Candace; Burtt, Noel; Gabriel, Stacey B.; Dandona, Sonny; Jarinova, Olga; Qu, Liming; Wilensky, Robert; Matthai, William; Hakonarson, Hakon H.; Devaney, Joe; Burnett, Mary Susan; Pichard, Augusto D.; Kent, Kenneth M.; Satler, Lowell; Lindsay, Joseph M.; Waksman, Ron; Knouff, Christopher W.; Waterworth, Dawn M.; Walker, Max C.; Epstein, Stephen E.; Rader, Daniel J.; Nelson, Christopher P.; Wright, Benjamin J.; Balmforth, Anthony J.; Ball, Stephen G.; Loehr, Laura R.; Rosamond, Wayne D.; Benjamin, Emelia; Haritunians, Talin; Couper, David; Murabito, Joanne; Wang, Ying A.; Stricker, Bruno H.; Chang, Patricia P.; Willerson, James T.; Felix, Stephan B.; Watzinger, Norbert; Aragam, Jayashri; Zweiker, Robert; Lind, Lars; Rodeheffer, Richard J.; Greiser, Karin Halina; Deckers, Jaap W.; Stritzke, Jan; Ingelsson, Erik; Kullo, Iftikhar; Haerting, Johannes; Reffelmann, Thorsten; Redfield, Margaret M.; Werdan, Karl; Mitchell, Gary F.; Arnett, Donna K.; Gottdiener, John S.; Blettner, Maria; Friedrich, Nele; Kovacs, Peter; Wild, Philipp S.; Froguel, Philippe; Rettig, Rainer; Mägi, Reedik; Biffar, Reiner; Schmidt, Reinhold; Middelberg, Rita P. S.; Carroll, Robert J.; Penninx, Brenda W.; Scott, Rodney J.; Katz, Ronit; Sedaghat, Sanaz; Wild, Sarah H.; Kardia, Sharon L. R.; Ulivi, Sheila; Hwang, Shih-Jen; Enroth, Stefan; Kloiber, Stefan; Trompet, Stella; Stengel, Benedicte; Hancock, Stephen J.; Turner, Stephen T.; Rosas, Sylvia E.; Stracke, Sylvia; Harris, Tamara B.; Zeller, Tanja; Zemunik, Tatijana; Lehtimäki, Terho; Illig, Thomas; Aspelund, Thor; Nikopensius, Tiit; Esko, Tonu; Tanaka, Toshiko; Gyllensten, Ulf; Völker, Uwe; Emilsson, Valur; Vitart, Veronique; Aalto, Ville; Gudnason, Vilmundur; Chouraki, Vincent; Chen, Wei-Min; Igl, Wilmar; März, Winfried; Koenig, Wolfgang; Lieb, Wolfgang; Loos, Ruth J. F.; Liu, Yongmei; Snieder, Harold; Pramstaller, Peter P.; Parsa, Afshin; O'Connell, Jeffrey R.; Susztak, Katalin; Hamet, Pavel; Tremblay, Johanne; de Boer, Ian H.; Böger, Carsten A.; Goessling, Wolfram; Chasman, Daniel I.; Köttgen, Anna; Kao, W. H. Linda; Fox, Caroline S.

2016-01-01

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways. PMID:26831199

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

PubMed

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

2008-01-01

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

Congruent population structure inferred from dispersal behaviour and intensive genetic surveys of the threatened Florida scrub-jay (Aphelocoma cœrulescens)

USGS Publications Warehouse

Coulon, A.; Fitzpatrick, J.W.; Bowman, R.; Stith, B.M.; Makarewich, C.A.; Stenzler, L.M.; Lovette, I.J.

2008-01-01

The delimitation of populations, defined as groups of individuals linked by gene flow, is possible by the analysis of genetic markers and also by spatial models based on dispersal probabilities across a landscape. We combined these two complimentary methods to define the spatial pattern of genetic structure among remaining populations of the threatened Florida scrub-jay, a species for which dispersal ability is unusually well-characterized. The range-wide population was intensively censused in the 1990s, and a metapopulation model defined population boundaries based on predicted dispersal-mediated demographic connectivity. We subjected genotypes from more than 1000 individual jays screened at 20 microsatellite loci to two Bayesian clustering methods. We describe a consensus method for identifying common features across many replicated clustering runs. Ten genetically differentiated groups exist across the present-day range of the Florida scrub-jay. These groups are largely consistent with the dispersal-defined metapopulations, which assume very limited dispersal ability. Some genetic groups comprise more than one metapopulation, likely because these genetically similar metapopulations were sundered only recently by habitat alteration. The combined reconstructions of population structure based on genetics and dispersal-mediated demographic connectivity provide a robust depiction of the current genetic and demographic organization of this species, reflecting past and present levels of dispersal among occupied habitat patches. The differentiation of populations into 10 genetic groups adds urgency to management efforts aimed at preserving what remains of genetic variation in this dwindling species, by maintaining viable populations of all genetically differentiated and geographically isolated populations.

Disentangling the heterogeneity of autism spectrum disorder through genetic findings

PubMed Central

Jeste, Shafali S.; Geschwind, Daniel H.

2014-01-01

Autism spectrum disorder (ASD) represents a heterogeneous group of disorders, which presents a substantial challenge to diagnosis and treatment. Over the past decade, considerable progress has been made in the identification of genetic risk factors for ASD that define specific mechanisms and pathways underlying the associated behavioural deficits. In this Review, we discuss how some of the latest advances in the genetics of ASD have facilitated parsing of the phenotypic heterogeneity of this disorder. We argue that only through such advances will we begin to define endophenotypes that can benefit from targeted, hypothesis-driven treatments. We review the latest technologies used to identify and characterize the genetics underlying ASD and then consider three themes—single-gene disorders, the gender bias in ASD, and the genetics of neurological comorbidities—that highlight ways in which we can use genetics to define the many phenotypes within the autism spectrum. We also present current clinical guidelines for genetic testing in ASD and their implications for prognosis and treatment. PMID:24468882

Physical break-down of the classical view on cancer cell invasion and metastasis.

PubMed

Mierke, Claudia T

2013-03-01

Eight classical hallmarks of cancer have been proposed and are well-defined by using biochemical or molecular genetic methods, but are not yet precisely defined by cellular biophysical processes. To define the malignant transformation of neoplasms and finally reveal the functional pathway, which enables cancer cells to promote cancer progression, these classical hallmarks of cancer require the inclusion of specific biomechanical properties of cancer cells and their microenvironment such as the extracellular matrix and embedded cells such as fibroblasts, macrophages or endothelial cells. Nonetheless a main novel ninth hallmark of cancer is still elusive in classical tumor biological reviews, which is the aspect of physics in cancer disease by the natural selection of an aggressive (highly invasive) subtype of cancer cells. The physical aspects can be analyzed by using state-of-the-art biophysical methods. Thus, this review will present current cancer research in a different light and will focus on novel physical methods to investigate the aggressiveness of cancer cells from a biophysicist's point of view. This may lead to novel insights into cancer disease and will overcome classical views on cancer. In addition, this review will discuss how physics of cancer can help to reveal whether cancer cells will invade connective tissue and metastasize. In particular, this review will point out how physics can improve, break-down or support classical approaches to examine tumor growth even across primary tumor boundaries, the invasion of single or collective cancer cells, transendothelial migration of cancer cells and metastasis in targeted organs. Finally, this review will show how physical measurements can be integrated into classical tumor biological analysis approaches. The insights into physical interactions between cancer cells, the primary tumor and the microenvironment may help to solve some "old" questions in cancer disease progression and may finally lead to novel approaches for development and improvement of cancer diagnostics and therapies. Copyright © 2013 Elsevier GmbH. All rights reserved.

Nonallelic heterogeneity in autosomal dominant retinitis pigmentosa with incomplete penetrance

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

Kim, S.K.; Berson, E.L.; Dryja, T.P.

1994-08-01

Retinitis pigmentosa is a group of retinal diseases in which photoreceptor cells throughout the retina degenerate. Although there is considerable genetic heterogeneity (autosomal dominant, autosomal recessive, and X-linked forms exist), there is a possibility that some clinically defined subtypes of the disease may be the result of mutations at the same locus. One possible clinically defined subtype is that of autosomal dominant retinitis pigmentosa (ADRP) with incomplete penetrance. Whereas in most families with ADRP, carriers can be clearly identified because of visual loss, ophthalmological findings, or abnormal electroretinograms (ERGs), in occasional families some obligate carriers are asymptomatic and have normalmore » or nearly normal ERGs even late in life. A recent paper reported the mapping of the diseases locus in one pedigree (designated adRP7) with ADRP with incomplete penetrance to chromosome 7p. To test the idea that ADRP with incomplete penetrance may be genetically homogeneous, we have evaluated whether a different family with incomplete penetrance also has a disease gene linked to the same region. 4 refs., 1 fig., 1 tab.« less

DNA methylation heterogeneity defines a disease spectrum in Ewing sarcoma

PubMed Central

Sheffield, Nathan C; Pierron, Gaelle; Klughammer, Johanna; Datlinger, Paul; Schönegger, Andreas; Schuster, Michael; Hadler, Johanna; Surdez, Didier; Guillemot, Delphine; Lapouble, Eve; Freneaux, Paul; Champigneulle, Jacqueline; Bouvier, Raymonde; Walder, Diana; Ambros, Ingeborg M; Hutter, Caroline; Sorz, Eva; Amaral, Ana T; de Álava, Enrique; Schallmoser, Katharina; Strunk, Dirk; Rinner, Beate; Liegl-Atzwanger, Bernadette; Huppertz, Berthold; Leithner, Andreas; de Pinieux, Gonzague; Terrier, Philippe; Laurence, Valérie; Michon, Jean; Ladenstein, Ruth; Holter, Wolfgang; Windhager, Reinhard; Dirksen, Uta; Ambros, Peter F; Delattre, Olivier; Kovar, Heinrich; Bock, Christoph; Tomazou, Eleni M

2018-01-01

Developmental tumors in children and young adults carry few genetic alterations, yet they have diverse clinical presentation. Focusing on Ewing sarcoma, we sought to establish the prevalence and characteristics of epigenetic heterogeneity in genetically homogeneous cancers. We performed genome-scale DNA methylation sequencing for a large cohort of Ewing sarcoma tumors and analyzed epigenetic heterogeneity on three levels: between cancers, between tumors, and within tumors. We observed consistent DNA hypomethylation at enhancers regulated by the disease-defining EWS-FLI1 fusion protein, thus establishing epigenomic enhancer reprogramming as a ubiquitous and characteristic feature of Ewing sarcoma. DNA methylation differences between tumors identified a continuous disease spectrum underlying Ewing sarcoma, which reflected the strength of an EWS-FLI1 regulatory signature and a continuum between mesenchymal and stem cell signatures. There was substantial epigenetic heterogeneity within tumors, particularly in patients with metastatic disease. In summary, our study provides a comprehensive assessment of epigenetic heterogeneity in Ewing sarcoma and thereby highlights the importance of considering nongenetic aspects of tumor heterogeneity in the context of cancer biology and personalized medicine. PMID:28134926

Endothelial Cell Implantation and Survival within Experimental Gliomas

NASA Astrophysics Data System (ADS)

Lal, Bachchu; Indurti, Ravi R.; Couraud, Pierre-Olivier; Goldstein, Gary W.; Laterra, John

1994-10-01

The delivery of therapeutic genes to primary brain neoplasms opens new opportunities for treating these frequently fatal tumors. Efficient gene delivery to tissues remains an important obstacle to therapy, and this problem has unique characteristics in brain tumors due to the blood-brain and blood-tumor barriers. The presence of endothelial mitogens and vessel proliferation within solid tumors suggests that genetically modified endothelial cells might efficiently transplant to brain tumors. Rat brain endothelial cells immortalized with the adenovirus E1A gene and further modified to express the β-galactosidase reporter were examined for their ability to survive implantation to experimental rat gliomas. Rats received 9L, F98, or C6 glioma cells in combination with endothelial cells intracranially to caudate/putamen or subcutaneously to flank. Implanted endothelial cells were identified by β-galactosidase histochemistry or by polymerase chain reaction in all tumors up to 35 days postimplantation, the latest time examined. Implanted endothelial cells appeared to cooperate in tumor vessel formation and expressed the brain-specific endothelial glucose transporter type 1 as identified by immunohistochemistry. The proliferation of implanted endothelial cells was supported by their increased number within tumors between postimplantation days 14 and 21 (P = 0.015) and by their expression of the proliferation antigen Ki67. These findings establish that genetically modified endothelial cells can be stably engrafted to growing gliomas and suggest that endothelial cell implantation may provide a means of delivering therapeutic genes to brain neoplasms and other solid tumors. In addition, endothelial implantation to brain may be useful for defining mechanisms of brain-specific endothelial differentiation.

Combinatorial gene editing in mammalian cells using ssODNs and TALENs

NASA Astrophysics Data System (ADS)

Strouse, Bryan; Bialk, Pawel; Niamat, Rohina A.; Rivera-Torres, Natalia; Kmiec, Eric B.

2014-01-01

The regulation of gene editing is being elucidated in mammalian cells and its potential as well as its limitations are becoming evident. ssODNs carry out gene editing by annealing to their complimentary sequence at the target site and acting as primers for replication fork extension. To effect a genetic change, a large amount of ssODN molecules must be introduced into cells and as such induce a Reduced Proliferation Phenotype (RPP), a phenomenon in which corrected cells do not proliferate. To overcome this limitation, we have used TAL-Effector Nucleases (TALENs) to increase the frequency, while reducing the amount of ssODN required to direct gene correction. This strategy resolves the problem and averts the serious effects of RPP. The efficiency of gene editing can be increased significantly if cells are targeted while they progress through S phase. Our studies define new reaction parameters that will help guide experimental strategies of gene editing.

A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells.

PubMed

Park, Sungjin; Szumlanski, Amy L; Gu, Fangwei; Guo, Feng; Nielsen, Erik

2011-07-17

In plants, cell shape is defined by the cell wall, and changes in cell shape and size are dictated by modification of existing cell walls and deposition of newly synthesized cell-wall material. In root hairs, expansion occurs by a process called tip growth, which is shared by root hairs, pollen tubes and fungal hyphae. We show that cellulose-like polysaccharides are present in root-hair tips, and de novo synthesis of these polysaccharides is required for tip growth. We also find that eYFP-CSLD3 proteins, but not CESA cellulose synthases, localize to a polarized plasma-membrane domain in root hairs. Using biochemical methods and genetic complementation of a csld3 mutant with a chimaeric CSLD3 protein containing a CESA6 catalytic domain, we provide evidence that CSLD3 represents a distinct (1→4)-β-glucan synthase activity in apical plasma membranes during tip growth in root-hair cells.

Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

PubMed

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R; Greenleaf, William J; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H

2015-07-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.

Transient Acquisition of Pluripotency During Somatic Cell Transdifferentiation with iPSC Reprogramming Factors

PubMed Central

Maza, Itay; Caspi, Inbal; Zviran, Asaf; Chomsky, Elad; Rais, Yoach; Viukov, Sergey; Geula, Shay; Buenrostro, Jason D.; Weinberger, Leehee; Krupalnik, Vladislav; Hanna, Suhair; Zerbib, Mirie; Dutton, James R.; Greenleaf, William J.; Massarwa, Rada; Novershtern, Noa; Hanna, Jacob H.

2015-01-01

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors1,2. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation3–6. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced transdifferentiation pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by different methods. PMID:26098448

The complex pathophysiology of acquired aplastic anaemia

PubMed Central

Zeng, Y; Katsanis, E

2015-01-01

Immune-mediated destruction of haematopoietic stem/progenitor cells (HSPCs) plays a central role in the pathophysiology of acquired aplastic anaemia (aAA). Dysregulated CD8+ cytotoxic T cells, CD4+ T cells including T helper type 1 (Th1), Th2, regulatory T cells and Th17 cells, natural killer (NK) cells and NK T cells, along with the abnormal production of cytokines including interferon (IFN)-γ, tumour necrosis factor (TNF)-α and transforming growth factor (TGF)-β, induce apoptosis of HSPCs, constituting a consistent and defining feature of severe aAA. Alterations in the polymorphisms of TGF-β, IFN-γ and TNF-α genes, as well as certain human leucocyte antigen (HLA) alleles, may account for the propensity to immune-mediated killing of HSPCs and/or ineffective haematopoiesis. Although the inciting autoantigens remain elusive, autoantibodies are often detected in the serum. In addition, recent studies provide genetic and molecular evidence that intrinsic and/or secondary deficits in HSPCs and bone marrow mesenchymal stem cells may underlie the development of bone marrow failure. PMID:25683099

Genetic Heterogeneity in Therapy-Naïve Synchronous Primary Breast Cancers and Their Metastases.

PubMed

Ng, Charlotte K Y; Bidard, Francois-Clement; Piscuoglio, Salvatore; Geyer, Felipe C; Lim, Raymond S; de Bruijn, Ino; Shen, Ronglai; Pareja, Fresia; Berman, Samuel H; Wang, Lu; Pierga, Jean-Yves; Vincent-Salomon, Anne; Viale, Agnes; Norton, Larry; Sigal, Brigitte; Weigelt, Britta; Cottu, Paul; Reis-Filho, Jorge S

2017-08-01

Purpose: Paired primary breast cancers and metachronous metastases after adjuvant treatment are reported to differ in their clonal composition and genetic alterations, but it is unclear whether these differences stem from the selective pressures of the metastatic process, the systemic therapies, or both. We sought to define the repertoire of genetic alterations in breast cancer patients with de novo metastatic disease who had not received local or systemic therapy. Experimental Design: Up to two anatomically distinct core biopsies of primary breast cancers and synchronous distant metastases from nine patients who presented with metastatic disease were subjected to high-depth whole-exome sequencing. Mutations, copy number alterations and their cancer cell fractions, and mutation signatures were defined using state-of-the-art bioinformatics methods. All mutations identified were validated with orthogonal methods. Results: Genomic differences were observed between primary and metastatic deposits, with a median of 60% (range 6%-95%) of shared somatic mutations. Although mutations in known driver genes including TP53, PIK3CA , and GATA3 were preferentially clonal in both sites, primary breast cancers and their synchronous metastases displayed spatial intratumor heterogeneity. Likely pathogenic mutations affecting epithelial-to-mesenchymal transition-related genes, including SMAD4, TCF7L2 , and TCF4 ( ITF2 ), were found to be restricted to or enriched in the metastatic lesions. Mutational signatures of trunk mutations differed from those of mutations enriched in the primary tumor or the metastasis in six cases. Conclusions: Synchronous primary breast cancers and metastases differ in their repertoire of somatic genetic alterations even in the absence of systemic therapy. Mutational signature shifts might contribute to spatial intratumor genetic heterogeneity. Clin Cancer Res; 23(15); 4402-15. ©2017 AACR . ©2017 American Association for Cancer Research.

Transcriptomic evidence for the evolution of shoot meristem function in sporophyte-dominant land plants through concerted selection of ancestral gametophytic and sporophytic genetic programs.

PubMed

Frank, Margaret H; Scanlon, Michael J

2015-02-01

Alternation of generations, in which the haploid and diploid stages of the life cycle are each represented by multicellular forms that differ in their morphology, is a defining feature of the land plants (embryophytes). Anciently derived lineages of embryophytes grow predominately in the haploid gametophytic generation from apical cells that give rise to the photosynthetic body of the plant. More recently evolved plant lineages have multicellular shoot apical meristems (SAMs), and photosynthetic shoot development is restricted to the sporophyte generation. The molecular genetic basis for this evolutionary shift from gametophyte-dominant to sporophyte-dominant life cycles remains a major question in the study of land plant evolution. We used laser microdissection and next generation RNA sequencing to address whether angiosperm meristem patterning genes expressed in the sporophytic SAM of Zea mays are expressed in the gametophytic apical cells, or in the determinate sporophytes, of the model bryophytes Marchantia polymorpha and Physcomitrella patens. A wealth of upregulated genes involved in stem cell maintenance and organogenesis are identified in the maize SAM and in both the gametophytic apical cell and sporophyte of moss, but not in Marchantia. Significantly, meiosis-specific genetic programs are expressed in bryophyte sporophytes, long before the onset of sporogenesis. Our data suggest that this upregulated accumulation of meiotic gene transcripts suppresses indeterminate cell fate in the Physcomitrella sporophyte, and overrides the observed accumulation of meristem patterning genes. A model for the evolution of indeterminate growth in the sporophytic generation through the concerted selection of ancestral meristem gene programs from gametophyte-dominant lineages is proposed. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Cell-of-Origin of Cancer versus Cancer Stem Cells: Assays and Interpretations.

PubMed

Rycaj, Kiera; Tang, Dean G

2015-10-01

A tumor originates from a normal cell that has undergone tumorigenic transformation as a result of genetic mutations. This transformed cell is the cell-of-origin for the tumor. In contrast, an established clinical tumor is sustained by subpopulations of self-renewing cancer cells operationally called cancer stem cells (CSC) that can generate, intraclonally, both tumorigenic and nontumorigenic cells. Identifying and characterizing tumor cell-of-origin and CSCs should help elucidate tumor cell heterogeneity, which, in turn, should help understand tumor cell responses to clinical treatments, drug resistance, tumor relapse, and metastatic spread. Both tumor transplantation and lineage-tracing assays have been helpful in characterizing these cancer cell populations, although each system has its strengths and caveats. In this article, we briefly review and summarize advantages and limitations of both assays in support of a combinatorial approach to accurately define the roles of both cancer-initiating and cancer-propagating cells. As an aside, we also wish to clarify the definitions of cancer cell-of-origin and CSCs, which are often interchangeably used by mistake. ©2015 American Association for Cancer Research.

Genome-Edited, TH-expressing Neuroblastoma Cells as a Disease Model for Dopamine-Related Disorders: A Proof-of-Concept Study on DJ-1-deficient Parkinsonism

PubMed Central

Prasuhn, Jannik; Mårtensson, Christoph U.; Krajka, Victor; Klein, Christine; Rakovic, Aleksandar

2018-01-01

Impairment of the dopaminergic (DA) system is a common cause of several movement disorders including Parkinson’s disease (PD), however, little is known about the underlying disease mechanisms. The recent development of stem-cell-based protocols for the generation of DA neurons partially solved this issue, however, this technology is costly and time-consuming. Commonly used cell lines, i.e., neuroblastoma (SHSY5Y) and PC12 cells are still widely used to investigate PD and significantly contributed to our understanding of mechanisms involved in development of the disease. However, they either do not express DA at all or require additional, only partially efficient differentiations in order to produce DA. Here we generated and characterized transgenic SH-SY5Y cells, ectopically expressing tyrosine hydroxylase (SHTH+), that can be used as a homogenous, DA-producing model to study alterations in DA metabolism and oxidative stress. We demonstrated that SHTH+ produce high levels of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) making this model suitable to investigate not only alterations in DA synthesis but also its turnover. We also provide evidence for the presence of other enzymes involved in DA synthesis and its turnover in these cells. Finally, we showed that these cells can easily be genetically modified using CRISPR/Cas9 technology in order to study genetically defined forms of movement disorders using DJ1-linked PD as a model. PMID:29379417

The role of self-defined race/ethnicity in population structure control.

PubMed

Liu, X-Q; Paterson, A D; John, E M; Knight, J A

2006-07-01

Population-based association studies are powerful tools for the genetic mapping of complex diseases. However, this method is sensitive to potential confounding by population structure. While statistical methods that use genetic markers to detect and control for population structure have been the focus of current literature, the utility of self-defined race/ethnicity in controlling for population structure has been controversial. In this study of 1334 individuals, who self-identified as either African American, European American or Hispanic, we demonstrated that when the true underlying genetic structure and the self-defined racial/ethnic groups were roughly in agreement with each other, the self-defined race/ethnicity information was useful in the control of population structure.

Lineage analysis of quiescent regenerative stem cells in the adult brain by genetic labelling reveals spatially restricted neurogenic niches in the olfactory bulb.

PubMed

Giachino, Claudio; Taylor, Verdon

2009-07-01

The subventricular zone (SVZ) of the lateral ventricles is the major neurogenic region in the adult mammalian brain, harbouring neural stem cells within defined niches. The identity of these stem cells and the factors regulating their fate are poorly understood. We have genetically mapped a population of Nestin-expressing cells during postnatal development to study their potential and fate in vivo. Taking advantage of the recombination characteristics of a nestin::CreER(T2) allele, we followed a subpopulation of neural stem cells and traced their fate in a largely unrecombined neurogenic niche. Perinatal nestin::CreER(T2)-expressing cells give rise to multiple glial cell types and neurons, as well as to stem cells of the adult SVZ. In the adult SVZ nestin::CreER(T2)-expressing neural stem cells give rise to several neuronal subtypes in the olfactory bulb (OB). We addressed whether the same population of neural stem cells play a role in SVZ regeneration. Following anti-mitotic treatment to eliminate rapidly dividing progenitors, relatively quiescent nestin::CreER(T2)-targeted cells are spared and contribute to SVZ regeneration, generating new proliferating precursors and neuroblasts. Finally, we have identified neurogenic progenitors clustered in ependymal-like niches within the rostral migratory stream (RMS) of the OB. These OB-RMS progenitors generate neuroblasts that, upon transplantation, graft, migrate and differentiate into granule and glomerular neurons. In summary, using conditional lineage tracing we have identified neonatal cells that are the source of neurogenic and regenerative neural stem cells in the adult SVZ and occupy a novel neurogenic niche in the OB.

Stochastic modeling indicates that aging and somatic evolution in the hematopoetic system are driven by non-cell-autonomous processes.

PubMed

Rozhok, Andrii I; Salstrom, Jennifer L; DeGregori, James

2014-12-01

Age-dependent tissue decline and increased cancer incidence are widely accepted to be rate-limited by the accumulation of somatic mutations over time. Current models of carcinogenesis are dominated by the assumption that oncogenic mutations have defined advantageous fitness effects on recipient stem and progenitor cells, promoting and rate-limiting somatic evolution. However, this assumption is markedly discrepant with evolutionary theory, whereby fitness is a dynamic property of a phenotype imposed upon and widely modulated by environment. We computationally modeled dynamic microenvironment-dependent fitness alterations in hematopoietic stem cells (HSC) within the Sprengel-Liebig system known to govern evolution at the population level. Our model for the first time integrates real data on age-dependent dynamics of HSC division rates, pool size, and accumulation of genetic changes and demonstrates that somatic evolution is not rate-limited by the occurrence of mutations, but instead results from aged microenvironment-driven alterations in the selective/fitness value of previously accumulated genetic changes. Our results are also consistent with evolutionary models of aging and thus oppose both somatic mutation-centric paradigms of carcinogenesis and tissue functional decline. In total, we demonstrate that aging directly promotes HSC fitness decline and somatic evolution via non-cell-autonomous mechanisms.

LRRC6 Mutation Causes Primary Ciliary Dyskinesia with Dynein Arm Defects

PubMed Central

Horani, Amjad; Ferkol, Thomas W.; Shoseyov, David; Wasserman, Mollie G.; Oren, Yifat S.; Kerem, Batsheva; Amirav, Israel; Cohen-Cymberknoh, Malena; Dutcher, Susan K.; Brody, Steven L.; Elpeleg, Orly; Kerem, Eitan

2013-01-01

Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD) remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses revealed a single common homozygous region on chromosome 8 and one candidate was conserved in organisms with motile cilia. Sequencing revealed a single novel mutation in LRRC6 (Leucine-rich repeat containing protein 6) that fit the model of autosomal recessive genetic transmission, leading to a change of a highly conserved amino acid from aspartic acid to histidine (Asp146His). LRRC6 was localized to the cytoplasm and was up-regulated during ciliogenesis in human airway epithelial cells in a Foxj1-dependent fashion. Nasal epithelial cells isolated from affected individuals and shRNA-mediated silencing in human airway epithelial cells, showed reduced LRRC6 expression, absent dynein arms, and slowed cilia beat frequency. Dynein arm proteins were either absent or mislocalized to the cytoplasm in airway epithelial cells from a primary ciliary dyskinesia subject. These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects. PMID:23527195

The genetic network controlling plasma cell differentiation.

PubMed

Nutt, Stephen L; Taubenheim, Nadine; Hasbold, Jhagvaral; Corcoran, Lynn M; Hodgkin, Philip D

2011-10-01

Upon activation by antigen, mature B cells undergo immunoglobulin class switch recombination and differentiate into antibody-secreting plasma cells, the endpoint of the B cell developmental lineage. Careful quantitation of these processes, which are stochastic, independent and strongly linked to the division history of the cell, has revealed that populations of B cells behave in a highly predictable manner. Considerable progress has also been made in the last few years in understanding the gene regulatory network that controls the B cell to plasma cell transition. The mutually exclusive transcriptomes of B cells and plasma cells are maintained by the antagonistic influences of two groups of transcription factors, those that maintain the B cell program, including Pax5, Bach2 and Bcl6, and those that promote and facilitate plasma cell differentiation, notably Irf4, Blimp1 and Xbp1. In this review, we discuss progress in the definition of both the transcriptional and cellular events occurring during late B cell differentiation, as integrating these two approaches is crucial to defining a regulatory network that faithfully reflects the stochastic features and complexity of the humoral immune response. 2011 Elsevier Ltd. All rights reserved.

CRISPR/Cas9 therapeutics: a cure for cancer and other genetic diseases.

PubMed

Khan, Faheem Ahmed; Pandupuspitasari, Nuruliarizki Shinta; Chun-Jie, Huang; Ao, Zhou; Jamal, Muhammad; Zohaib, Ali; Khan, Farhan Ahmed; Hakim, Muthia Raihana; ShuJun, Zhang

2016-08-09

Cancer is caused by a series of alterations in genome and epigenome mostly resulting in activation of oncogenes or inactivation of cancer suppressor genes. Genetic engineering has become pivotal in the treatment of cancer and other genetic diseases, especially the formerly-niche use of clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9. In defining its superior use, we have followed the recent advances that have been made in producing CRISPR/Cas9 as a therapy of choice. We also provide important genetic mutations where CRISPRs can be repurposed to create adaptive immunity to fight carcinomas and edit genetic mutations causing it. Meanwhile, challenges to CRISPR technology are also discussed with emphasis on ability of pathogens to evolve against CRISPRs. We follow the recent developments on the function of CRISPRs with different carriers which can efficiently deliver it to target cells; furthermore, analogous technologies are also discussed along CRISPRs, including zinc-finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs). Moreover, progress in clinical applications of CRISPR therapeutics is reviewed; in effect, patients can have lower morbidity and/or mortality from the therapeutic method with least possible side-effects.

CRISPR/Cas9 therapeutics: a cure for cancer and other genetic diseases

PubMed Central

Khan, Faheem Ahmed; Pandupuspitasari, Nuruliarizki Shinta; Chun-Jie, Huang; Ao, Zhou; Jamal, Muhammad; Zohaib, Ali; Khan, Farhan Ahmed; Hakim, Muthia Raihana; ShuJun, Zhang

2016-01-01

Cancer is caused by a series of alterations in genome and epigenome mostly resulting in activation of oncogenes or inactivation of cancer suppressor genes. Genetic engineering has become pivotal in the treatment of cancer and other genetic diseases, especially the formerly-niche use of clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9. In defining its superior use, we have followed the recent advances that have been made in producing CRISPR/Cas9 as a therapy of choice. We also provide important genetic mutations where CRISPRs can be repurposed to create adaptive immunity to fight carcinomas and edit genetic mutations causing it. Meanwhile, challenges to CRISPR technology are also discussed with emphasis on ability of pathogens to evolve against CRISPRs. We follow the recent developments on the function of CRISPRs with different carriers which can efficiently deliver it to target cells; furthermore, analogous technologies are also discussed along CRISPRs, including zinc-finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs). Moreover, progress in clinical applications of CRISPR therapeutics is reviewed; in effect, patients can have lower morbidity and/or mortality from the therapeutic method with least possible side-effects. PMID:27250031

Functional genomics platform for pooled screening and mammalian genetic interaction maps

PubMed Central

Kampmann, Martin; Bassik, Michael C.; Weissman, Jonathan S.

2014-01-01

Systematic genetic interaction maps in microorganisms are powerful tools for identifying functional relationships between genes and defining the function of uncharacterized genes. We have recently implemented this strategy in mammalian cells as a two-stage approach. First, genes of interest are robustly identified in a pooled genome-wide screen using complex shRNA libraries. Second, phenotypes for all pairwise combinations of hit genes are measured in a double-shRNA screen and used to construct a genetic interaction map. Our protocol allows for rapid pooled screening under various conditions without a requirement for robotics, in contrast to arrayed approaches. Each stage of the protocol can be implemented in ~2 weeks, with additional time for analysis and generation of reagents. We discuss considerations for screen design, and present complete experimental procedures as well as a full computational analysis suite for identification of hits in pooled screens and generation of genetic interaction maps. While the protocols outlined here were developed for our original shRNA-based approach, they can be applied more generally, including to CRISPR-based approaches. PMID:24992097

Imaging dynamic redox processes with genetically encoded probes.

PubMed

Ezeriņa, Daria; Morgan, Bruce; Dick, Tobias P

2014-08-01

Redox signalling plays an important role in many aspects of physiology, including that of the cardiovascular system. Perturbed redox regulation has been associated with numerous pathological conditions; nevertheless, the causal relationships between redox changes and pathology often remain unclear. Redox signalling involves the production of specific redox species at specific times in specific locations. However, until recently, the study of these processes has been impeded by a lack of appropriate tools and methodologies that afford the necessary redox species specificity and spatiotemporal resolution. Recently developed genetically encoded fluorescent redox probes now allow dynamic real-time measurements, of defined redox species, with subcellular compartment resolution, in intact living cells. Here we discuss the available genetically encoded redox probes in terms of their sensitivity and specificity and highlight where uncertainties or controversies currently exist. Furthermore, we outline major goals for future probe development and describe how progress in imaging methodologies will improve our ability to employ genetically encoded redox probes in a wide range of situations. This article is part of a special issue entitled "Redox Signalling in the Cardiovascular System." Copyright © 2014 Elsevier Ltd. All rights reserved.

The links between hypertrophy, reproductive potential and longevity in the Saccharomyces cerevisiae yeast.

PubMed

Molon, Mateusz; Zadrag-Tecza, Renata

2016-01-01

The yeast Saccharomyces cerevisiae has long been used as a model organism for studying the basic mechanisms of aging. However, the main problem with the use of this unicellular fungus is the unit of "longevity". For all organisms, lifespan is expressed in units of time, while in the case of yeast it is defined by the number of daughter cells produced. Additionally, in yeast the phenotypic effects of mutations often show a clear dependence on the genetic background, suggesting the need for an analysis of strains representing different genetic backgrounds. Our results confirm the data presented in earlier papers that the reproductive potential is strongly associated with an increase in cell volume per generation. An excessive cell volume results in the loss of reproductive capacity. These data clearly support the hypertrophy hypothesis. The time of life of all analysed mutants, with the exception of sch9D, is the same as in the case of the wild-type strain. Interestingly, the 121% increase of the fob1D mutant's reproductive potential compared to the sfp1D mutant does not result in prolongation of the mutant's time of life (total lifespan).

Isolation and Characterization of Chromosome-Gain and Increase-in-Ploidy Mutants in Yeast

PubMed Central

Chan, CSM.; Botstein, D.

1993-01-01

We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an increase-in-ploidy (ipl) phenotype, which, in each case, cosegregates with a temperature-sensitive growth phenotype. Four new alleles of CDC31, which is required for spindle pole body duplication, were also recovered from this screen. Temperature-shift experiments with ipl1 cells show that they suffer severe nondisjunction at 37°. Similar experiments with ipl2 cells show that they gain entire sets of chromosomes and become arrested as unbudded cells at 37°. Molecular cloning and genetic mapping show that IPL1 is a newly identified gene, whereas IPL2 is allelic to BEM2, which is required for normal bud growth. PMID:8293973

Synthetic biology and metabolic engineering.

PubMed

Stephanopoulos, Gregory

2012-11-16

Metabolic engineering emerged 20 years ago as the discipline occupied with the directed modification of metabolic pathways for the microbial synthesis of various products. As such, it deals with the engineering (design, construction, and optimization) of native as well as non-natural routes of product synthesis, aided in this task by the availability of synthetic DNA, the core enabling technology of synthetic biology. The two fields, however, only partially overlap in their interest in pathway engineering. While fabrication of biobricks, synthetic cells, genetic circuits, and nonlinear cell dynamics, along with pathway engineering, have occupied researchers in the field of synthetic biology, the sum total of these areas does not constitute a coherent definition of synthetic biology with a distinct intellectual foundation and well-defined areas of application. This paper reviews the origins of the two fields and advances two distinct paradigms for each of them: that of unit operations for metabolic engineering and electronic circuits for synthetic biology. In this context, metabolic engineering is about engineering cell factories for the biological manufacturing of chemical and pharmaceutical products, whereas the main focus of synthetic biology is fundamental biological research facilitated by the use of synthetic DNA and genetic circuits.

A Western blot-based investigation of the yeast secretory pathway designed for an intermediate-level undergraduate cell biology laboratory.

PubMed

Hood-Degrenier, Jennifer K

2008-01-01

The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor.

Reprogramming cancer cells: overview & current progress.

PubMed

Lim, Kian Lam; Teoh, Hoon Koon; Choong, Pei Feng; Teh, Hui Xin; Cheong, Soon Keng; Kamarul, Tunku

2016-07-01

Cancer is a disease with genetic and epigenetic origins, and the possible effects of reprogramming cancer cells using the defined sets of transcription factors remain largely uninvestigated. In the handful of publications available so far, findings have shown that reprogramming cancer cells changed the characteristics of the cells to differ from the parental cancer cells. These findings indicated the possibility of utilizing reprogramming technology to create a disease model in the laboratory to be used in studying the molecular pathogenesis or for drug screening of a particular cancer model. Despite numerous methods employed in generating induced pluripotent stem cells (iPSCs) from cancer cells only a few studies have successfully reprogrammed malignant human cells. In this review we will provide an overview on i) methods to reprogram cancer cells, ii) characterization of the reprogrammed cancer cells, and iii) the differential effects of reprogramming on malignancy, epigenetics and response of the cancer cells to chemotherapeutic agents. Continued technical progress in cancer cell reprogramming technology will be instrumental for more refined in vitro disease models and ultimately for the development of directed and personalized therapy for cancer patients in the future.

Hematopoietic Stem Cells Are the Major Source of Multilineage Hematopoiesis in Adult Animals.

PubMed

Sawai, Catherine M; Babovic, Sonja; Upadhaya, Samik; Knapp, David J H F; Lavin, Yonit; Lau, Colleen M; Goloborodko, Anton; Feng, Jue; Fujisaki, Joji; Ding, Lei; Mirny, Leonid A; Merad, Miriam; Eaves, Connie J; Reizis, Boris

2016-09-20

Hematopoietic stem cells (HSCs) sustain long-term reconstitution of hematopoiesis in transplantation recipients, yet their role in the endogenous steady-state hematopoiesis remains unclear. In particular, recent studies suggested that HSCs provide a relatively minor contribution to immune cell development in adults. We directed transgene expression in a fraction of HSCs that maintained reconstituting activity during serial transplantations. Inducible genetic labeling showed that transgene-expressing HSCs gave rise to other phenotypic HSCs, confirming their top position in the differentiation hierarchy. The labeled HSCs rapidly contributed to committed progenitors of all lineages and to mature myeloid cells and lymphocytes, but not to B-1a cells or tissue macrophages. Importantly, labeled HSCs gave rise to more than two-thirds of all myeloid cells and platelets in adult mice, and this contribution could be accelerated by an induced interferon response. Thus, classically defined HSCs maintain immune cell development in the steady state and during systemic cytokine responses. Copyright © 2016 Elsevier Inc. All rights reserved.

ATM and MET kinases are synthetic lethal with non-genotoxic activation of p53

PubMed Central

Sullivan, Kelly D.; Padilla-Just, Nuria; Henry, Ryan E.; Porter, Christopher C.; Kim, Jihye; Tentler, John J.; Eckhardt, S. Gail; Tan, Aik Choon; DeGregori, James; Espinosa, Joaquín M.

2012-01-01

The p53 tumor suppressor orchestrates alternative stress responses including cell cycle arrest and apoptosis, but the mechanisms defining cell fate upon p53 activation are poorly understood. Several small molecule activators of p53 have been developed, including Nutlin-3, but their therapeutic potential is limited by the fact that they induce reversible cell cycle arrest in most cancer cell types. We report here the results of a ‘Synthetic Lethal with Nutlin-3’ genome-wide shRNA screen, which revealed that the ATM and MET kinases govern cell fate choice upon p53 activation. Genetic or pharmacological interference with ATM or MET activity converts the cellular response from cell cycle arrest into apoptosis in diverse cancer cell types without affecting expression of key p53 target genes. ATM and MET inhibitors enable Nutlin-3 to kill tumor spheroids. These results identify novel pathways controlling the cellular response to p53 activation and aid in the design of p53-based therapies. PMID:22660439

Analysis of mutational spectra by denaturant capillary electrophoresis

PubMed Central

Ekstrøm, Per O.; Khrapko, Konstantin; Li-Sucholeiki, Xiao-Cheng; Hunter, Ian W.; Thilly, William G.

2009-01-01

Numbers and kinds of point mutant within DNA from cells, tissues and human population may be discovered for nearly any 75–250bp DNA sequence. High fidelity DNA amplification incorporating a thermally stable DNA “clamp” is followed by separation by denaturing capillary electrophoresis (DCE). DCE allows for peak collection and verification sequencing. DCE in a mode of cycling temperature, e.g.+/− 5°C, CyDCE, permits high resolution of mutant sequences using computer defined analytes without preliminary optimization experiments. DNA sequencers have been modified to permit higher throughput CyDCE and a massively parallel,~25,000 capillary system, has been designed for pangenomic scans in large human populations. DCE has been used to define quantitative point mutational spectra for study a wide variety of genetic phenomena: errors of DNA polymerases, mutations induced in human cells by chemicals and irradiation, testing of human gene-common disease associations and the discovery of origins of point mutations in human development and carcinogenesis. PMID:18600220

FlyMAD: rapid thermogenetic control of neuronal activity in freely walking Drosophila.

PubMed

Bath, Daniel E; Stowers, John R; Hörmann, Dorothea; Poehlmann, Andreas; Dickson, Barry J; Straw, Andrew D

2014-07-01

Rapidly and selectively modulating the activity of defined neurons in unrestrained animals is a powerful approach in investigating the circuit mechanisms that shape behavior. In Drosophila melanogaster, temperature-sensitive silencers and activators are widely used to control the activities of genetically defined neuronal cell types. A limitation of these thermogenetic approaches, however, has been their poor temporal resolution. Here we introduce FlyMAD (the fly mind-altering device), which allows thermogenetic silencing or activation within seconds or even fractions of a second. Using computer vision, FlyMAD targets an infrared laser to freely walking flies. As a proof of principle, we demonstrated the rapid silencing and activation of neurons involved in locomotion, vision and courtship. The spatial resolution of the focused beam enabled preferential targeting of neurons in the brain or ventral nerve cord. Moreover, the high temporal resolution of FlyMAD allowed us to discover distinct timing relationships for two neuronal cell types previously linked to courtship song.

PRUNE is crucial for normal brain development and mutated in microcephaly with neurodevelopmental impairment.

PubMed

Zollo, Massimo; Ahmed, Mustafa; Ferrucci, Veronica; Salpietro, Vincenzo; Asadzadeh, Fatemeh; Carotenuto, Marianeve; Maroofian, Reza; Al-Amri, Ahmed; Singh, Royana; Scognamiglio, Iolanda; Mojarrad, Majid; Musella, Luca; Duilio, Angela; Di Somma, Angela; Karaca, Ender; Rajab, Anna; Al-Khayat, Aisha; Mohan Mohapatra, Tribhuvan; Eslahi, Atieh; Ashrafzadeh, Farah; Rawlins, Lettie E; Prasad, Rajniti; Gupta, Rashmi; Kumari, Preeti; Srivastava, Mona; Cozzolino, Flora; Kumar Rai, Sunil; Monti, Maria; Harlalka, Gaurav V; Simpson, Michael A; Rich, Philip; Al-Salmi, Fatema; Patton, Michael A; Chioza, Barry A; Efthymiou, Stephanie; Granata, Francesca; Di Rosa, Gabriella; Wiethoff, Sarah; Borgione, Eugenia; Scuderi, Carmela; Mankad, Kshitij; Hanna, Michael G; Pucci, Piero; Houlden, Henry; Lupski, James R; Crosby, Andrew H; Baple, Emma L

2017-04-01

PRUNE is a member of the DHH (Asp-His-His) phosphoesterase protein superfamily of molecules important for cell motility, and implicated in cancer progression. Here we investigated multiple families from Oman, India, Iran and Italy with individuals affected by a new autosomal recessive neurodevelopmental and degenerative disorder in which the cardinal features include primary microcephaly and profound global developmental delay. Our genetic studies identified biallelic mutations of PRUNE1 as responsible. Our functional assays of disease-associated variant alleles revealed impaired microtubule polymerization, as well as cell migration and proliferation properties, of mutant PRUNE. Additionally, our studies also highlight a potential new role for PRUNE during microtubule polymerization, which is essential for the cytoskeletal rearrangements that occur during cellular division and proliferation. Together these studies define PRUNE as a molecule fundamental for normal human cortical development and define cellular and clinical consequences associated with PRUNE mutation. © The Author (2017). Published by Oxford University Press on behalf of the Guarantors of Brain.

HLA-B*57 and IFNL4-Related Polymorphisms Are Associated With Protection Against HIV-1 Disease Progression in Controllers

PubMed Central

Dominguez-Molina, Beatriz; Tarancon-Diez, Laura; Hua, Stephane; Abad-Molina, Cristina; Rodriguez-Gallego, Esther; Machmach, Kawthar; Vidal, Francesc; Tural, Cristina; Moreno, Santiago; Goñi, María José; Ramírez de Arellano, Elena; del Val, Margarita; Gonzalez-Escribano, María Francisca; Del Romero, Jorge; Rodriguez, Carmen; Capa, Laura; Viciana, Pompeyo; Alcamí, José; Yu, Xu G.; Walker, Bruce D.; Leal, Manuel; Lichterfeld, Mathias

2017-01-01

Abstract Background. Human immunodeficiency virus type 1 (HIV-1) controllers maintain HIV-1 viremia at low levels (normally <2000 HIV-RNA copies/mL) without antiretroviral treatment. However, some HIV-1 controllers have evidence of immunologic progression with marked CD4+ T-cell decline. We investigated host genetic factors associated with protection against CD4+ T-cell loss in HIV-1 controllers. Methods. We analyzed the association of interferon-lambda 4 (IFNL4)–related polymorphisms and human leukocyte antigen (HLA)-B haplotypes within long-term nonprogressor HIV-1 controllers (LTNP-Cs; defined by maintaining CD4+ T-cells counts >500 cells/mm3 for more than 7 years after HIV-1 diagnosis) vs non-LTNP-Cs who developed CD4+ T-cell counts <500 cells/mm3. Both a Spanish study cohort (n = 140) and an international validation cohort (n = 914) were examined. Additionally, in a subgroup of individuals, HIV-1–specific T-cell responses and soluble cytokines were analyzed. Results. HLA-B*57 was independently associated with the LTNP-C phenotype (odds ratio [OR], 3.056 [1.029–9.069]; P = .044 and OR, 1.924 [1.252–2.957]; P = .003) while IFNL4 genotypes represented independent factors for becoming non-LTNP-C (TT/TT, ss469415590; OR, 0.401 [0.171–0.942]; P = .036 or A/A, rs12980275; OR, 0.637 [0.434–0.934]; P = .021) in the Spanish and validation cohorts, respectively, after adjusting for sex, age at HIV-1 diagnosis, IFNL4-related polymorphisms, and different HLA-B haplotypes. LTNP-Cs showed lower plasma induced protein 10 (P = .019) and higher IFN-γ (P = .02) levels than the HIV-1 controllers with diminished CD4+ T-cell numbers. Moreover, LTNP-Cs exhibited higher quantities of interleukin (IL)2+CD57- and IFN-γ +CD57- HIV-1–specific CD8+ T cells (P = .002 and .041, respectively) than non-LTNP-Cs. Conclusions. We defined genetic markers able to segregate stable HIV-1 controllers from those who experience CD4+ T-cell decline. These findings allow for identification of HIV-1 controllers at risk for immunologic progression and provide avenues for personalized therapeutic interventions and precision medicine for optimizing clinical care of these individuals. PMID:27986689

Unisexual and Heterosexual Meiotic Reproduction Generate Aneuploidy and Phenotypic Diversity De Novo in the Yeast Cryptococcus neoformans

PubMed Central

Li, Wenjun; Floyd-Averette, Anna; Mieczkowski, Piotr; Dietrich, Fred S.; Heitman, Joseph

2013-01-01

Aneuploidy is known to be deleterious and underlies several common human diseases, including cancer and genetic disorders such as trisomy 21 in Down's syndrome. In contrast, aneuploidy can also be advantageous and in fungi confers antifungal drug resistance and enables rapid adaptive evolution. We report here that sexual reproduction generates phenotypic and genotypic diversity in the human pathogenic yeast Cryptococcus neoformans, which is globally distributed and commonly infects individuals with compromised immunity, such as HIV/AIDS patients, causing life-threatening meningoencephalitis. C. neoformans has a defined a-α opposite sexual cycle; however, >99% of isolates are of the α mating type. Interestingly, α cells can undergo α-α unisexual reproduction, even involving genotypically identical cells. A central question is: Why would cells mate with themselves given that sex is costly and typically serves to admix preexisting genetic diversity from genetically divergent parents? In this study, we demonstrate that α-α unisexual reproduction frequently generates phenotypic diversity, and the majority of these variant progeny are aneuploid. Aneuploidy is responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Phenotypic/genotypic changes were not observed following asexual mitotic reproduction. Aneuploidy was also detected in progeny from a-α opposite-sex congenic mating; thus, both homothallic and heterothallic sexual reproduction can generate phenotypic diversity de novo. Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling de novo genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments. PMID:24058295

Efficacy of BET bromodomain inhibition in Kras-mutant non-small cell lung cancer

PubMed Central

Shimamura, Takeshi; Chen, Zhao; Soucheray, Margaret; Carretero, Julian; Kikuchi, Eiki; Tchaicha, Jeremy H.; Gao, Yandi; Cheng, Katherine A.; Cohoon, Travis J.; Qi, Jun; Akbay, Esra; Kimmelman, Alec C.; Kung, Andrew L.; Bradner, James E.; Wong, Kwok-Kin

2013-01-01

Purpose Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as the dependency of mutant Kras tumors on MYC function. Unfortunately, drug-like small-molecule inhibitors of KRAS and MYC have yet to be realized. The recent discovery, in hematologic malignancies, that BET bromodomain inhibition impairs MYC expression and MYC transcriptional function established the rationale of targeting KRAS-driven NSCLC with BET inhibition. Experimental Design We performed functional assays to evaluate the effects of JQ1 in genetically defined NSCLC cells lines harboring KRAS and/or LKB1 mutations. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. Effects of bromodomain inhibition on transcriptional pathways were explored and validated by expression analysis. Results While JQ1 is broadly active in NSCLC cells, activity of JQ1 in mutant KRAS NSCLC is abrogated by concurrent alteration or genetic knock-down of LKB1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1. Conclusion Bromodomain inhibition comprises a promising therapeutic strategy for KRAS mutant NSCLC with wild-type LKB1, via inhibition of MYC function. Clinical studies of BET bromodomain inhibitors in aggressive NSCLC will be actively pursued. PMID:24045185

Placenta and Placental Derivatives in Regenerative Therapies: Experimental Studies, History, and Prospects

PubMed Central

Prokopyuk, Volodymyr; Pogozhykh, Denys

2018-01-01

Placental structures, capable to persist in a genetically foreign organism, are a natural model of allogeneic engraftment carrying a number of distinctive properties. In this review, the main features of the placenta and its derivatives such as structure, cellular composition, immunological and endocrine aspects, and the ability to invasion and deportation are discussed. These features are considered from a perspective that determines the placental material as a unique source for regenerative cell therapies and a lesson for immunological tolerance. A historical overview of clinical applications of placental extracts, cells, and tissue components is described. Empirically accumulated data are summarized and compared with modern research. Furthermore, we define scopes and outlooks of application of placental cells and tissues in the rapidly progressing field of regenerative medicine. PMID:29535770

Defining Causative Factors Contributing in the Activation of Hedgehog Signaling in Diffuse Large B-Cell Lymphoma

PubMed Central

Ramirez, Elisa; Singh, Rajesh R; Kunkalla, Kranthi; Liu, Yadong; Qu, Changju; Cain, Christine; Multani, Asha S.; Lennon, Patrick A; Jackacky, Jared; Ho, Michael; Dawud, Sity; Gu, Jun; Yang, Su; Hu, Peter C; Vega, Francisco

2012-01-01

Hedgehog (Hh) signaling pathway is activated in diffuse large B-cell lymphoma (DLBCL). Genetic abnormalities that explain activation of Hh signaling in DLBCL are unknown. We investigate the presence of amplifications of Hh genes that might result in activation of this pathway in DLBCL. Our data showed few extra copies of GLI1 and SMO due to chromosomal aneuploidies in a subset of DLBCL cell lines. We also showed that pharmacologic inhibition of PI3K/AKT and NF-KB pathways resulted in decreased expression of GLI1 and Hh ligands. In conclusion, our data support the hypothesis that aberrant activation of Hh signaling in DLBCL mainly results from integration of deregulated oncogenic signaling inputs converging into Hh signaling. PMID:22809693

Identification of evolutionary hotspots based on genetic data from multiple terrestrial and aquatic taxa and gap analysis of hotspots in protected lands encompassed by the South Atlantic Landscape Conservation Cooperative.

USGS Publications Warehouse

Robinson, J.; Snider, M.; Duke, J.; Moyer, G.R.

2014-01-01

 The southeastern United States is a recognized hotspot of biodiversity for a variety of aquatic taxa, including fish, amphibians, and mollusks. Unfortunately, the great diversity of the area is accompanied by a large proportion of species at risk of extinction . Gap analysis was employed to assess the representation of evolutionary hotspots in protected lands w h ere an evolutionary hotspot was defined as an area with high evolutionary potential and measured by atypical patterns of genetic divergence, genetic diversity, and to a lesser extent genetic similarity across multiple terrestrial or aquatic taxa. A survey of the primary literature produced 16 terrestrial and 14 aquatic genetic datasets for estimation of genetic divergence and diversity. Relative genetic diversity and divergence values for each terrestrial and aquatic dataset were used for interpolation of multispecies genetic surfaces and subsequent visualization using ArcGIS. The multispecies surfaces interpolated from relative divergences and diversity data identified numerous evolutionary hotspots for both terrestrial and aquatic taxa , many of which were afforded some current protection. For instance, 14% of the cells identified as hotspots of aquatic diversity were encompassed by currently protected areas. Additionally, 25% of the highest 1% of terrestrial diversity cells were afforded some level of protection. In contrast, areas of high and low divergence among species, and areas of high variance in diversity were poorly represented in the protected lands. Of particular interest were two areas that were consistently identified by several different measures as important from a conservation perspective. These included an area encompassing the panhandle of Florida and southern Georgia near the Apalachicola National Forest (displaying varying levels of genetic divergence and greater than average levels of genetic diversity) and a large portion of the coastal regions of North and South Carolina (displaying low genetic divergence and greater than average levels of genetic diversity) . Our results show the utility o f genetic data sets for identifying cross - species patterns of genetic  diversity and divergence (i.e., evolutionary hotspots) in aquatic and terrestrial environments for use in conservation design and delivery across the southeastern United States. 

Commensurate distances and similar motifs in genetic congruence and protein interaction networks in yeast

PubMed Central

Ye, Ping; Peyser, Brian D; Spencer, Forrest A; Bader, Joel S

2005-01-01

Background In a genetic interaction, the phenotype of a double mutant differs from the combined phenotypes of the underlying single mutants. When the single mutants have no growth defect, but the double mutant is lethal or exhibits slow growth, the interaction is termed synthetic lethality or synthetic fitness. These genetic interactions reveal gene redundancy and compensating pathways. Recently available large-scale data sets of genetic interactions and protein interactions in Saccharomyces cerevisiae provide a unique opportunity to elucidate the topological structure of biological pathways and how genes function in these pathways. Results We have defined congruent genes as pairs of genes with similar sets of genetic interaction partners and constructed a genetic congruence network by linking congruent genes. By comparing path lengths in three types of networks (genetic interaction, genetic congruence, and protein interaction), we discovered that high genetic congruence not only exhibits correlation with direct protein interaction linkage but also exhibits commensurate distance with the protein interaction network. However, consistent distances were not observed between genetic and protein interaction networks. We also demonstrated that congruence and protein networks are enriched with motifs that indicate network transitivity, while the genetic network has both transitive (triangle) and intransitive (square) types of motifs. These results suggest that robustness of yeast cells to gene deletions is due in part to two complementary pathways (square motif) or three complementary pathways, any two of which are required for viability (triangle motif). Conclusion Genetic congruence is superior to genetic interaction in prediction of protein interactions and function associations. Genetically interacting pairs usually belong to parallel compensatory pathways, which can generate transitive motifs (any two of three pathways needed) or intransitive motifs (either of two pathways needed). PMID:16283923

A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates.

PubMed

Plouhinec, Jean-Louis; Medina-Ruiz, Sofía; Borday, Caroline; Bernard, Elsa; Vert, Jean-Philippe; Eisen, Michael B; Harland, Richard M; Monsoro-Burq, Anne H

2017-10-01

During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research.

A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates

PubMed Central

Borday, Caroline; Bernard, Elsa; Vert, Jean-Philippe; Eisen, Michael B.; Harland, Richard M.

2017-01-01

During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research. PMID:29049289

Medical privacy and the disclosure of personal medical information: the beliefs and experiences of those with genetic and other clinical conditions.

PubMed

Kass, Nancy E; Hull, Sara Chandros; Natowicz, Marvin R; Faden, Ruth R; Plantinga, Laura; Gostin, Lawrence O; Slutsman, Julia

2004-07-30

There has been heightened legislative attention to medical privacy and to protections from genetic discrimination, without large-scale studies to document privacy concerns or analysis of whether experiences differ by whether the condition is genetic (defined here as a single-gene disorder) or non-genetic. To determine whether experiences regarding privacy, disclosure, and consequences of disclosure differ by whether one's medical condition is genetic, we conducted a descriptive study with one-time, structured quantitative and qualitative interviews. We interviewed approximately 100 adults or parents of children with each of the following medical conditions: sickle cell disease, cystic fibrosis, diabetes, and HIV, and 200 adults with or at risk for breast cancer or colon cancer. The percentages of the total 597 respondents experiencing positive or negative consequences of disclosure and the degree to which experiences differed by whether the condition was genetic were the outcomes of interest. Seventy-four percent were glad and 13% regretted others knew about their condition; these findings did not differ significantly by genetic vs. non-genetic condition. Reports of job and health insurance discrimination were not uncommon for the overall study population (19 and 27%, respectively) but were more likely among those with genetic conditions (30 and 37%, respectively). Legislation and other policy-making should target the needs of persons with all conditions and not focus exclusively on genetic discrimination, given that experiences and concerns generally do not differ based on the genetic etiology of the condition. Copyright 2004 Wiley-Liss, Inc.

Human Induced Hepatic Lineage-Oriented Stem Cells: Autonomous Specification of Human iPS Cells toward Hepatocyte-Like Cells without Any Exogenous Differentiation Factors

PubMed Central

Yanagi, Satoshi; Kato, Chika; Takashima, Ryokichi; Kobayashi, Eiji; Hagiwara, Keitaro; Ochiya, Takahiro

2015-01-01

Preparing targeted cells for medical applications from human induced pluripotent stem cells (hiPSCs) using growth factors, compounds, or gene transfer has been challenging. Here, we report that human induced hepatic lineage-oriented stem cells (hiHSCs) were generated and expanded as a new type of hiPSC under non-typical coculture with feeder cells in a chemically defined hiPSC medium at a very high density. Self-renewing hiHSCs expressed markers of both human embryonic stem cells (hESCs) and hepatocytes. Those cells were highly expandable, markedly enhancing gene expression of serum hepatic proteins and cytochrome P450 enzymes with the omission of FGF-2 from an undefined hiPSC medium. The hepatic specification of hiHSCs was not attributable to the genetic and epigenetic backgrounds of the starting cells, as they were established from distinct donors and different types of cells. Approximately 90% of hiHSCs autonomously differentiated to hepatocyte-like cells, even in a defined minimum medium without any of the exogenous growth factors necessary for hepatic specification. After 12 days of this culture, the differentiated cells significantly enhanced gene expression of serum hepatic proteins (ALB, SERPINA1, TTR, TF, FABP1, FGG, AGT, RBP4, and AHSG), conjugating enzymes (UGT2B4, UGT2B7, UGT2B10, GSTA2, and GSTA5), transporters (SULT2A1, SLC13A5, and SLCO2B1), and urea cycle-related enzymes (ARG1 and CPS1). In addition, the hepatocyte-like cells performed key functions of urea synthesis, albumin secretion, glycogen storage, indocyanine green uptake, and low-density lipoprotein uptake. The autonomous hepatic specification of hiHSCs was due to their culture conditions (coculture with feeder cells in a defined hiPSC medium at a very high density) in self-renewal rather than in differentiation. These results suggest the feasibility of preparing large quantities of hepatocytes as a convenient and inexpensive hiPSC differentiation. Our study also suggests the necessity of optimizing culture conditions to generate other specific lineage-oriented hiPSCs, allowing for a very simple differentiation. PMID:25875613

[Medulloblastoma. Pathology].

PubMed

Siegfried, A; Delisle, M-B

2018-04-24

Medulloblastomas, embryonal neuroepithelial tumors developed in the cerebellum or brain stem, are mainly observed in childhood. The treatment of WHO-Grade IV tumors depends on stratifications that are usually based on postoperative data, histopathological subtype, tumor extension and presence of MYC or NMYC amplifications. Recently, molecular biology studies, based on new technologies (i.e. sequencing, transcriptomic, methylomic) have introduced genetic subtypes integrated into the latest WHO-2016 neuropathological classification. According to this classification, the three genetic groups WNT, SHH, with or without mutated TP53 gene, and non-WNT/non-SHH, comprising subgroups 3 and 4, are recalled in this review. The contribution of immunohistochemistry to define these groups is specified. The four histopathological groups are detailed in comparison to the WHO-2007 classification and the molecular data: classic medulloblastoma, desmoplastic/nodular medulloblastoma, medulloblastoma with extensive nodularity, and large cell/anaplastic medulloblastoma. The groups defined on genetic and histopathological grounds are not strictly concordant. Depending on the age of the patients, their correlations are different, as well as their role in the management and prognosis of these tumors. Other embryonal tumors, for which new classifications are in progress and gliomas may be confused with a medulloblastoma and the elements of the differential diagnosis of these entities are discussed. This evolution in classification fully justifies ongoing structuring procedures such as histopathological review (RENOCLIP) and the organization of molecular biology platforms. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Diverse Genetic Regulon of the Virulence-Associated Transcriptional Regulator MucR in Brucella abortus 2308

PubMed Central

Caswell, Clayton C.; Elhassanny, Ahmed E. M.; Planchin, Emilie E.; Roux, Christelle M.; Weeks-Gorospe, Jenni N.; Ficht, Thomas A.; Dunman, Paul M.

2013-01-01

The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella. Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 (nolR), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [nolR]), and in Brucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants. PMID:23319565

Perivascular epithelioid cell tumors of the uterine cervix.

PubMed

Kudela, E; Biringer, K; Kasajova, P; Nachajova, M; Adamkov, M

2016-08-01

The World Health Organization (WHO) defines PEComas as mesenchymal tumors composed of histologically and immunohistochemically distinctive perivascular cells. Uterus is the most common site of a subgroup of PEComas not otherwise specified(NOS). PEComas of the uterine cervix are extremely rare, and only thirteen cases have been described in the English literature to date. In this review, we summarize the available data concerning diagnostics, immunohistochemical analysis, genetics and treatment of cervical PEComas. Radical hysterectomy with bilateral salpingooophorectomy is the best surgical approach available. Adjuvant therapy in its present form is not efficient. Therefore, further studies are needed to evaluate the newest treatment strategies. Copyright © 2016 Elsevier GmbH. All rights reserved.

Adoptive immunotherapy for the treatment of glioblastoma: progress and possibilities.

PubMed

Kuramitsu, Shunichiro; Yamamichi, Akane; Ohka, Fumiharu; Motomura, Kazuya; Hara, Masahito; Natsume, Atsushi

2016-12-01

Patients with glioblastoma have a very poor prognosis. Adoptive cellular therapy (ACT) is defined as the collection of circulating or tumor-infiltrating lymphocytes, their selection, modification, expansion and activation, and their re-administration to patients in order to induce antitumor activity. Although various ACTs have been attempted, most failed to improve the outcome. Immune checkpoint blockade antibodies and T cell engineering with tumor-specific chimeric antigen receptors suggest the emergence of a new era of immunotherapy. Here, we summarize approaches with ACTs using genetically modified T cells, which have been improved by enhancing their antitumor activity, and discuss strategies to develop these therapies. The mechanisms by which gliomas modulate and evade the immune system are also discussed.

Capturing neutral and adaptive genetic diversity for conservation in a highly structured tree species.

PubMed

Rodríguez-Quilón, Isabel; Santos-Del-Blanco, Luis; Serra-Varela, María Jesús; Koskela, Jarkko; González-Martínez, Santiago C; Alía, Ricardo

2016-10-01

Preserving intraspecific genetic diversity is essential for long-term forest sustainability in a climate change scenario. Despite that, genetic information is largely neglected in conservation planning, and how conservation units should be defined is still heatedly debated. Here, we use maritime pine (Pinus pinaster Ait.), an outcrossing long-lived tree with a highly fragmented distribution in the Mediterranean biodiversity hotspot, to prove the importance of accounting for genetic variation, of both neutral molecular markers and quantitative traits, to define useful conservation units. Six gene pools associated to distinct evolutionary histories were identified within the species using 12 microsatellites and 266 single nucleotide polymorphisms (SNPs). In addition, height and survival standing variation, their genetic control, and plasticity were assessed in a multisite clonal common garden experiment (16 544 trees). We found high levels of quantitative genetic differentiation within previously defined neutral gene pools. Subsequent cluster analysis and post hoc trait distribution comparisons allowed us to define 10 genetically homogeneous population groups with high evolutionary potential. They constitute the minimum number of units to be represented in a maritime pine dynamic conservation program. Our results uphold that the identification of conservation units below the species level should account for key neutral and adaptive components of genetic diversity, especially in species with strong population structure and complex evolutionary histories. The environmental zonation approach currently used by the pan-European genetic conservation strategy for forest trees would be largely improved by gradually integrating molecular and quantitative trait information, as data become available. © 2016 by the Ecological Society of America.

A Transcriptome Atlas of Physcomitrella patens Provides Insights into the Evolution and Development of Land Plants.

PubMed

Ortiz-Ramírez, Carlos; Hernandez-Coronado, Marcela; Thamm, Anna; Catarino, Bruno; Wang, Mingyi; Dolan, Liam; Feijó, José A; Becker, Jörg D

2016-02-01

Identifying the genetic mechanisms that underpin the evolution of new organ and tissue systems is an aim of evolutionary developmental biology. Comparative functional genetic studies between angiosperms and bryophytes can define those genetic changes that were responsible for developmental innovations. Here, we report the generation of a transcriptome atlas covering most phases in the life cycle of the model bryophyte Physcomitrella patens, including detailed sporophyte developmental progression. We identified a comprehensive set of sporophyte-specific transcription factors, and found that many of these genes have homologs in angiosperms that function in developmental processes such as flowering and shoot branching. Deletion of the PpTCP5 transcription factor results in development of supernumerary sporangia attached to a single seta, suggesting that it negatively regulates branching in the moss sporophyte. Given that TCP genes repress branching in angiosperms, we suggest that this activity is ancient. Finally, comparison of P. patens and Arabidopsis thaliana transcriptomes led us to the identification of a conserved core of transcription factors expressed in tip-growing cells. We identified modifications in the expression patterns of these genes that could account for developmental differences between P. patens tip-growing cells and A. thaliana pollen tubes and root hairs. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

[Gene therapy for inherited retinal dystrophies].

PubMed

Côco, Monique; Han, Sang Won; Sallum, Juliana Maria Ferraz

2009-01-01

The inherited retinal dystrophies comprise a large number of disorders characterized by a slow and progressive retinal degeneration. They are the result of mutations in genes that express in either the photoreceptor cells or the retinal pigment epithelium. The mode of inheritance can be autosomal dominant, autosomal recessive, X linked recessive, digenic or mitochondrial DNA inherited. At the moment, there is no treatment for these conditions and the patients can expect a progressive loss of vision. Accurate genetic counseling and support for rehabilitation are indicated. Research into the molecular and genetic basis of disease is continually expanding and improving the prospects for rational treatments. In this way, gene therapy, defined as the introduction of exogenous genetic material into human cells for therapeutic purposes, may ultimately offer the greatest treatment for the inherited retinal dystrophies. The eye is an attractive target for gene therapy because of its accessibility, immune privilege and translucent media. A number of retinal diseases affecting the eye have known gene defects. Besides, there is a well characterized animal model for many of these conditions. Proposals for clinical trials of gene therapy for inherited retinal degenerations owing to defects in the gene RPE65, have recently received ethical approval and the obtained preliminary results brought large prospects in the improvement on patient's quality of life.

Transgenic mouse models in the study of reproduction: insights into GATA protein function.

PubMed

Tevosian, Sergei G

2014-07-01

For the past 2 decades, transgenic technology in mice has allowed for an unprecedented insight into the transcriptional control of reproductive development and function. The key factor among the mouse genetic tools that made this rapid advance possible is a conditional transgenic approach, a particularly versatile method of creating gene deletions and substitutions in the mouse genome. A centerpiece of this strategy is an enzyme, Cre recombinase, which is expressed from defined DNA regulatory elements that are active in the tissue of choice. The regulatory DNA element (either genetically engineered or natural) assures Cre expression only in predetermined cell types, leading to the guided deletion of genetically modified (flanked by loxP or 'floxed' by loxP) gene loci. This review summarizes and compares the studies in which genes encoding GATA family transcription factors were targeted either globally or by Cre recombinases active in the somatic cells of ovaries and testes. The conditional gene loss experiments require detailed knowledge of the spatial and temporal expression of Cre activity, and the challenges in interpreting the outcomes are highlighted. These studies also expose the complexity of GATA-dependent regulation of gonadal gene expression and suggest that gene function is highly context dependent. © 2014 Society for Reproduction and Fertility.

Multiple levels of redundant processes inhibit Caenorhabditis elegans vulval cell fates.

PubMed

Andersen, Erik C; Saffer, Adam M; Horvitz, H Robert

2008-08-01

Many mutations cause obvious abnormalities only when combined with other mutations. Such synthetic interactions can be the result of redundant gene functions. In Caenorhabditis elegans, the synthetic multivulva (synMuv) genes have been grouped into multiple classes that redundantly inhibit vulval cell fates. Animals with one or more mutations of the same class undergo wild-type vulval development, whereas animals with mutations of any two classes have a multivulva phenotype. By varying temperature and genetic background, we determined that mutations in most synMuv genes within a single synMuv class enhance each other. However, in a few cases no enhancement was observed. For example, mutations that affect an Mi2 homolog and a histone methyltransferase are of the same class and do not show enhancement. We suggest that such sets of genes function together in vivo and in at least some cases encode proteins that interact physically. The approach of genetic enhancement can be applied more broadly to identify potential protein complexes as well as redundant processes or pathways. Many synMuv genes are evolutionarily conserved, and the genetic relationships we have identified might define the functions not only of synMuv genes in C. elegans but also of their homologs in other organisms.

Multiple Levels of Redundant Processes Inhibit Caenorhabditis elegans Vulval Cell Fates

PubMed Central

Andersen, Erik C.; Saffer, Adam M.; Horvitz, H. Robert

2008-01-01

Many mutations cause obvious abnormalities only when combined with other mutations. Such synthetic interactions can be the result of redundant gene functions. In Caenorhabditis elegans, the synthetic multivulva (synMuv) genes have been grouped into multiple classes that redundantly inhibit vulval cell fates. Animals with one or more mutations of the same class undergo wild-type vulval development, whereas animals with mutations of any two classes have a multivulva phenotype. By varying temperature and genetic background, we determined that mutations in most synMuv genes within a single synMuv class enhance each other. However, in a few cases no enhancement was observed. For example, mutations that affect an Mi2 homolog and a histone methyltransferase are of the same class and do not show enhancement. We suggest that such sets of genes function together in vivo and in at least some cases encode proteins that interact physically. The approach of genetic enhancement can be applied more broadly to identify potential protein complexes as well as redundant processes or pathways. Many synMuv genes are evolutionarily conserved, and the genetic relationships we have identified might define the functions not only of synMuv genes in C. elegans but also of their homologs in other organisms. PMID:18689876

Defining a genetic ideotype for crop improvement.

PubMed

Trethowan, Richard M

2014-01-01

While plant breeders traditionally base selection on phenotype, the development of genetic ideotypes can help focus the selection process. This chapter provides a road map for the establishment of a refined genetic ideotype. The first step is an accurate definition of the target environment including the underlying constraints, their probability of occurrence, and impact on phenotype. Once the environmental constraints are established, the wealth of information on plant physiological responses to stresses, known gene information, and knowledge of genotype ×environment and gene × environment interaction help refine the target ideotype and form a basis for cross prediction.Once a genetic ideotype is defined the challenge remains to build the ideotype in a plant breeding program. A number of strategies including marker-assisted recurrent selection and genomic selection can be used that also provide valuable information for the optimization of genetic ideotype. However, the informatics required to underpin the realization of the genetic ideotype then becomes crucial. The reduced cost of genotyping and the need to combine pedigree, phenotypic, and genetic data in a structured way for analysis and interpretation often become the rate-limiting steps, thus reducing genetic gain. Systems for managing these data and an example of ideotype construction for a defined environment type are discussed.

Multifunctional glial support by Semper cells in the Drosophila retina

PubMed Central

Charlton-Perkins, Mark A.

2017-01-01

Glial cells play structural and functional roles central to the formation, activity and integrity of neurons throughout the nervous system. In the retina of vertebrates, the high energetic demand of photoreceptors is sustained in part by Müller glia, an intrinsic, atypical radial glia with features common to many glial subtypes. Accessory and support glial cells also exist in invertebrates, but which cells play this function in the insect retina is largely undefined. Using cell-restricted transcriptome analysis, here we show that the ommatidial cone cells (aka Semper cells) in the Drosophila compound eye are enriched for glial regulators and effectors, including signature characteristics of the vertebrate visual system. In addition, cone cell-targeted gene knockdowns demonstrate that such glia-associated factors are required to support the structural and functional integrity of neighboring photoreceptors. Specifically, we show that distinct support functions (neuronal activity, structural integrity and sustained neurotransmission) can be genetically separated in cone cells by down-regulating transcription factors associated with vertebrate gliogenesis (pros/Prox1, Pax2/5/8, and Oli/Olig1,2, respectively). Further, we find that specific factors critical for glial function in other species are also critical in cone cells to support Drosophila photoreceptor activity. These include ion-transport proteins (Na/K+-ATPase, Eaat1, and Kir4.1-related channels) and metabolic homeostatic factors (dLDH and Glut1). These data define genetically distinct glial signatures in cone/Semper cells that regulate their structural, functional and homeostatic interactions with photoreceptor neurons in the compound eye of Drosophila. In addition to providing a new high-throughput model to study neuron-glia interactions, the fly eye will further help elucidate glial conserved "support networks" between invertebrates and vertebrates. PMID:28562601

Roles of specific Kv channel types in repolarization of the action potential in genetically identified subclasses of pyramidal neurons in mouse neocortex

PubMed Central

Pathak, Dhruba; Guan, Dongxu

2016-01-01

The action potential (AP) is a fundamental feature of excitable cells that serves as the basis for long-distance signaling in the nervous system. There is considerable diversity in the appearance of APs and the underlying repolarization mechanisms in different neuronal types (reviewed in Bean BP. Nat Rev Neurosci 8: 451–465, 2007), including among pyramidal cell subtypes. In the present work, we used specific pharmacological blockers to test for contributions of Kv1, Kv2, or Kv4 channels to repolarization of single APs in two genetically defined subpopulations of pyramidal cells in layer 5 of mouse somatosensory cortex (etv1 and glt) as well as pyramidal cells from layer 2/3. These three subtypes differ in AP properties (Groh A, Meyer HS, Schmidt EF, Heintz N, Sakmann B, Krieger P. Cereb Cortex 20: 826–836, 2010; Guan D, Armstrong WE, Foehring RC. J Neurophysiol 113: 2014–2032, 2015) as well as laminar position, morphology, and projection targets. We asked what the roles of Kv1, Kv2, and Kv4 channels are in AP repolarization and whether the underlying mechanisms are pyramidal cell subtype dependent. We found that Kv4 channels are critically involved in repolarizing neocortical pyramidal cells. There are also pyramidal cell subtype-specific differences in the role for Kv1 channels. Only Kv4 channels were involved in repolarizing the narrow APs of glt cells. In contrast, in etv1 cells and layer 2/3 cells, the broader APs are partially repolarized by Kv1 channels in addition to Kv4 channels. Consistent with their activation in the subthreshold range, Kv1 channels also regulate AP voltage threshold in all pyramidal cell subtypes. PMID:26864770

Identification of cells initiating human melanomas.

PubMed

Schatton, Tobias; Murphy, George F; Frank, Natasha Y; Yamaura, Kazuhiro; Waaga-Gasser, Ana Maria; Gasser, Martin; Zhan, Qian; Jordan, Stefan; Duncan, Lyn M; Weishaupt, Carsten; Fuhlbrigge, Robert C; Kupper, Thomas S; Sayegh, Mohamed H; Frank, Markus H

2008-01-17

Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies and solid cancers. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5- bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ subpopulations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5- progeny, whereas ABCB5- tumour populations give rise, at lower rates, exclusively to ABCB5- cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy.

Centronuclear myopathies: genotype-phenotype correlation and frequency of defined genetic forms in an Italian cohort.

PubMed

Fattori, Fabiana; Maggi, Lorenzo; Bruno, Claudio; Cassandrini, Denise; Codemo, Valentina; Catteruccia, Michela; Tasca, Giorgio; Berardinelli, Angela; Magri, Francesca; Pane, Marika; Rubegni, Anna; Santoro, Lucio; Ruggiero, Lucia; Fiorini, Patrizio; Pini, Antonella; Mongini, Tiziana; Messina, Sonia; Brisca, Giacomo; Colombo, Irene; Astrea, Guja; Fiorillo, Chiara; Bragato, Cinzia; Moroni, Isabella; Pegoraro, Elena; D'Apice, Maria Rosaria; Alfei, Enrico; Mora, Marina; Morandi, Lucia; Donati, Alice; Evilä, Anni; Vihola, Anna; Udd, Bjarne; Bernansconi, Pia; Mercuri, Eugenio; Santorelli, Filippo Maria; Bertini, Enrico; D'Amico, Adele

2015-07-01

Centronuclear myopathies (CNMs) are a group of clinically and genetically heterogeneous muscle disorders. To date, mutation in 7 different genes has been reported to cause CNMs but 30 % of cases still remain genetically undefined. Genetic investigations are often expensive and time consuming. Clinical and morphological clues are needed to facilitate genetic tests and to choose the best approach for genetic screening. We aimed to describe genotype-phenotype correlation in an Italian cohort of patients affected by CNMs, to define the relative frequencies of its defined genetic forms and to draw a diagnostic algorithm to address genetic investigations. We recruited patients with CNMs from all the Italian tertiary neuromuscular centers following clinical and histological criteria. All selected patients were screened for the four 'canonical' genes related to CNMs: MTM1, DNM2, RYR1 and BIN1. Pathogenetic mutations were found in 38 of the 54 screened patients (70 %), mostly in patients with congenital onset (25 of 30 patients, 83 %): 15 in MTM1, 6 in DNM2, 3 in RYR1 and one in TTN. Among the 13 patients with a childhood-adolescence onset, mutations were found in 6 patients (46 %), all in DNM2. In the group of the 11 patients with adult onset, mutations were identified in 7 patients (63 %), again in DNM2, confirming that variants in this gene are relatively more common in late-onset phenotypes. The present study provides the relative molecular frequency of centronuclear myopathy and of its genetically defined forms in Italy and also proposes a diagnostic algorithm to be used in clinical practice to address genetic investigations.

Etiology and early pathogenesis of malignant testicular germ cell tumors: towards possibilities for preinvasive diagnosis

PubMed Central

Elzinga-Tinke, Jenny E; Dohle, Gert R; Looijenga, Leendert HJ

2015-01-01

Malignant testicular germ cell tumors (TGCT) are the most frequent cancers in Caucasian males (20–40 years) with an 70% increasing incidence the last 20 years, probably due to combined action of (epi)genetic and (micro)environmental factors. It is expected that TGCT have carcinoma in situ (CIS) as their common precursor, originating from an embryonic germ cell blocked in its maturation process. The overall cure rate of TGCT is more than 90%, however, men surviving TGCT can present long-term side effects of systemic cancer treatment. In contrast, men diagnosed and treated for CIS only continue to live without these long-term side effects. Therefore, early detection of CIS has great health benefits, which will require an informative screening method. This review described the etiology and early pathogenesis of TGCT, as well as the possibilities of early detection and future potential of screening men at risk for TGCT. For screening, a well-defined risk profile based on both genetic and environmental risk factors is needed. Since 2009, several genome wide association studies (GWAS) have been published, reporting on single-nucleotide polymorphisms (SNPs) with significant associations in or near the genes KITLG, SPRY4, BAK1, DMRT1, TERT, ATF7IP, HPGDS, MAD1L1, RFWD3, TEX14, and PPM1E, likely to be related to TGCT development. Prenatal, perinatal, and postnatal environmental factors also influence the onset of CIS. A noninvasive early detection method for CIS would be highly beneficial in a clinical setting, for which specific miRNA detection in semen seems to be very promising. Further research is needed to develop a well-defined TGCT risk profile, based on gene-environment interactions, combined with noninvasive detection method for CIS. PMID:25791729

Vaccination for treatment and prevention of cancer in animal models.

PubMed

Cavallo, Federica; Offringa, Rienk; van der Burg, Sjoerd H; Forni, Guido; Melief, Cornelis J M

2006-01-01

Two approaches to immunological intervention in tumor-host interactions in mouse models are discussed in this review. The first is described with reference to experiments in which CD8(+) T lymphocytes are used to kill established transplantable tumors. Peptides and their optimal presentation by dendritic cells and intervention in immune regulatory mechanisms are the key issues for efficient induction of T-killer cell-mediated tumor eradication. The time frame of tumor therapy and the threat imposed by tumor growth in transplantable models and cancer patients require the induction of a robust T-cell reaction. Prevention of the progression of small preneoplastic lesions, on the other hand, requires the significant and prolonged immune protection sought in the second approach. This is based on antibody production and the coordinated activation of multiple low-avidity cell-mediated mechanisms elicited by DNA vaccination in genetically modified cancer-prone mice, transgenic for a mutant Her-2/neu growth factor receptor expressed at the plasma membrane surface of preneoplastic mammary gland epithelial cells. Vaccination with appropriate DNA formulations results in prolonged immune inhibition of the progression of preneoplastic mammary lesions but is ineffective against established tumors. The use of molecularly defined adjuvants and intervention in immune regulatory mechanisms are critical in both the elicitation of an effective T-cell mediated reaction required for tumor debulking in the first set of models and the induction by vaccination of a sustained immune memory able to prevent the expansion of preneoplastic lesions in genetically cancer-prone mice.

Regulation of cell-fate determination in Dictyostelium.

PubMed

Brown, J M; Firtel, R A

1999-12-15

A key step in the development of all multicellular organisms is the differentiation of specialized cell types. The eukaryotic microorganism Dictyostelium discoideum provides a unique experimental system for studying cell-type determination and spatial patterning in a developing multicellular organism. Unlike metazoans, which become multicellular by undergoing many rounds of cell division after fertilization of an egg, the social amoeba Dictyostelium achieves multicellularity by the aggregation of approximately 10(5) cells in response to nutrient depletion. Following aggregation, cell-type differentiation and morphogenesis result in a multicellular organism with only a few cell types that exhibit a defined patterning along the anterior-posterior axis of the organism. Analysis of the mechanisms that control these processes is facilitated by the relative simplicity of Dictyostelium development and the availability of molecular, genetic, and cell biological tools. Interestingly, analysis has shown that many molecules that play integral roles in the development of higher eukaryotes, such as PKA, STATs, and GSK-3, are also essential for cell-type differentiation and patterning in Dictyostelium. The role of these and other signaling pathways in the induction, maintenance, and patterning of cell types during Dictyostelium development is discussed.

Genetic correlations of mid-infrared-predicted milk fatty acid groups with milk production traits.

PubMed

Fleming, A; Schenkel, F S; Malchiodi, F; Ali, R A; Mallard, B; Sargolzaei, M; Jamrozik, J; Johnston, J; Miglior, F

2018-05-01

The objective of this research was to estimate the genetic correlations between milk mid-infrared-predicted fatty acid groups and production traits in first-parity Canadian Holsteins. Contents of short-chain, medium-chain, long-chain, saturated, and unsaturated fatty acid groupings in milk samples can be predicted using mid-infrared spectral data for cows enrolled in milk recording programs. Predicted fatty acid group contents were obtained for 49,127 test-day milk samples from 10,029 first-parity Holstein cows in 810 herds. Milk yield, fat and protein yield, fat and protein percentage, fat-to-protein ratio, and somatic cell score were also available for these test days. Genetic parameters were estimated for the fatty acid groups and production traits using multiple-trait random regression test day models by Bayesian methods via Gibbs sampling. Three separate 8- or 9-trait analyses were performed, including the 5 fatty acid groups with different combinations of the production traits. Posterior standard deviations ranged from <0.001 to 0.01. Average daily genetic correlations were negative and similar to each other for the fatty acid groups with milk yield (-0.62 to -0.59) and with protein yield (-0.32 to -0.25). Weak and positive average daily genetic correlations were found between somatic cell score and the fatty acid groups (from 0.25 to 0.36). Stronger genetic correlations with fat yield, fat and protein percentage, and fat-to-protein ratio were found with medium-chain and saturated fatty acid groups compared with those with long-chain and unsaturated fatty acid groups. Genetic correlations were very strong between the fatty acid groups and fat percentage, ranging between 0.88 for unsaturated and 0.99 for saturated fatty acids. Daily genetic correlations from 5 to 305 d in milk with milk, protein yield and percentage, and somatic cell score traits showed similar patterns for all fatty acid groups. The daily genetic correlations with fat yield at the beginning of lactation were decreasing for long-chain and unsaturated fatty acid groups and increasing for short-chain fatty acids. Genetic correlations between fat percentage and fatty acids were increasing at the beginning of lactation for short- and medium-chain and saturated fatty acids, but slightly decreasing for long-chain and unsaturated fatty acid groups. These results can be used in defining fatty acid traits and breeding objectives. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

OncoSimulR: genetic simulation with arbitrary epistasis and mutator genes in asexual populations.

PubMed

Diaz-Uriarte, Ramon

2017-06-15

OncoSimulR implements forward-time genetic simulations of biallelic loci in asexual populations with special focus on cancer progression. Fitness can be defined as an arbitrary function of genetic interactions between multiple genes or modules of genes, including epistasis, restrictions in the order of accumulation of mutations, and order effects. Mutation rates can differ among genes, and can be affected by (anti)mutator genes. Also available are sampling from simulations (including single-cell sampling), plotting the genealogical relationships of clones and generating and plotting fitness landscapes. Implemented in R and C ++, freely available from BioConductor for Linux, Mac and Windows under the GNU GPL license. Version 2.5.9 or higher available from: http://www.bioconductor.org/packages/devel/bioc/html/OncoSimulR.html . GitHub repository at: https://github.com/rdiaz02/OncoSimul. ramon.diaz@iib.uam.es. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

Integrated Patient-Derived Models Delineate Individualized Therapeutic Vulnerabilities of Pancreatic Cancer.

PubMed

Witkiewicz, Agnieszka K; Balaji, Uthra; Eslinger, Cody; McMillan, Elizabeth; Conway, William; Posner, Bruce; Mills, Gordon B; O'Reilly, Eileen M; Knudsen, Erik S

2016-08-16

Pancreatic ductal adenocarcinoma (PDAC) harbors the worst prognosis of any common solid tumor, and multiple failed clinical trials indicate therapeutic recalcitrance. Here, we use exome sequencing of patient tumors and find multiple conserved genetic alterations. However, the majority of tumors exhibit no clearly defined therapeutic target. High-throughput drug screens using patient-derived cell lines found rare examples of sensitivity to monotherapy, with most models requiring combination therapy. Using PDX models, we confirmed the effectiveness and selectivity of the identified treatment responses. Out of more than 500 single and combination drug regimens tested, no single treatment was effective for the majority of PDAC tumors, and each case had unique sensitivity profiles that could not be predicted using genetic analyses. These data indicate a shortcoming of reliance on genetic analysis to predict efficacy of currently available agents against PDAC and suggest that sensitivity profiling of patient-derived models could inform personalized therapy design for PDAC. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Horror Autoinflammaticus: The Molecular Pathophysiology of Autoinflammatory Disease*

PubMed Central

Masters, Seth L.; Simon, Anna; Aksentijevich, Ivona; Kastner, Daniel L.

2010-01-01

The autoinflammatory diseases are characterized by seemingly unprovoked episodes of inflammation, without high-titer autoantibodies or antigen-specific T cells. The concept was proposed ten years ago with the identification of the genes underlying hereditary periodic fever syndromes. This nosology has taken root because of the dramatic advances in our knowledge of the genetic basis of both mendelian and complex autoinflammatory diseases, and with the recognition that these illnesses derive from genetic variants of the innate immune system. Herein we propose an updated classification scheme based on the molecular insights garnered over the past decade, supplanting a clinical classification that has served well but is opaque to the genetic, immunologic, and therapeutic interrelationships now before us. We define six categories of autoinflammatory disease: IL-1β activation disorders (inflammasomopathies), NF-κB activation syndromes, protein misfolding disorders, complement regulatory diseases, disturbances in cytokine signaling, and macrophage activation syndromes. A system based on molecular pathophysiology will bring greater clarity to our discourse while catalyzing new hypotheses both at the bench and at the bedside. PMID:19302049

A genetically engineered live attenuated vaccine of Coccidioides posadasii protects BALB/c mice against coccidioidomycosis.

PubMed

Xue, Jianmin; Chen, Xia; Selby, Dale; Hung, Chiung-Yu; Yu, Jieh-Juen; Cole, Garry T

2009-08-01

Coccidioidomycosis (also known as San Joaquin Valley fever) is an occupational disease. Workers exposed to outdoor dust which contains spores of the soil-inhabiting fungus have a significantly increased risk of respiratory infection. In addition, people with compromised T-cell immunity, the elderly, and certain racial groups, particularly African-Americans and Filipinos, who live in regions of endemicity in the southwestern United States have an elevated incidence of symptomatic infection caused by inhalation of spores of Coccidioides posadasii or Coccidioides immitis. Recurring epidemics and escalation of medical costs have helped to motivate production of a vaccine against valley fever. The major focus has been the development of a defined, T-cell-reactive, recombinant protein vaccine. However, none of the products described to date have provided full protection to coccidioidal disease-susceptible BALB/c mice. Here we describe the first genetically engineered, live, attenuated vaccine that protects both BALB/c and C57BL/6 mice against coccidioidomycosis. Two chitinase genes (CTS2 and CTS3) were disrupted to yield the attenuated strain, which was unable to endosporulate and was no longer infectious. Vaccinated survivors mounted an immune response characterized by production of both T-helper-1- and T-helper-2-type cytokines. Histology revealed well-formed granulomas and markedly diminished inflammation. Significantly fewer organisms were observed in the lungs of survivors than in those of nonvaccinated mice. Additional investigations are required to further define the nature of the live, attenuated vaccine-induced immunity against Coccidioides infection.

Tissue and cellular rigidity and mechanosensitive signaling activation in Alexander disease.

PubMed

Wang, Liqun; Xia, Jing; Li, Jonathan; Hagemann, Tracy L; Jones, Jeffrey R; Fraenkel, Ernest; Weitz, David A; Zhang, Su-Chun; Messing, Albee; Feany, Mel B

2018-05-15

Glial cells have increasingly been implicated as active participants in the pathogenesis of neurological diseases, but critical pathways and mechanisms controlling glial function and secondary non-cell autonomous neuronal injury remain incompletely defined. Here we use models of Alexander disease, a severe brain disorder caused by gain-of-function mutations in GFAP, to demonstrate that misregulation of GFAP leads to activation of a mechanosensitive signaling cascade characterized by activation of the Hippo pathway and consequent increased expression of A-type lamin. Importantly, we use genetics to verify a functional role for dysregulated mechanotransduction signaling in promoting behavioral abnormalities and non-cell autonomous neurodegeneration. Further, we take cell biological and biophysical approaches to suggest that brain tissue stiffness is increased in Alexander disease. Our findings implicate altered mechanotransduction signaling as a key pathological cascade driving neuronal dysfunction and neurodegeneration in Alexander disease, and possibly also in other brain disorders characterized by gliosis.

Motifs, modules and games in bacteria.

PubMed

Wolf, Denise M; Arkin, Adam P

2003-04-01

Global explorations of regulatory network dynamics, organization and evolution have become tractable thanks to high-throughput sequencing and molecular measurement of bacterial physiology. From these, a nascent conceptual framework is developing, that views the principles of regulation in term of motifs, modules and games. Motifs are small, repeated, and conserved biological units ranging from molecular domains to small reaction networks. They are arranged into functional modules, genetically dissectible cellular functions such as the cell cycle, or different stress responses. The dynamical functioning of modules defines the organism's strategy to survive in a game, pitting cell against cell, and cell against environment. Placing pathway structure and dynamics into an evolutionary context begins to allow discrimination between those physical and molecular features that particularize a species to its surroundings, and those that provide core physiological function. This approach promises to generate a higher level understanding of cellular design, pathway evolution and cellular bioengineering.

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist.

PubMed

Sanchez, Elisa; Huse, Morgan

2018-04-25

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

Motifs, modules and games in bacteria

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

Wolf, Denise M.; Arkin, Adam P.

2003-04-01

Global explorations of regulatory network dynamics, organization and evolution have become tractable thanks to high-throughput sequencing and molecular measurement of bacterial physiology. From these, a nascent conceptual framework is developing, that views the principles of regulation in term of motifs, modules and games. Motifs are small, repeated, and conserved biological units ranging from molecular domains to small reaction networks. They are arranged into functional modules, genetically dissectible cellular functions such as the cell cycle, or different stress responses. The dynamical functioning of modules defines the organism's strategy to survive in a game, pitting cell against cell, and cell against environment.more » Placing pathway structure and dynamics into an evolutionary context begins to allow discrimination between those physical and molecular features that particularize a species to its surroundings, and those that provide core physiological function. This approach promises to generate a higher level understanding of cellular design, pathway evolution and cellular bioengineering.« less

Progress in understanding the pathogenesis of Langerhans cell histiocytosis: back to Histiocytosis X?

PubMed Central

Berres, Marie-Luise; Merad, Miriam; Allen, Carl E.

2016-01-01

Summary Langerhans cell histiocytosis (LCH), the most common histiocytic disorder, is characterized by the accumulation of CD1A+/CD207+ mononuclear phagocytes within granulomatous lesions that can affect nearly all organ systems. Historically, LCH has been presumed to arise from transformed or pathologically activated epidermal dendritic cells called Langerhans cells. However, new evidence supports a model in which LCH occurs as a consequence of a misguided differentiation programme of myeloid dendritic cell precursors. Genetic, molecular and functional data implicate activation of the ERK signalling pathway at critical stages in myeloid differentiation as an essential and universal driver of LCH pathology. Based on these findings, we propose that LCH should be re-defined as an inflammatory myeloid neoplasia. Increased understanding of LCH pathogenesis will provide opportunities to optimize and personalize therapy through improved risk-stratification, targeted therapy and assessment of therapy response based on specific molecular features and origin of the pathological myeloid cells. PMID:25430560

TGF-β in inflammatory bowel disease: a key regulator of immune cells, epithelium, and the intestinal microbiota.

PubMed

Ihara, Sozaburo; Hirata, Yoshihiro; Koike, Kazuhiko

2017-07-01

Inflammatory bowel disease (IBD) is defined as chronic intestinal inflammation, and includes ulcerative colitis and Crohn's disease. Multiple factors are involved in the pathogenesis of IBD, and the condition is characterized by aberrant mucosal immune reactions to intestinal microbes in genetically susceptible hosts. Transforming growth factor-β (TGF-β) is an immune-suppressive cytokine produced by many cell types and activated by integrins. Active TGF-β binds to its receptor and regulates mucosal immune reactions through the TGF-β signaling pathway. Dysregulated TGF-β signaling is observed in the intestines of IBD patients. TGF-β signal impairment in specific cell types, such as T-cells and dendritic cells, results in spontaneous colitis in mouse models. In addition, specific intestinal microbes contribute to immune homeostasis by modulating TGF-β production. In this review, we describe the role of TGF-β in intestinal immunity, focusing on immune cells, epithelium, and intestinal microbes. In addition, we present potential therapeutic strategies for IBD that target TGF-β.

Robotic Automation of In Vivo Two-Photon Targeted Whole-Cell Patch-Clamp Electrophysiology.

PubMed

Annecchino, Luca A; Morris, Alexander R; Copeland, Caroline S; Agabi, Oshiorenoya E; Chadderton, Paul; Schultz, Simon R

2017-08-30

Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is normally performed "blind," meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Dissecting mitochondrial apoptosis pathways by gain-of-function cell culture screens.

PubMed

Grimm, Stefan

2013-05-01

While more primitive organism such as Caenorhabditis elegans and Drosophila melanogaster feature a limited, and by now probably mostly known, array of basic cell death factors, the mammalian cell is replete with additional regulators of the cell's demise. This abundance of apoptosis mediators has made it imperative to set up a systematic inventory of mammalian cell death genes. Genetic screens in this biological system have recently uncovered the rich diversity of cell death signalling and have in particular highlighted mitochondria as an organelle loaded with apoptosis regulators. Many of the screens that have addressed this utilised the novel technique of RNA interference but some also looked at gain-of-functions with transfected cDNAs. Here we give an overview of the rationale for the latter approach, present the genes discovered by this strategy and in particular describe the involvement of mitochondria and their signalling pathways defined by those genes. Copyright © 2012 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Characterization and In Vivo Testing of Mesenchymal Stem Cells Derived from Human Embryonic Stem Cells

PubMed Central

Gruenloh, William; Kambal, Amal; Sondergaard, Claus; McGee, Jeannine; Nacey, Catherine; Kalomoiris, Stefanos; Pepper, Karen; Olson, Scott; Fierro, Fernando

2011-01-01

Mesenchymal stem cells (MSCs) have been shown to contribute to the recovery of tissues through homing to injured areas, especially to hypoxic, apoptotic, or inflamed areas and releasing factors that hasten endogenous repair. In some cases genetic engineering of the MSC is desired, since they are excellent delivery vehicles. We have derived MSCs from the human embryonic stem cell (hESC) line H9 (H9-MSCs). They expressed CD105, CD90, CD73, and CD146, and lacked expression of CD45, CD34, CD14, CD31, and HLA-DR, the hESC pluripotency markers SSEA-4 and Tra-1-81, and the hESC early differentiation marker SSEA-1. Marrow-derived MSCs showed a similar phenotype. H9-MSCs did not form teratoma in our initial studies, whereas the parent H9 line did so robustly. H9-MSCs differentiated into bone, cartilage, and adipocytes in vitro, and displayed increased migration under hypoxic conditions. Finally, using a hindlimb ischemia model, H9-MSCs were shown to home to the hypoxic muscle, but not the contralateral limb, by 48 h after IV injection. In summary, we have defined methods for differentiation of hESCs into MSCs and have defined their characteristics and in vivo migratory properties. PMID:21275830

Mechanisms underlying cellular responses of cells from haemopoietic tissue to low

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

Kadhim, Munira A

2012-08-22

The above studies will provide fundamental mechanistic information relating genetic predisposition to important low dose phenomena, and will aid in the development of Department of Energy policy, as well as radiation risk policy for the public and the workplace. We believe the proposed studies accurately reflect the goals of the DOE low dose program. To accurately define the risks associated with human exposure to relevant environmental doses of low LET ionizing radiation, it is necessary to completely understand the biological effects at very low doses (i.e. less than 0.1 Gy), including the lowest possible dose, that of a single electronmore » track traversal. At such low doses, a range of studies have shown responses in biological systems which are not related to the direct interaction of radiation tracks with DNA. The role of these "non-targeted responses in critical tissues is poorly understood and little is known regarding the underlying mechanisms. Although critical for dosimetry and risk assessment, the role of individual genetic susceptibility in radiation risk is not satisfactorily defined at present. The aim of the proposed grant is to critically evaluate non-targeted effects of ionizing radiation with a focus on the induction of genomic instability (GI) in key stem cell populations from haemopoietic tissue. Using stem cells from two mouse strains (CBA/CaH and C57BL/6J) known to differ in their susceptibility to radiation effects, we plan to carefully dissect the role of genetic predisposition in these models on genomic instability. We will specifically focus on the effects of low doses of low LET radiation, down to the dose of 10mGy (0.01Gy) X-rays. Using conventional X-ray and we will be able to assess the role of genetic variation under various conditions at a range of doses down to the very low dose of 0.01Gy. Irradiations will be carried out using facilities in routine operation for such studies. Mechanistic studies of instability in different cell lineages will include the role of cytokines which have been shown to be in the initiation of instability. These studies also aim to uncover the possible mechanism of the initiation, perpetuation and delayed pathways of the instability response using relevant biological endpoints i.e. chromosomal instability, apoptosis induction, cytokine and gene array analysis. Integral to these studies will be an assessment of the role of genetic susceptibility in these responses, using CBA/CaH and C57BL/6J mice. The overall results suggest that low dose low LET X-irradiation induced delayed GI in both CBA/CaH and C57BL/6J haemopoeitic tissue. Using several biological approaches, some key strain and dose-specific differences have been identified in radiation-induced signalling in the initiation and perpetuation of the instability process. Furthermore, the induction of non-targeted radiation effects and genetic dependency may be linked to the use of alternative signalling pathways and mechanisms which have potential implications on evaluation of non-targeted effects in radiation risk assessment.« less

Syndromic autism spectrum disorders: moving from a clinically defined to a molecularly defined approach

PubMed Central

Fernandez, Bridget A.; Scherer, Stephen W.

2017-01-01

Autism spectrum disorder (ASD) encompasses a group of neurodevelopmental conditions diagnosed solely on the basis of behavioral assessments that reveal social deficits. Progress has been made in understanding its genetic underpinnings, but most ASD-associated genetic variants, which include copy number variants (CNVs) and mutations in ASD-risk genes, account for no more than 1 % of ASD cases. This high level of genetic heterogeneity leads to challenges obtaining and interpreting genetic testing in clinical settings. The traditional definition of syndromic ASD is a disorder with a clinically defined pattern of somatic abnormalities and a neurobehavioral phenotype that may include ASD. Most have a known genetic cause. Examples include fragile X syndrome and tuberous sclerosis complex. We propose dividing syndromic autism into the following two groups: (i) ASD that occurs in the context of a clinically defined syndrome-recognizing these disorders depends on the familiarity of the clinician with the features of the syndrome, and the diagnosis is typically confirmed by targeted genetic testing (eg, mutation screening of FMR1); (ii) ASD that occurs as a feature of a molecularly defined syndrome-for this group of patients, ASD-associated variants are identified by genome-wide testing that is not hypothesis driven (eg, microarray, whole exome sequencing). These ASD groups cannot be easily clinically defined because patients with a given variant have variable somatic abnormalities (dysmorphism and birth defects). In this article, we review common diagnoses from the above categories and suggest a testing strategy for patients, guided by determining whether the individual has essential or complex ASD; patients in the latter group have multiple morphologic anomalies on physical examination. Finally, we recommend that the syndromic versus nonsyndromic designation ultimately be replaced by classification of ASD according to its genetic etiology, which will inform about the associated spectrum and penetrance of neurobehavioral and somatic manifestations. PMID:29398931

Role of Resident Stem Cells in Vessel Formation and Arteriosclerosis.

PubMed

Zhang, Li; Issa Bhaloo, Shirin; Chen, Ting; Zhou, Bin; Xu, Qingbo

2018-05-25

Vascular, resident stem cells are present in all 3 layers of the vessel wall; they play a role in vascular formation under physiological conditions and in remodeling in pathological situations. Throughout development and adult early life, resident stem cells participate in vessel formation through vasculogenesis and angiogenesis. In adults, the vascular stem cells are mostly quiescent in their niches but can be activated in response to injury and participate in endothelial repair and smooth muscle cell accumulation to form neointima. However, delineation of the characteristics and of the migration and differentiation behaviors of these stem cells is an area of ongoing investigation. A set of genetic mouse models for cell lineage tracing has been developed to specifically address the nature of these cells and both migration and differentiation processes during physiological angiogenesis and in vascular diseases. This review summarizes the current knowledge on resident stem cells, which has become more defined and refined in vascular biology research, thus contributing to the development of new potential therapeutic strategies to promote endothelial regeneration and ameliorate vascular disease development. © 2018 The Authors.

Generation and genetic engineering of human induced pluripotent stem cells using designed zinc finger nucleases.

PubMed

Ramalingam, Sivaprakash; London, Viktoriya; Kandavelou, Karthikeyan; Cebotaru, Liudmila; Guggino, William; Civin, Curt; Chandrasegaran, Srinivasan

2013-02-15

Zinc finger nucleases (ZFNs) have become powerful tools to deliver a targeted double-strand break at a pre-determined chromosomal locus in order to insert an exogenous transgene by homology-directed repair. ZFN-mediated gene targeting was used to generate both single-allele chemokine (C-C motif) receptor 5 (CCR5)-modified human induced pluripotent stem cells (hiPSCs) and biallele CCR5-modified hiPSCs from human lung fibroblasts (IMR90 cells) and human primary cord blood mononuclear cells (CBMNCs) by site-specific insertion of stem cell transcription factor genes flanked by LoxP sites into the endogenous CCR5 locus. The Oct4 and Sox2 reprogramming factors, in combination with valproic acid, induced reprogramming of human lung fibroblasts to form CCR5-modified hiPSCs, while 5 factors, Oct4/Sox2/Klf4/Lin28/Nanog, induced reprogramming of CBMNCs. Subsequent Cre recombinase treatment of the CCR5-modified IMR90 hiPSCs resulted in the removal of the Oct4 and Sox2 transgenes. Further genetic engineering of the single-allele CCR5-modified IMR90 hiPSCs was achieved by site-specific addition of the large CFTR transcription unit to the remaining CCR5 wild-type allele, using CCR5-specific ZFNs and a donor construct containing tdTomato and CFTR transgenes flanked by CCR5 homology arms. CFTR was expressed efficiently from the endogenous CCR5 locus of the CCR5-modified tdTomato/CFTR hiPSCs. These results suggest that it might be feasible to use ZFN-evoked strategies to (1) generate precisely targeted genetically well-defined patient-specific hiPSCs, and (2) then to reshape their function by targeted addition and expression of therapeutic genes from the CCR5 chromosomal locus for autologous cell-based transgene-correction therapy to treat various recessive monogenic human diseases in the future.

Cell type-specific hypersensitivity to oxidative damage in CSB and XPA mice.

PubMed

de Waard, Harm; de Wit, Jan; Gorgels, Theo G M F; van den Aardweg, Gerard; Andressoo, Jaan Olle; Vermeij, Marcel; van Steeg, Harry; Hoeijmakers, Jan H J; van der Horst, Gijsbertus T J

2003-01-02

Mutations in the CSB gene cause Cockayne syndrome (CS), a rare inherited disorder, characterized by UV-sensitivity, severe neurodevelopmental and progeroid symptoms. CSB functions in the transcription-coupled repair (TCR) sub-pathway of nucleotide excision repair (NER), responsible for the removal of UV-induced and other helix-distorting lesions from the transcribed strand of active genes. Several lines of evidence support the notion that the CSB TCR defect extends to other non-NER type transcription-blocking lesions, notably various kinds of oxidative damage, which may provide an explanation for part of the severe CS phenotype. We used genetically defined mouse models to examine the relationship between the CSB defect and sensitivity to oxidative damage in different cell types and at the level of the intact organism. The main conclusions are: (1) CSB(-/-) mouse embryo fibroblasts (MEFs) exhibit a clear hypersensitivity to ionizing radiation, extending the findings in genetically heterogeneous human CSB fibroblasts to another species. (2) CSB(-/-) MEFs are highly sensitive to paraquat, strongly indicating that the increased cytotoxicity is due to oxidative damage. (3) The hypersenstivity is independent of genetic background and directly related to the CSB defect and is not observed in totally NER-deficient XPA MEFs. (4) Wild type embryonic stem (ES) cells display an increased sensitivity to ionizing radiation compared to fibroblasts. Surprisingly, the CSB deficiency has only a very minor additional effect on ES cell sensitivity to oxidative damage and is comparable to that of an XPA defect, indicating cell type-specific differences in the contribution of TCR and NER to cellular survival. (5) Similar to ES cells, CSB and XPA mice both display a minor sensitivity to whole-body X-ray exposure. This suggests that the response of an intact organism to radiation is largely determined by the sensitivity of stem cells, rather than differentiated cells. These findings establish the role of transcription-coupled repair in resistance to oxidative damage and reveal a cell- and organ-specific impact of this repair pathway to the clinical phenotype of CS and XP.

Coupling of Peripheral Tolerance to Endogenous Interleukin 10 Promotes Effective Modulation of Myelin-Activated T Cells and Ameliorates Experimental Allergic Encephalomyelitis

PubMed Central

Legge, Kevin L.; Min, Booki; Bell, J. Jeremiah; Caprio, Jacque C.; Li, Lequn; Gregg, Randal K.; Zaghouani, Habib

2000-01-01

Several immune-based approaches are being considered for modulation of inflammatory T cells and amelioration of autoimmune diseases. The most recent strategies include simulation of peripheral self-tolerance by injection of adjuvant free antigen, local delivery of cytokines by genetically altered T cells, and interference with the function of costimulatory molecules. Although promising results have been obtained from these studies that define mechanisms of T cell modulation, efficacy, practicality, and toxicity, concerns remain unsolved, thereby justifying further investigations to define alternatives for effective downregulation of aggressive T cells. In prior studies, we demonstrated that an immunoglobulin (Ig) chimera carrying the encephalitogenic proteolipid protein (PLP)1 peptide corresponding to amino acid sequence 139–151 of PLP, Ig-PLP1, is presented to T cells ∼100-fold better than free PLP1. Here, we demonstrate that aggregation endows Ig-PLP1 with an additional feature, namely, induction of interleukin (IL)-10 production by macrophages and dendritic cells, both of which are antigen-presenting cells (APCs). These functions synergize in vivo and drive effective modulation of autoimmunity. Indeed, it is shown that animals with ongoing active experimental allergic encephalomyelitis dramatically reduce the severity of their paralysis when treated with adjuvant free aggregated Ig-PLP1. Moreover, IL-10 displays bystander antagonism on unrelated autoreactive T cells, allowing for reversal of disease involving multiple epitopes. Therefore, aggregated Ig-PLP1 likely brings together a peripheral T cell tolerance mechanism emanating from peptide presentation by APCs expressing suboptimal costimulatory molecules and IL-10 bystander suppression to drive a dual-modal T cell modulation system effective for reversal of autoimmunity involving several epitopes and diverse T cell specificities. PMID:10859329

Acentriolar mitosis activates a p53-dependent apoptosis pathway in the mouse embryo

PubMed Central

Bazzi, Hisham; Anderson, Kathryn V.

2014-01-01

Centrosomes are the microtubule-organizing centers of animal cells that organize interphase microtubules and mitotic spindles. Centrioles are the microtubule-based structures that organize centrosomes, and a defined set of proteins, including spindle assembly defective-4 (SAS4) (CPAP/CENPJ), is required for centriole biogenesis. The biological functions of centrioles and centrosomes vary among animals, and the functions of mammalian centrosomes have not been genetically defined. Here we use a null mutation in mouse Sas4 to define the cellular and developmental functions of mammalian centrioles in vivo. Sas4-null embryos lack centrosomes but survive until midgestation. As expected, Sas4−/− mutants lack primary cilia and therefore cannot respond to Hedgehog signals, but other developmental signaling pathways are normal in the mutants. Unlike mutants that lack cilia, Sas4−/− embryos show widespread apoptosis associated with global elevated expression of p53. Cell death is rescued in Sas4−/− p53−/− double-mutant embryos, demonstrating that mammalian centrioles prevent activation of a p53-dependent apoptotic pathway. Expression of p53 is not activated by abnormalities in bipolar spindle organization, chromosome segregation, cell-cycle profile, or DNA damage response, which are normal in Sas4−/− mutants. Instead, live imaging shows that the duration of prometaphase is prolonged in the mutants while two acentriolar spindle poles are assembled. Independent experiments show that prolonging spindle assembly is sufficient to trigger p53-dependent apoptosis. We conclude that a short delay in the prometaphase caused by the absence of centrioles activates a previously undescribed p53-dependent cell death pathway in the rapidly dividing cells of the mouse embryo. PMID:24706806

The molecular genetics of eyelid tumors: recent advances and future directions.

PubMed

Milman, Tatyana; McCormick, Steven A

2013-02-01

Unprecedented recent advances in the molecular genetics of cutaneous malignancies have markedly improved our ability to diagnose, treat, and counsel patients with skin tumors. This review provides an update on molecular genetics of periocular cutaneous basal cell carcinoma, squamous cell carcinoma, sebaceous carcinoma, Merkel cell carcinoma, and malignant melanoma and describes how the knowledge of molecular genetics is translated into clinical practice. A literature search of peer-reviewed and indexed publications from 1965 to 2012 using the PubMed search engine was performed. Key terms included: molecular genetics, eyelid, basal cell carcinoma, squamous cell carcinoma, sebaceous adenoma, sebaceous epithelioma, sebaceoma, sebaceous carcinoma, Merkel cell carcinoma, and melanoma. Seminal articles prior to 1965 were selected from primary sources and reviews from the initial search. Articles were chosen based on pertinence to clinical, genetic, and therapeutic topics reviewed in this manuscript. We reviewed the literature regarding the advances in molecular genetics of cutaneous basal cell carcinoma, squamous cell carcinoma, sebaceous neoplasia, Merkel cell carcinoma, and malignant melanoma, and possible future directions towards diagnosing and treating cutaneous tumors at the genetic level. Cell culture experiments, animal models, and molecular genetic studies on the patients' tumor tissues helped to elucidate genetic aberrations in these lesions. Cell culture experiments, animal studies and, ultimately, clinical trials provided means to test and develop novel therapeutic strategies, namely targeted therapy directed at specific molecular genetic defects. While remarkable progress has been made in this process, the complexity of the molecular genetics of skin tumors makes complete elucidation of the genetic mechanisms and the search for ideal therapies challenging. The recent studies focusing on molecular genetics of cutaneous malignancies show promising results, thereby improving our ability to diagnose, treat and counsel patients with these lesions. Future studies will hopefully help unravel further molecular mechanisms involved in cutaneous neoplasia and provide insights into novel preventative and therapeutic modalities.

Cloned ferrets produced by somatic cell nuclear transfer.

PubMed

Li, Ziyi; Sun, Xingshen; Chen, Juan; Liu, Xiaoming; Wisely, Samantha M; Zhou, Qi; Renard, Jean-Paul; Leno, Gregory H; Engelhardt, John F

2006-05-15

Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (approximately 3-4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink.

Visualization of Calcium Dynamics in Kidney Proximal Tubules

PubMed Central

Szebényi, Kornélia; Füredi, András; Kolacsek, Orsolya; Csohány, Rózsa; Prókai, Ágnes; Kis-Petik, Katalin; Szabó, Attila; Bősze, Zsuzsanna; Bender, Balázs; Tóvári, József; Enyedi, Ágnes; Orbán, Tamás I.

2015-01-01

Intrarenal changes in cytoplasmic calcium levels have a key role in determining pathologic and pharmacologic responses in major kidney diseases. However, cell-specific delivery of calcium-sensitive probes in vivo remains problematic. We generated a transgenic rat stably expressing the green fluorescent protein-calmodulin–based genetically encoded calcium indicator (GCaMP2) predominantly in the kidney proximal tubules. The transposon-based method used allowed the generation of homozygous transgenic rats containing one copy of the transgene per allele with a defined insertion pattern, without genetic or phenotypic alterations. We applied in vitro confocal and in vivo two-photon microscopy to examine basal calcium levels and ligand- and drug-induced alterations in these levels in proximal tubular epithelial cells. Notably, renal ischemia induced a transient increase in cellular calcium, and reperfusion resulted in a secondary calcium load, which was significantly decreased by systemic administration of specific blockers of the angiotensin receptor and the Na-Ca exchanger. The parallel examination of in vivo cellular calcium dynamics and renal circulation by fluorescent probes opens new possibilities for physiologic and pharmacologic investigations. PMID:25788535

Identification of the meiotic life cycle stage of Trypanosoma brucei in the tsetse fly

PubMed Central

Peacock, Lori; Ferris, Vanessa; Sharma, Reuben; Sunter, Jack; Bailey, Mick; Carrington, Mark; Gibson, Wendy

2011-01-01

Elucidating the mechanism of genetic exchange is fundamental for understanding how genes for such traits as virulence, disease phenotype, and drug resistance are transferred between pathogen strains. Genetic exchange occurs in the parasitic protists Trypanosoma brucei, T. cruzi, and Leishmania major, but the precise cellular mechanisms are unknown, because the process has not been observed directly. Here we exploit the identification of homologs of meiotic genes in the T. brucei genome and demonstrate that three functionally distinct, meiosis-specific proteins are expressed in the nucleus of a single specific cell type, defining a previously undescribed developmental stage occurring within the tsetse fly salivary gland. Expression occurs in clonal and mixed infections, indicating that the meiotic program is an intrinsic but hitherto cryptic part of the developmental cycle of trypanosomes. In experimental crosses, expression of meiosis-specific proteins usually occurred before cell fusion. This is evidence of conventional meiotic division in an excavate protist, and the functional conservation of the meiotic machinery in these divergent organisms underlines the ubiquity and basal evolution of meiosis in eukaryotes. PMID:21321215

Activating Akt1 mutations alter DNA double strand break repair and radiosensitivity

PubMed Central

Oeck, S.; Al-Refae, K.; Riffkin, H.; Wiel, G.; Handrick, R.; Klein, D.; Iliakis, G.; Jendrossek, V.

2017-01-01

The survival kinase Akt has clinical relevance to radioresistance. However, its contributions to the DNA damage response, DNA double strand break (DSB) repair and apoptosis remain poorly defined and often contradictory. We used a genetic approach to explore the consequences of genetic alterations of Akt1 for the cellular radiation response. While two activation-associated mutants with prominent nuclear access, the phospho-mimicking Akt1-TDSD and the clinically relevant PH-domain mutation Akt1-E17K, accelerated DSB repair and improved survival of irradiated Tramp-C1 murine prostate cancer cells and Akt1-knockout murine embryonic fibroblasts in vitro, the classical constitutively active membrane-targeted myrAkt1 mutant had the opposite effects. Interestingly, DNA-PKcs directly phosphorylated Akt1 at S473 in an in vitro kinase assay but not vice-versa. Pharmacological inhibition of DNA-PKcs or Akt restored radiosensitivity in tumour cells expressing Akt1-E17K or Akt1-TDSD. In conclusion, Akt1-mediated radioresistance depends on its activation state and nuclear localization and is accessible to pharmacologic inhibition. PMID:28209968

Cell plasticity and heterogeneity in cancer.

PubMed

Marjanovic, Nemanja D; Weinberg, Robert A; Chaffer, Christine L

2013-01-01

Heterogeneity within a given cancer arises from diverse cell types recruited to the tumor and from genetic and/or epigenetic differences amongst the cancer cells themselves. These factors conspire to create a disease with various phenotypes. There are 2 established models of cancer development and progression to metastatic disease. These are the clonal evolution and cancer stem cell models. The clonal evolution theory suggests that successive mutations accumulating in a given cell generate clonal outgrowths that thrive in response to microenvironmental selection pressures, dictating the phenotype of the tumor. The alternative cancer stem cell (CSC) model suggests that cancer cells with similar genetic backgrounds can be hierarchically organized according to their tumorigenic potential. Accordingly, CSCs reside at the apex of the hierarchy and are thought to possess the majority of a cancer's tumor-initiating and metastatic ability. A defining feature of this model is its apparent unidirectional nature, whereby CSCs undergo symmetric division to replenish the CSC pool and irreversible asymmetric division to generate daughter cells (non-CSCs) with low tumorigenic potential. However, evolving evidence supports a new model of tumorigenicity, in which considerable plasticity exists between the non-CSC and CSC compartments, such that non-CSCs can reacquire a CSC phenotype. These findings suggest that some tumors may adhere to a plastic CSC model, in which bidirectional conversions are common and essential components of tumorigenicity. Accumulating evidence surrounding the plasticity of cancer cells, in particular, suggests that aggressive CSCs can be created de novo within a tumor. Given the current focus on therapeutic targeting of CSCs, we discuss the implications of non-CSC-to-CSC conversions on the development of future therapies. © 2012 American Association for Clinical Chemistry

Long-term in vivo provision of antigen-specific T cell immunity by programming hematopoietic stem cells

NASA Astrophysics Data System (ADS)

Yang, Lili; Baltimore, David

2005-03-01

A method to genetically program mouse hematopoietic stem cells to develop into functional CD8 or CD4 T cells of defined specificity in vivo is described. For this purpose, a bicistronic retroviral vector was engineered that efficiently delivers genes for both and chains of T cell receptor (TCR) to hematopoietic stem cells. When modified cell populations were used to reconstruct the hematopoietic lineages of recipient mice, significant percentages of antigen-specific CD8 or CD4 T cells were observed. These cells expressed normal surface markers and responded to peptide antigen stimulation by proliferation and cytokine production. Moreover, they could mature into memory cells after peptide stimulation. Using TCRs specific for a model tumor antigen, we found that the recipient mice were able to partially resist a challenge with tumor cells carrying the antigen. By combining cells modified with CD8- and CD4-specific TCRs, and boosting with dendritic cells pulsed with cognate peptides, complete suppression of tumor could be achieved and even tumors that had become established would regress and be eliminated after dendritic cell/peptide immunization. This methodology of "instructive immunotherapy" could be developed for controlling the growth of human tumors and attacking established pathogens.

Progenitor cell domains in the developing and adult pancreas

PubMed Central

Kopp, Janel L; Dubois, Claire L; Hao, Ergeng; Thorel, Fabrizio; Herrera, Pedro L

2011-01-01

Unlike organs with defined stem cell compartments, such as the intestine, the pancreas has limited capacity to regenerate. The question of whether the adult pancreas harbors facultative stem/progenitor cells has been a prime subject of debate. Cumulative evidence from recent genetic lineage tracing studies, in which specific cell populations were marked and traced in adult mice, suggests that endocrine and acinar cells are no longer generated from progenitors in the adult pancreas. These studies further indicate that adult pancreatic ductal cells are not a source for endocrine cells following pancreatic injury, as previously suggested. Our own studies have shown that adult ductal cells reinitiate expression of some endocrine progenitor markers, including Ngn3, after injury by partial duct ligation (PDL), but that these cells do not undergo endocrine cell differentiation. Here, we present additional evidence that endocrine cells do not arise from ducts following β-cell ablation by streptozotocin or by a diphtheria toxin-expressing transgene or when β-cell ablation is combined with PDL. In this review, we discuss findings from recent lineage tracing studies of embryonic and adult pancreatic ductal cells. Based upon the combined evidence from these studies, we propose that multipotency is associated with a specific transcriptional signature. PMID:21558806

Molecular-genetic profiling and high-throughput in vitro drug screening in NUT midline carcinoma—an aggressive and fatal disease

PubMed Central

Stirnweiss, Anja; Oommen, Joyce; Kotecha, Rishi S.; Kees, Ursula R.; Beesley, Alex H.

2017-01-01

NUT midline carcinoma (NMC) is a rare and aggressive cancer, with survival typically less than seven months, that can arise in people of any age. Genetically, NMC is defined by the chromosomal fusion of NUTM1 with a chromatin-binding partner, typically the bromodomain-containing protein BRD4. However, little is known about other genetic aberrations in this disease. In this study, we used a unique panel of cell lines to describe the molecular-genetic features of NMC. Next-generation sequencing identified a recurring high-impact mutation in the DNA-helicase gene RECQL5 in 75% of lines studied, and biological signals from mutation-signature and network analyses consistent with a general failure in DNA-repair. A high-throughput drug screen confirmed that microtubule inhibitors, topoisomerase inhibitors and anthracyclines are highly cytotoxic in the majority of NMC lines, and that cell lines expressing the BRD4-NUTM1 (exon11:exon2) variant are an order of magnitude more responsive to bromodomain inhibitors (iBETs) on average than those with other BRD4-NUTM1 translocation variants. We also identified a highly significant correlation between iBET and aurora kinase inhibitor efficacy in this study. Integration of exome sequencing, transcriptome, and drug sensitivity profiles suggested that aberrant activity of the nuclear receptor co-activator NCOA3 may correlate with poor response to iBETs. In conclusion, our data emphasize the heterogeneity of NMC and highlights genetic aberrations that could be explored to improve therapeutic strategies. The novel finding of a recurring RECQL5 mutation, together with recent reports of chromoplexy in this disease, suggests that DNA-repair pathways are likely to play a central role in NMC tumorigenesis. PMID:29348827

MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity.

PubMed

Chu, Daniel K W; Hui, Kenrie P Y; Perera, Ranawaka A P M; Miguel, Eve; Niemeyer, Daniela; Zhao, Jincun; Channappanavar, Rudragouda; Dudas, Gytis; Oladipo, Jamiu O; Traoré, Amadou; Fassi-Fihri, Ouafaa; Ali, Abraham; Demissié, Getnet F; Muth, Doreen; Chan, Michael C W; Nicholls, John M; Meyerholz, David K; Kuranga, Sulyman A; Mamo, Gezahegne; Zhou, Ziqi; So, Ray T Y; Hemida, Maged G; Webby, Richard J; Roger, Francois; Rambaut, Andrew; Poon, Leo L M; Perlman, Stanley; Drosten, Christian; Chevalier, Veronique; Peiris, Malik

2018-03-20

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa as well as in the Arabian Peninsula, zoonotic disease appears confined to the Arabian Peninsula. MERS-CoVs from Africa have hitherto been poorly studied. We genetically and phenotypically characterized MERS-CoV from dromedaries sampled in Morocco, Burkina Faso, Nigeria, and Ethiopia. Viruses from Africa (clade C) are phylogenetically distinct from contemporary viruses from the Arabian Peninsula (clades A and B) but remain antigenically similar in microneutralization tests. Viruses from West (Nigeria, Burkina Faso) and North (Morocco) Africa form a subclade, C1, that shares clade-defining genetic signatures including deletions in the accessory gene ORF4b Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung. BF785 replicated to lower titer in lungs of human DPP4-transduced mice. A reverse genetics-derived recombinant MERS-CoV (EMC) lacking ORF4b elicited higher type I and III IFN responses than the isogenic EMC virus in Calu-3 cells. However, ORF4b deletions may not be the major determinant of the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to zoonotic potential. There is an urgent need for studies of MERS-CoV at the animal-human interface. Copyright © 2018 the Author(s). Published by PNAS.

Remodeling the zonula adherens in response to tension and the role of afadin in this response

PubMed Central

Acharya, Bipul R.; Peyret, Grégoire; Fardin, Marc-Antoine; Mège, René-Marc; Ladoux, Benoit; Yap, Alpha S.; Fanning, Alan S.

2016-01-01

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions. PMID:27114502

The complex pathophysiology of acquired aplastic anaemia.

PubMed

Zeng, Y; Katsanis, E

2015-06-01

Immune-mediated destruction of haematopoietic stem/progenitor cells (HSPCs) plays a central role in the pathophysiology of acquired aplastic anaemia (aAA). Dysregulated CD8(+) cytotoxic T cells, CD4(+) T cells including T helper type 1 (Th1), Th2, regulatory T cells and Th17 cells, natural killer (NK) cells and NK T cells, along with the abnormal production of cytokines including interferon (IFN)-γ, tumour necrosis factor (TNF)-α and transforming growth factor (TGF)-β, induce apoptosis of HSPCs, constituting a consistent and defining feature of severe aAA. Alterations in the polymorphisms of TGF-β, IFN-γ and TNF-α genes, as well as certain human leucocyte antigen (HLA) alleles, may account for the propensity to immune-mediated killing of HSPCs and/or ineffective haematopoiesis. Although the inciting autoantigens remain elusive, autoantibodies are often detected in the serum. In addition, recent studies provide genetic and molecular evidence that intrinsic and/or secondary deficits in HSPCs and bone marrow mesenchymal stem cells may underlie the development of bone marrow failure. © 2015 British Society for Immunology.

Evidence that Meningeal Mast Cells Can Worsen Stroke Pathology in Mice

PubMed Central

Arac, Ahmet; Grimbaldeston, Michele A.; Nepomuceno, Andrew R.B.; Olayiwola, Oluwatobi; Pereira, Marta P.; Nishiyama, Yasuhiro; Tsykin, Anna; Goodall, Gregory J.; Schlecht, Ulrich; Vogel, Hannes; Tsai, Mindy; Galli, Stephen J.; Bliss, Tonya M.; Steinberg, Gary K.

2015-01-01

Stroke is the leading cause of adult disability and the fourth most common cause of death in the United States. Inflammation is thought to play an important role in stroke pathology, but the factors that promote inflammation in this setting remain to be fully defined. An understudied but important factor is the role of meningeal-located immune cells in modulating brain pathology. Although different immune cells traffic through meningeal vessels en route to the brain, mature mast cells do not circulate but are resident in the meninges. With the use of genetic and cell transfer approaches in mice, we identified evidence that meningeal mast cells can importantly contribute to the key features of stroke pathology, including infiltration of granulocytes and activated macrophages, brain swelling, and infarct size. We also obtained evidence that two mast cell-derived products, interleukin-6 and, to a lesser extent, chemokine (C-C motif) ligand 7, can contribute to stroke pathology. These findings indicate a novel role for mast cells in the meninges, the membranes that envelop the brain, as potential gatekeepers for modulating brain inflammation and pathology after stroke. PMID:25134760

Switches for multiple behavioral states and a viral-based approach to non-invasive whole-brain cargo delivery (Conference Presentation)

NASA Astrophysics Data System (ADS)

Gradinaru, Viviana

2017-05-01

Over the past years we have worked on: (1) Viral-based approaches to non-invasive whole-brain cargo delivery: Genetically-encoded tools that can be used to visualize, monitor, and modulate mammalian neurons are revolutionizing neuroscience. These tools are particularly powerful in rodents and invertebrate models where intersectional transgenic strategies are available to restrict their expression to defined cell populations. However, use of genetic tools in non-transgenic animals is often hindered by the lack of vectors capable of safe, efficient, and specific delivery to the desired cellular targets. To begin to address these challenges, we have developed an in vivo Cre-based selection platform (CREATE) for identifying adeno-associated viruses (AAVs) that more efficiently transduce genetically defined cell populations. Our platform's novelty and power arises from the additional selective pressure imparted by a recovery step that amplifies only those capsid variants that have functionally transduced a genetically-defined, Cre-expressing target cell population. The Cre-dependent capsid recovery works within heterogeneous tissue samples without the need for additional steps such as selective capsid recovery approaches that require cell sorting or secondary adenovirus infection. As a first test of the CREATE platform, we selected for viruses that transduced the brain after intravascular delivery and found a novel vector, AAV-PHP.B, that is 40- to 90-fold more efficient at transducing the brain than the current standard, AAV9. AAV-PHP.B transduces most neuronal types and glia across the brain. We also demonstrate here how whole-body tissue clearing can facilitate transduction maps of systemically delivered genes. Since CNS disorders are notoriously challenging due to the restrictive nature of the blood brain barrier our discovery that recombinant vectors can be engineered to overcome this barrier is enabling for the whole field. With the exciting advances in gene editing via the CRISPR-Cas, RNA interference and gene replacement strategies, the availability of potent gene delivery methods provided by vectors such as our reported AAV-PHP.B is key to advancing the field of genome engineering. • Deverman BE, Pravdo P, Simpson B, Banerjee A, Kumar, S.R., Chan K, Wu WL, Yang B, Gradinaru V. Cre-Dependent Capsid Selection Yields AAVs for Global Gene Transfer to the Adult Brain. Nature Biotechnol. 2016 Feb 34(2):204-9. PMID: 26829320 • Yang B, Treweek JB, Kulkarni RP, Deverman BE, Chen CK, Lubeck E, Shah S, Cai L, Gradinaru V. Single-cell phenotyping within transparent intact tissue through whole-body clearing. Cell. 2014 Aug 14;158(4):945-58. PMCID: PMC4153367. (2) The mesopontine tegmentum, including the pedunculopontine and laterodorsal tegmental nuclei (PPN and LDT), provides major cholinergic inputs to midbrain and regulates locomotion and reward. To delineate the underlying projection-specific circuit mechanisms, we employed optogenetics to control mesopontine cholinergic neurons at somata and at divergent projections within distinct midbrain areas. Bidirectional manipulation of PPN cholinergic cell bodies exerted opposing effects on locomotor behavior and reinforcement learning. These motor and reward effects were separable via limiting photostimulation to PPN cholinergic terminals in the ventral substantia nigra pars compacta (vSNc) or to the ventral tegmental area (VTA), respectively. LDT cholinergic neurons also form connections with vSNc and VTA neurons; however, although photo-excitation of LDT cholinergic terminals in the VTA caused positive reinforcement, LDT-to-vSNc modulation did not alter locomotion or reward. Therefore, the selective targeting of projection-specific mesopontine cholinergic pathways may offer increased benefit in treating movement and addiction disorders. • Xiao C, Cho JR, Zhou C, Treweek BJ, Chan K, McKinney SL, Yang B, and Gradinaru V. Cholinergic Mesopontine Signals Govern Locomotion and Reward Through Dissociable Midbrain Pathways. Neuron 2016 Apr 20;90(2)33-47. PMCID: PMC4840478

Perspectives of gene expression profiling for diagnosis and therapy in haematological malignancies.

PubMed

Bacher, Ulrike; Kohlmann, Alexander; Haferlach, Torsten

2009-05-01

Considering the heterogeneity of leukaemias and the widening spectrum of therapeutic strategies, novel diagnostic methods are urgently needed for haematological malignancies. For a decade, gene expression profiling (GEP) has been applied in leukaemia research. Thus, various studies demonstrated worldwide that the majority of genetically defined leukaemia subtypes are accurately predictable by GEP, for example, with respect to reciprocal rearrangements in acute myeloid leukaemia (AML). Moreover, novel prognostically relevant gene classifiers were developed as, for example, in normal karyotype AML. Considering the lymphatic malignancies, GEP studies defined novel clinically relevant subtypes in diffuse large B cell lymphoma (DLBCL), and improved the discrimination of Burkitt lymphoma and DLBCL cases, overcoming considerable overlaps of these entities that exist from morphological and genetic perspectives. Treatment-specific sensitivity assays are being developed for targeted drugs such as farnesyl transferase inhibitors in AML or imatinib in BCR-ABL1 positive acute lymphoblastic leukaemia (ALL). Irrespectively of these proceedings, an introduction of the microarray technology in haematological practice requires diagnostic algorithms and strategies for interaction with currently established diagnostic techniques. Large multicentre studies such as the MILE Study (Microarray Innovations in LEukemia) aim at translating this methodology into clinical routine workflows and to catalyze this process.

Synthetic transitions: towards a new synthesis

PubMed Central

Solé, Ricard

2016-01-01

The evolution of life in our biosphere has been marked by several major innovations. Such major complexity shifts include the origin of cells, genetic codes or multicellularity to the emergence of non-genetic information, language or even consciousness. Understanding the nature and conditions for their rise and success is a major challenge for evolutionary biology. Along with data analysis, phylogenetic studies and dedicated experimental work, theoretical and computational studies are an essential part of this exploration. With the rise of synthetic biology, evolutionary robotics, artificial life and advanced simulations, novel perspectives to these problems have led to a rather interesting scenario, where not only the major transitions can be studied or even reproduced, but even new ones might be potentially identified. In both cases, transitions can be understood in terms of phase transitions, as defined in physics. Such mapping (if correct) would help in defining a general framework to establish a theory of major transitions, both natural and artificial. Here, we review some advances made at the crossroads between statistical physics, artificial life, synthetic biology and evolutionary robotics. This article is part of the themed issue ‘The major synthetic evolutionary transitions’. PMID:27431516

Towards defining the role of glycans as hardware in information storage and transfer: basic principles, experimental approaches and recent progress.

PubMed

Solís, D; Jiménez-Barbero, J; Kaltner, H; Romero, A; Siebert, H C; von der Lieth, C W; Gabius, H J

2001-01-01

The term 'code' in biological information transfer appears to be tightly and hitherto exclusively connected with the genetic code based on nucleotides and translated into functional activities via proteins. However, the recent appreciation of the enormous coding capacity of oligosaccharide chains of natural glycoconjugates has spurred to give heed to a new concept: versatile glycan assembly by the genetically encoded glycosyltransferases endows cells with a probably not yet fully catalogued array of meaningful messages. Enciphered by sugar receptors such as endogenous lectins the information of code words established by a series of covalently linked monosaccharides as letters for example guides correct intra- and intercellular routing of glycoproteins, modulates cell proliferation or migration and mediates cell adhesion. Evidently, the elucidation of the structural frameworks and the recognition strategies within the operation of the sugar code poses a fascinating conundrum. The far-reaching impact of this recognition mode on the level of cells, tissues and organs has fueled vigorous investigations to probe the subtleties of protein-carbohydrate interactions. This review presents information on the necessarily concerted approach using X-ray crystallography, molecular modeling, nuclear magnetic resonance spectroscopy, thermodynamic analysis and engineered ligands and receptors. This part of the treatise is flanked by exemplarily chosen insights made possible by these techniques. Copyright 2001 S. Karger AG, Basel

Successful application of preimplantation genetic diagnosis for beta-thalassaemia and sickle cell anaemia in Italy.

PubMed

Chamayou, S; Alecci, C; Ragolia, C; Giambona, A; Siciliano, S; Maggio, A; Fichera, M; Guglielmino, A

2002-05-01

In Italy, the autosomal recessive diseases beta-thalassaemia and sickle cell anaemia are so widespread that in some regions they can be defined as 'social diseases'. In this study, nine clinical applications of preimplantation genetic diagnosis (PGD) were performed for beta-thalassaemia and sickle cell anaemia on seven Sicilian couples and carriers of beta-globin gene mutations. The studied mutations were: Cd39, HbS, IVS1 nt1, IVS1 nt6 and IVS1 nt110. ICSI was performed with partner's sperm on 131 out of 147 retrieved oocytes, and this resulted in 72 zygotes; 32 embryos were successfully biopsied on day 3. The biopsied blastomeres were lysed and the beta-globin alleles amplified by nested PCR. The mutation diagnosis was performed by restriction enzyme digestion and reverse dot-blot. The amplification efficacy was 97.2%. The genotype study of non-transferred and surplus embryos showed that the allele drop-out rate was 8.6%. Seventeen embryos were transferred in utero on day 4. All couples received an embryo transfer; of the four pregnancies obtained, three resulted in live births and one miscarried at 11 weeks. Prenatal diagnosis at the 11th week and miscarriage material analysis confirmed the PGD results. These studies represent the first successful application of PGD for beta-thalassaemia and sickle cell anaemia in Italy.

Membrane Protected Apoptotic Trophoblast Microparticles Contain Nucleic Acids

PubMed Central

Orozco, Aaron F.; Jorgez, Carolina J.; Horne, Cassandra; Marquez-Do, Deborah A.; Chapman, Matthew R.; Rodgers, John R.; Bischoff, Farideh Z.; Lewis, Dorothy E.

2008-01-01

Microparticles (MPs) that circulate in blood may be a source of DNA for molecular analyses, including prenatal genetic diagnoses. Because MPs are heterogeneous in nature, however, further characterization is important before use in clinical settings. One key question is whether DNA is either bound to aggregates of blood proteins and lipid micelles or intrinsically associated with MPs from dying cells. To test the latter hypothesis, we asked whether MPs derived in vitro from dying cells were similar to those in maternal plasma. JEG-3 cells model extravillous trophoblasts, which predominate during the first trimester of pregnancy when prenatal diagnosis is most relevant. MPs were derived from apoptosis and increased over 48 hours. Compared with necrotic MPs, DNA in apoptotic MPs was more fragmented and resistant to plasma DNases. Membrane-specific dyes indicated that apoptotic MPs had more membranous material, which protects nucleic acids, including RNA. Flow cytometry showed that MPs derived from dying cells displayed light scatter and DNA staining similar to MPs found in maternal plasma. Quantification of maternal MPs using characteristics defined by MPs generated in vitro revealed a significant increase of DNA+ MPs in the plasma of women with preeclampsia compared with plasma from women with normal pregnancies. Apoptotic MPs are therefore a likely source of stable DNA that could be enriched for both early genetic diagnosis and monitoring of pathological pregnancies. PMID:18974299

The future of human embryonic stem cell research: addressing ethical conflict with responsible scientific research.

PubMed

Gilbert, David M

2004-05-01

Embryonic stem (ES) cells have almost unlimited regenerative capacity and can potentially generate any body tissue. Hence they hold great promise for the cure of degenerative human diseases. But their derivation and the potential for misuse have raised a number of ethical issues. These ethical issues threaten to paralyze pubic funding for ES cell research, leaving experimentation in the hands of the private sector and precluding the public's ability to monitor practices, research alternatives, and effectively address the very ethical issues that are cause for concern in the first place. With new technology being inevitable, and the potential for abuse high, government must stay involved if the public is to play a role in shaping the direction of research. In this essay, I will define levels of ethical conflict that can be delineated by the anticipated advances in technology. From the urgent need to derive new ES cell lines with existing technology, to the most far-reaching goal of deriving genetically identical tissues from an adult patients cells, technology-specific ethical dilemmas can be defined and addressed. This staged approach provides a solid ethical framework for moving forward with ES cell research. Moreover, by anticipating the moral conflicts to come, one can predict the types of scientific advances that could overcome these conflicts, and appropriately direct federal funding toward these goals to offset potentially less responsible research directives that will inevitably go forward via private or foreign funding.

Impact of genetic variants on haematopoiesis in patients with thrombocytopenia absent radii (TAR) syndrome.

PubMed

Manukjan, Georgi; Bösing, Hendrik; Schmugge, Markus; Strauß, Gabriele; Schulze, Harald

2017-11-01

Thrombocytopenia absent radii (TAR) syndrome is clearly defined by the combination of radial aplasia and reduced platelet counts. The genetics of TAR syndrome has recently been resolved and comprises a microdeletion on Chromosome 1 including the RBM8A gene and a single nucleotide polymorphism (SNP) either at the 5' untranslated region (5'UTR) or within the first intron of RBM8A. Although phenotypically readily diagnosed after birth, the genetic determination of particular SNPs in TAR syndrome harbours valuable information to evaluate disease severity and treatment decisions. Here, we present clinical data in a cohort of 38 patients and observed that platelet counts in individuals with 5'UTR SNP are significantly lower compared to patients bearing the SNP in intron 1. Moreover, elevated haemoglobin values could only be assessed in patients with 5'UTR SNP whereas white blood cell count is unaffected, indicating that frequently observed anaemia in TAR patients could also be SNP-dependent whereas leucocytosis does not correlate with genetic background. However, this report on a large cohort provides an overview of important haematological characteristics in TAR patients, facilitating evaluation of the various traits in this disease and indicating the importance of genetic validation for TAR syndrome. © 2017 John Wiley & Sons Ltd.

Aging, Rejuvenation, and Epigenetic Reprogramming: Resetting the Aging Clock

PubMed Central

Rando, Thomas A.; Chang, Howard Y.

2012-01-01

The underlying cause of aging remains one of the central mysteries of biology. Recent studies in several different systems suggest that not only may the rate of aging be modified by environmental and genetic factors, but also that the aging clock can be reversed, restoring characteristics of youthfulness to aged cells and tissues. This Review focuses on the emerging biology of rejuvenation through the lens of epigenetic reprogramming. By defining youthfulness and senescence as epigenetic states, a framework for asking new questions about the aging process emerges. PMID:22265401

Thought waves remotely affect the performance (output voltage) of photoelectric cells

NASA Astrophysics Data System (ADS)

Cao, Dayong; Cao, Daqing

2012-02-01

In our experiments, thought waves have been shown to be capable of changing (affecting) the output voltage of photovoltaic cells located from as far away as 1-3 meters. There are no wires between brain and photoelectric cell and so it is presumed only the thought waves act on the photoelectric cell. In continual rotations, the experiments tested different solar cells, measuring devices and lamps, and the experiments were done in different labs. The first experiment was conducted on Oct 2002. Tests are ongoing. Conclusions and assumptions include: 1) the slow thought wave has the energy of space-time as defined by C1.00007: The mass, energy, space and time systemic theory- MEST. Every process releases a field effect electrical vibration which be transmitted and focussed in particular paths; 2) the thought wave has the information of the order of tester; 3) the brain (with the physical system of MEST) and consciousness (with the spirit system of the mind, consciousness, emotion and desire-MECD) can produce the information (a part of them as the Genetic code); 4) through some algorithms such as ACO Ant Colony Optimization and EA Evolutionary Algorithm (or genetic algorithm) working in RAM, human can optimize the information. This Optimizational function is the intelligence; 5) In our experiments, not only can thought waves affect the voltage of the output photoelectric signals by its energy, but they can also selectively increase or decrease those photoelectric currents through remote consciousness interface and a conscious-brain information technology.

Heightening energetic stress selectively targets LKB1-deficient non-small cell lung cancers

PubMed Central

Momcilovic, Milica; McMickle, Robert; Abt, Evan; Seki, Atsuko; Simko, Sarah A.; Magyar, Clara; Stout, David B.; Fishbein, Michael C.; Walser, Tonya C.; Dubinett, Steven M.; Shackelford, David B.

2015-01-01

Inactivation of the LKB1 tumor suppressor is a frequent event in non-small cell lung carcinoma (NSCLC) leading to the activation of mammalian target of rapamycin complex 1 (mTORC1) and sensitivity to the metabolic stress inducer phenformin. In this study, we explored the combinatorial use of phenformin with the mTOR catalytic kinase inhibitor MLN0128 as a treatment strategy for NSCLC bearing co-mutations in the LKB1 and KRAS genes. NSCLC is a genetically and pathologically heterogeneous disease, giving rise to lung tumors of varying histologies that include adenocarcinomas (ADCs) and squamous cell carcinomas (SCCs). We demonstrate that phenformin in combination with MLN0128 induced a significant therapeutic response in KRAS/LKB1 mutant human cell lines and genetically engineered mouse models of NSCLC that develop both ADCs and SCCs. Specifically, we found that KRAS/LKB1 mutant lung ADCs responded strongly to phenformin + MLN0128 treatment, but the response of SCCs to single or combined treatment with MLN0128 was more attenuated due to acquired resistance to mTOR inhibition through modulation of the AKT-GSK signaling axis. Combinatorial use of the mTOR inhibitor and AKT inhibitor MK2206 robustly inhibited the growth and viability of squamous lung tumors thus providing an effective strategy to overcome resistance. Taken together, our findings define new personalized therapeutic strategies that may be rapidly translated into clinical use for the treatment of KRAS/LKB1 mutant adenocarcinomas and squamous cell tumors. PMID:26574479

Heightening Energetic Stress Selectively Targets LKB1-Deficient Non-Small Cell Lung Cancers.

PubMed

Momcilovic, Milica; McMickle, Robert; Abt, Evan; Seki, Atsuko; Simko, Sarah A; Magyar, Clara; Stout, David B; Fishbein, Michael C; Walser, Tonya C; Dubinett, Steven M; Shackelford, David B

2015-11-15

Inactivation of the LKB1 tumor suppressor is a frequent event in non-small cell lung carcinoma (NSCLC) leading to the activation of mTOR complex 1 (mTORC1) and sensitivity to the metabolic stress inducer phenformin. In this study, we explored the combinatorial use of phenformin with the mTOR catalytic kinase inhibitor MLN0128 as a treatment strategy for NSCLC bearing comutations in the LKB1 and KRAS genes. NSCLC is a genetically and pathologically heterogeneous disease, giving rise to lung tumors of varying histologies that include adenocarcinomas and squamous cell carcinomas (SCC). We demonstrate that phenformin in combination with MLN0128 induced a significant therapeutic response in KRAS/LKB1-mutant human cell lines and genetically engineered mouse models of NSCLC that develop both adenocarcinomas and SCCs. Specifically, we found that KRAS/LKB1-mutant lung adenocarcinomas responded strongly to phenformin + MLN0128 treatment, but the response of SCCs to single or combined treatment with MLN0128 was more attenuated due to acquired resistance to mTOR inhibition through modulation of the AKT-GSK signaling axis. Combinatorial use of the mTOR inhibitor and AKT inhibitor MK2206 robustly inhibited the growth and viability of squamous lung tumors, thus providing an effective strategy to overcome resistance. Taken together, our findings define new personalized therapeutic strategies that may be rapidly translated into clinical use for the treatment of KRAS/LKB1-mutant adenocarcinomas and squamous cell tumors. ©2015 American Association for Cancer Research.

A Decrease in Ambient Temperature Induces Post-Mitotic Enlargement of Palisade Cells in North American Lake Cress.

PubMed

Amano, Rumi; Nakayama, Hokuto; Morohoshi, Yurika; Kawakatsu, Yaichi; Ferjani, Ali; Kimura, Seisuke

2015-01-01

In order to maintain organs and structures at their appropriate sizes, multicellular organisms orchestrate cell proliferation and post-mitotic cell expansion during morphogenesis. Recent studies using Arabidopsis leaves have shown that compensation, which is defined as post-mitotic cell expansion induced by a decrease in the number of cells during lateral organ development, is one example of such orchestration. Some of the basic molecular mechanisms underlying compensation have been revealed by genetic and chimeric analyses. However, to date, compensation had been observed only in mutants, transgenics, and γ-ray-treated plants, and it was unclear whether it occurs in plants under natural conditions. Here, we illustrate that a shift in ambient temperature could induce compensation in Rorippa aquatica (Brassicaceae), a semi-aquatic plant found in North America. The results suggest that compensation is a universal phenomenon among angiosperms and that the mechanism underlying compensation is shared, in part, between Arabidopsis and R. aquatica.

A Decrease in Ambient Temperature Induces Post-Mitotic Enlargement of Palisade Cells in North American Lake Cress

PubMed Central

Morohoshi, Yurika; Kawakatsu, Yaichi; Ferjani, Ali; Kimura, Seisuke

2015-01-01

In order to maintain organs and structures at their appropriate sizes, multicellular organisms orchestrate cell proliferation and post-mitotic cell expansion during morphogenesis. Recent studies using Arabidopsis leaves have shown that compensation, which is defined as post-mitotic cell expansion induced by a decrease in the number of cells during lateral organ development, is one example of such orchestration. Some of the basic molecular mechanisms underlying compensation have been revealed by genetic and chimeric analyses. However, to date, compensation had been observed only in mutants, transgenics, and γ-ray–treated plants, and it was unclear whether it occurs in plants under natural conditions. Here, we illustrate that a shift in ambient temperature could induce compensation in Rorippa aquatica (Brassicaceae), a semi-aquatic plant found in North America. The results suggest that compensation is a universal phenomenon among angiosperms and that the mechanism underlying compensation is shared, in part, between Arabidopsis and R. aquatica. PMID:26569502

Specification of regional intestinal stem cell identity during Drosophila metamorphosis.

PubMed

Driver, Ian; Ohlstein, Benjamin

2014-05-01

In the adult Drosophila midgut the bone morphogenetic protein (BMP) signaling pathway is required to specify and maintain the acid-secreting region of the midgut known as the copper cell region (CCR). BMP signaling is also involved in the modulation of intestinal stem cell (ISC) proliferation in response to injury. How ISCs are able to respond to the same signaling pathway in a regionally different manner is currently unknown. Here, we show that dual use of the BMP signaling pathway in the midgut is possible because BMP signals are only capable of transforming ISC and enterocyte identity during a defined window of metamorphosis. ISC heterogeneity is established prior to adulthood and then maintained in cooperation with regional signals from surrounding tissue. Our data provide a conceptual framework for how other tissues maintained by regional stem cells might be patterned and establishes the pupal and adult midgut as a novel genetic platform for identifying genes necessary for regional stem cell specification and maintenance.

Oncology meets immunology: the cancer-immunity cycle.

PubMed

Chen, Daniel S; Mellman, Ira

2013-07-25

The genetic and cellular alterations that define cancer provide the immune system with the means to generate T cell responses that recognize and eradicate cancer cells. However, elimination of cancer by T cells is only one step in the Cancer-Immunity Cycle, which manages the delicate balance between the recognition of nonself and the prevention of autoimmunity. Identification of cancer cell T cell inhibitory signals, including PD-L1, has prompted the development of a new class of cancer immunotherapy that specifically hinders immune effector inhibition, reinvigorating and potentially expanding preexisting anticancer immune responses. The presence of suppressive factors in the tumor microenvironment may explain the limited activity observed with previous immune-based therapies and why these therapies may be more effective in combination with agents that target other steps of the cycle. Emerging clinical data suggest that cancer immunotherapy is likely to become a key part of the clinical management of cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

Altered Cytochrome c Display Precedes Apoptotic Cell Death in Drosophila

PubMed Central

Varkey, Johnson; Chen, Po; Jemmerson, Ronald; Abrams, John M.

1999-01-01

Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation and apoptosis. We found that an overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoked apoptogenic cytochrome c display and, surprisingly, caspase activity was necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation was triggered by mitochondria from apoptotic cells but identical preparations from healthy cells were inactive. Our observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases. PMID:10037791

The potential for crizotinib in non-small cell lung cancer: a perspective review

PubMed Central

Bang, Yung-Jue

2011-01-01

Tyrosine kinases have a crucial role as key regulators of signaling pathways that influence cell differentiation and growth. Dysregulation of tyrosine kinase-mediated signaling is understood to be an important oncogenic driver. Genetic rearrangements involving the tyrosine kinase anaplastic lymphoma kinase (ALK) gene occur in non-small cell lung cancer (NSCLC), anaplastic large cell lymphomoas, inflammatory myofibroblastic tumors, and other cancers. Cells with abnormal ALK signaling are sensitive to ALK inhibitors such as crizotinib. This review will highlight the discovery of the fusion between echinoderm microtubule-associated protein-like 4 (EML4) and ALK as an oncogenic driver, recognition of other ALK gene rearrangements in NSCLC, and the confirmation that crizotinib is an effective treatment for patients with ALK-positive NSCLC. Work is underway to further define the role for crizotinib in the treatment of ALK-positive lung cancer and other cancers and to investigate the molecular mechanisms for resistance to ALK inhibition with crizotinib. PMID:22084642

microRNA-206 promotes skeletal muscle regeneration and delays progression of Duchenne muscular dystrophy in mice

PubMed Central

Liu, Ning; Williams, Andrew H.; Maxeiner, Johanna M.; Bezprozvannaya, Svetlana; Shelton, John M.; Richardson, James A.; Bassel-Duby, Rhonda; Olson, Eric N.

2012-01-01

Skeletal muscle injury activates adult myogenic stem cells, known as satellite cells, to initiate proliferation and differentiation to regenerate new muscle fibers. The skeletal muscle–specific microRNA miR-206 is upregulated in satellite cells following muscle injury, but its role in muscle regeneration has not been defined. Here, we show that miR-206 promotes skeletal muscle regeneration in response to injury. Genetic deletion of miR-206 in mice substantially delayed regeneration induced by cardiotoxin injury. Furthermore, loss of miR-206 accelerated and exacerbated the dystrophic phenotype in a mouse model of Duchenne muscular dystrophy. We found that miR-206 acts to promote satellite cell differentiation and fusion into muscle fibers through suppressing a collection of negative regulators of myogenesis. Our findings reveal an essential role for miR-206 in satellite cell differentiation during skeletal muscle regeneration and indicate that miR-206 slows progression of Duchenne muscular dystrophy. PMID:22546853

One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

DOE PAGES

Herricks, Thurston; Dilworth, David J.; Mast, Fred D.; ...

2016-11-16

Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAYmore » extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.« less

One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

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

Herricks, Thurston; Dilworth, David J.; Mast, Fred D.

Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAYmore » extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.« less

Reconstituting development of pancreatic intraepithelial neoplasia from primary human pancreas duct cells

PubMed Central

Lee, Jonghyeob; Snyder, Emily R.; Liu, Yinghua; Gu, Xueying; Wang, Jing; Flowers, Brittany M.; Kim, Yoo Jung; Park, Sangbin; Szot, Gregory L.; Hruban, Ralph H.; Longacre, Teri A.; Kim, Seung K.

2017-01-01

Development of systems that reconstitute hallmark features of human pancreatic intraepithelial neoplasia (PanINs), the precursor to pancreatic ductal adenocarcinoma, could generate new strategies for early diagnosis and intervention. However, human cell-based PanIN models with defined mutations are unavailable. Here, we report that genetic modification of primary human pancreatic cells leads to development of lesions resembling native human PanINs. Primary human pancreas duct cells harbouring oncogenic KRAS and induced mutations in CDKN2A, SMAD4 and TP53 expand in vitro as epithelial spheres. After pancreatic transplantation, mutant clones form lesions histologically similar to native PanINs, including prominent stromal responses. Gene expression profiling reveals molecular similarities of mutant clones with native PanINs, and identifies potential PanIN biomarker candidates including Neuromedin U, a circulating peptide hormone. Prospective reconstitution of human PanIN development from primary cells provides experimental opportunities to investigate pancreas cancer development, progression and early-stage detection. PMID:28272465

Clonal architecture of secondary acute myeloid leukemia defined by single-cell sequencing.

PubMed

Hughes, Andrew E O; Magrini, Vincent; Demeter, Ryan; Miller, Christopher A; Fulton, Robert; Fulton, Lucinda L; Eades, William C; Elliott, Kevin; Heath, Sharon; Westervelt, Peter; Ding, Li; Conrad, Donald F; White, Brian S; Shao, Jin; Link, Daniel C; DiPersio, John F; Mardis, Elaine R; Wilson, Richard K; Ley, Timothy J; Walter, Matthew J; Graubert, Timothy A

2014-07-01

Next-generation sequencing has been used to infer the clonality of heterogeneous tumor samples. These analyses yield specific predictions-the population frequency of individual clones, their genetic composition, and their evolutionary relationships-which we set out to test by sequencing individual cells from three subjects diagnosed with secondary acute myeloid leukemia, each of whom had been previously characterized by whole genome sequencing of unfractionated tumor samples. Single-cell mutation profiling strongly supported the clonal architecture implied by the analysis of bulk material. In addition, it resolved the clonal assignment of single nucleotide variants that had been initially ambiguous and identified areas of previously unappreciated complexity. Accordingly, we find that many of the key assumptions underlying the analysis of tumor clonality by deep sequencing of unfractionated material are valid. Furthermore, we illustrate a single-cell sequencing strategy for interrogating the clonal relationships among known variants that is cost-effective, scalable, and adaptable to the analysis of both hematopoietic and solid tumors, or any heterogeneous population of cells.

ECUT: Energy Conversion and Utilization Technologies program - Biocatalysis research activity

NASA Technical Reports Server (NTRS)

Wilcox, R.

1984-01-01

The activities of the Biocatalysis Research Activity are organized into the Biocatalysis and Molecular Modeling work elements and a supporting planning and analysis function. In the Biocatalysis work element, progress is made in developing a method for stabilizing genetically engineered traits in microorganisms, refining a technique for monitoring cells that are genetically engineered, and identifying strains of fungi for highly efficient preprocessing of biomass for optimizing the efficiency of bioreactors. In the Molecular Modeling work element, a preliminary model of the behavior of enzymes is developed. A preliminary investigation of the potential for synthesizing enzymes for use in electrochemical processes is completed. Contact with industry and universities is made to define key biocatalysis technical issues and to broaden the range of potential participants in the activity. Analyses are conducted to identify and evaluate potential concepts for future research funding.

How animal models of leukaemias have already benefited patients.

PubMed

Ablain, Julien; Nasr, Rihab; Zhu, Jun; Bazarbachi, Ali; Lallemand-Breittenbach, Valérie; de Thé, Hugues

2013-04-01

The relative genetic simplicity of leukaemias, the development of which likely relies on a limited number of initiating events has made them ideal for disease modelling, particularly in the mouse. Animal models provide incomparable insights into the mechanisms of leukaemia development and allow exploration of the molecular pillars of disease maintenance, an aspect often biased in cell lines or ex vivo systems. Several of these models, which faithfully recapitulate the characteristics of the human disease, have been used for pre-clinical purposes and have been instrumental in predicting therapy response in patients. We plea for a wider use of genetically defined animal models in the design of clinical trials, with a particular focus on reassessment of existing cancer or non-cancer drugs, alone or in combination. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Adoptive T-cell therapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

PubMed

Fujiwara, Hiroshi

2014-02-01

The functional properties of the adoptive immune response mediated by effector T lymphocytes are decisively regulated by their T-cell receptors (TCRs). Transfer of genes encoding target antigen-specific receptors enables polyclonal T cells to redirect toward cancer cells and virally infected cells expressing those defined antigens. Using this technology, a large population of redirected T cells displaying uniform therapeutic properties has been produced, powerfully advancing their clinical application as "cellular drugs" for adoptive immunotherapy against cancer. Clinically, anticancer adoptive immunotherapy using these genetically engineered T cells has an impressive and proven track record. Notable examples include the dramatic benefit of chimeric antigen receptor gene-modified T cells redirected towards B-cell lineage antigen CD19 in patients with chronic lymphocytic leukemia, and the impressive outcomes in the use of TCR gene-modified T cells redirected towards NY-ESO-1, a representative cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. In this review, we briefly overview the current status of this treatment option in the context of hematological malignancy, and discuss a number of challenges that still pose an obstacle to the full effectiveness of this strategy.

Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 1: background to spermatogenesis, spermatogonia, and spermatocytes.

PubMed

Hermo, Louis; Pelletier, R-Marc; Cyr, Daniel G; Smith, Charles E

2010-04-01

Spermatogenesis, a study of germ cell development, is a long, orderly, and well-defined process occurring in seminiferous tubules of the testis. It is a temporal event whereby undifferentiated spermatogonial germ cells evolve into maturing spermatozoa over a period of several weeks. Spermatogenesis is characterized by three specific functional phases: proliferation, meiosis, and differentiation, and it involves spermatogonia, spermatocytes, and spermatids. Germ cells at steps of development form various cellular associations or stages, with 6, 12, and 14 specific stages being identified in human, mouse, and rat, respectively. The stages evolve over time in a given area of the seminiferous tubule forming a cycle of the seminiferous epithelium that has a well-defined duration for a given species. In this part, we discuss the proliferation and meiotic phase whereby spermatogonia undergo several mitotic divisions to form spermatocytes that undergo two meiotic divisions to form haploid spermatids. In the rat, spermatogonia can be subdivided into several classes: stem cells (A(s)), proliferating cells (A(pr), A(al)), and differentiating cells (A(1)-A(4), In, B). They are dependent on a specific microenvironment (niche) contributed by Sertoli, myoid, and Leydig cells for proper development. Spermatogonia possess several surface markers whereby they can be identified from each other. During meiosis, spermatocytes undergo chromosomal pairing, synapsis, and genetic exchange as well as transforming into haploid cells following meiosis. The meiotic cells form specific structural entities such as the synaptonemal complex and sex body. Many genes involved in spermatogonial renewal and the meiotic process have been identified and shown to be essential for this event. Copyright 2009 Wiley-Liss, Inc.

Identification of cells initiating human melanomas

PubMed Central

Schatton, Tobias; Murphy, George F.; Frank, Natasha Y.; Yamaura, Kazuhiro; Waaga-Gasser, Ana Maria; Gasser, Martin; Zhan, Qian; Jordan, Stefan; Duncan, Lyn M.; Weishaupt, Carsten; Fuhlbrigge, Robert C.; Kupper, Thomas S.; Sayegh, Mohamed H.; Frank, Markus H.

2012-01-01

Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies1,2 and solid cancers3–6. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs 7, 8) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5+ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5+ melanoma cells possess greater tumorigenic capacity than ABCB5− bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5+ sub-populations for self-renewal and differentiation, because ABCB5+ cancer cells generate both ABCB5+ and ABCB5− progeny, whereas ABCB5− tumour populations give rise, at lower rates, exclusively to ABCB5− cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5+ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy. PMID:18202660

Piecemeal Buildup of the Genetic Code, Ribosomes, and Genomes from Primordial tRNA Building Blocks

PubMed Central

Caetano-Anollés, Derek; Caetano-Anollés, Gustavo

2016-01-01

The origin of biomolecular machinery likely centered around an ancient and central molecule capable of interacting with emergent macromolecular complexity. tRNA is the oldest and most central nucleic acid molecule of the cell. Its co-evolutionary interactions with aminoacyl-tRNA synthetase protein enzymes define the specificities of the genetic code and those with the ribosome their accurate biosynthetic interpretation. Phylogenetic approaches that focus on molecular structure allow reconstruction of evolutionary timelines that describe the history of RNA and protein structural domains. Here we review phylogenomic analyses that reconstruct the early history of the synthetase enzymes and the ribosome, their interactions with RNA, and the inception of amino acid charging and codon specificities in tRNA that are responsible for the genetic code. We also trace the age of domains and tRNA onto ancient tRNA homologies that were recently identified in rRNA. Our findings reveal a timeline of recruitment of tRNA building blocks for the formation of a functional ribosome, which holds both the biocatalytic functions of protein biosynthesis and the ability to store genetic memory in primordial RNA genomic templates. PMID:27918435

Piecemeal Buildup of the Genetic Code, Ribosomes, and Genomes from Primordial tRNA Building Blocks.

PubMed

Caetano-Anollés, Derek; Caetano-Anollés, Gustavo

2016-12-02

The origin of biomolecular machinery likely centered around an ancient and central molecule capable of interacting with emergent macromolecular complexity. tRNA is the oldest and most central nucleic acid molecule of the cell. Its co-evolutionary interactions with aminoacyl-tRNA synthetase protein enzymes define the specificities of the genetic code and those with the ribosome their accurate biosynthetic interpretation. Phylogenetic approaches that focus on molecular structure allow reconstruction of evolutionary timelines that describe the history of RNA and protein structural domains. Here we review phylogenomic analyses that reconstruct the early history of the synthetase enzymes and the ribosome, their interactions with RNA, and the inception of amino acid charging and codon specificities in tRNA that are responsible for the genetic code. We also trace the age of domains and tRNA onto ancient tRNA homologies that were recently identified in rRNA. Our findings reveal a timeline of recruitment of tRNA building blocks for the formation of a functional ribosome, which holds both the biocatalytic functions of protein biosynthesis and the ability to store genetic memory in primordial RNA genomic templates.

Modifiers and mechanisms of multi-system polyglutamine neurodegenerative disorders: lessons from fly models.

PubMed

Mallik, Moushami; Lakhotia, Subhash C

2010-12-01

Polyglutamine (polyQ) diseases, resulting from a dynamic expansion of glutamine repeats in a polypeptide, are a class of genetically inherited late onset neurodegenerative disorders which, despite expression of the mutated gene widely in brain and other tissues, affect defined subpopulations of neurons in a disease-specific manner. We briefly review the different polyQ-expansion-induced neurodegenerative disorders and the advantages of modelling them in Drosophila. Studies using the fly models have successfully identified a variety of genetic modifiers and have helped in understanding some of the molecular events that follow expression of the abnormal polyQ proteins. Expression of the mutant polyQ proteins causes, as a consequence of intra-cellular and inter-cellular networking, mis-regulation at multiple steps like transcriptional and posttranscriptional regulations, cell signalling, protein quality control systems (protein folding and degradation networks), axonal transport machinery etc., in the sensitive neurons, resulting ultimately in their death. The diversity of genetic modifiers of polyQ toxicity identified through extensive genetic screens in fly and other models clearly reflects a complex network effect of the presence of the mutated protein. Such network effects pose a major challenge for therapeutic applications.

Novel genetic association of TNF-α-238 and PDCD1-7209 polymorphisms with long-term non-progressive HIV-1 infection.

PubMed

Nasi, Milena; Riva, Agostino; Borghi, Vanni; D'Amico, Roberto; Del Giovane, Cinzia; Casoli, Claudio; Galli, Massimo; Vicenzi, Elisa; Gibellini, Lara; De Biasi, Sara; Clerici, Mario; Mussini, Cristina; Cossarizza, Andrea; Pinti, Marcello

2013-10-01

About 2-5% of HIV-1-infected subjects, defined as long-term non-progressors (LTNPs), remain immunologically stable for a long time without treatment. The factors governing this condition are known only in part, and include genetic factors. Thus, we studied 20 polymorphisms of 15 genes encoding proinflammatory and immunoregulatory cytokines, chemokines and their receptors, genes involved in apoptosis, and the gene HCP5. We analyzed 47 Caucasian LTNPs infected for >9 years, compared with 131 HIV-1-infected Caucasian patients defined as 'usual progressors'. The genotypes were determined by methods based upon PCR, and the statistical analysis was performed by univariate logistic regression. The well-known CCR5Δ32 del32 allele, the cell death-related TNF-α-238 A and PDCD1-7209 T alleles, and HCP5 rs2395029 G, a non-coding protein associated with the HLA-B*5701, were found positively associated with the LTNP condition. No association was observed for other single nucleotide polymorphisms (SDF-1-801, IL-10-592, MCP-1-2518, CX3CR1 V249I, CCR2V64I, RANTES-403, IL-2-330, IL-1β-511, IL-4-590, FASL IVS3nt-169, FAS-670, FAS-1377, FASL IVS2nt-124, PDCD1-7146, MMP-7-181, and MMP7-153). The novel genetic associations between allelic variants of genes TNF-α-238 and PDCD1-7209 with the LTNP condition underline the importance of host genetic factors in the progression of HIV-1 infection and in immunological preservation. Copyright © 2013 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Molecular Basis for Electron Flow Within Metal-and Electrode-Reducing Biofilms

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

Bond, Daniel R.

2016-11-01

Electrochemical, spectral, genetic, and biochemical techniques were developed to reveal that a diverse suite of redox proteins and structural macromolecules outside the cell work together to move electrons long distances between Geobacter cells to metals and electrodes. In this project, we greatly expanded the known participants in the electron transfer pathway of Geobacter. For example, in addition to well-studied pili, polysaccharides contribute to anchoring, different cytochromes are required under different conditions, strategies change with redox potential, and the localization of these components can change depending on where cells are located in a biofilm. By inventing new electrodes compatible with real-timemore » spectral measurements, we were able to visualize the redox status of biofilms in action, leading to a hypothesis that long-distance electron transfer is ultimately limiting in these systems and redox potentials change within biofilms. The goals of this project were met, as we were able to 1) identify new elements crucial to the expression, assembly and function of the extracellular electron transfer phenotype 2) expand spectral and electrochemical techniques to define the mechanism and route of electron transfer through the matrix, and 3) combine this knowledge to build the next generation of genetic tools for study of this complex process.« less

WASP and SCAR are evolutionarily conserved in actin-filled pseudopod-based motility

PubMed Central

2017-01-01

Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode and identify its phenotypic and molecular markers. In this study, we focus on a widely dispersed migration mode characterized by dynamic actin-filled pseudopods that we call “α-motility.” Mining genomic data reveals a clear trend: only organisms with both WASP and SCAR/WAVE—activators of branched actin assembly—make actin-filled pseudopods. Although SCAR has been shown to drive pseudopod formation, WASP’s role in this process is controversial. We hypothesize that these genes collectively represent a genetic signature of α-motility because both are used for pseudopod formation. WASP depletion from human neutrophils confirms that both proteins are involved in explosive actin polymerization, pseudopod formation, and cell migration. WASP and WAVE also colocalize to dynamic signaling structures. Moreover, retention of WASP together with SCAR correctly predicts α-motility in disease-causing chytrid fungi, which we show crawl at >30 µm/min with actin-filled pseudopods. By focusing on one migration mode in many eukaryotes, we identify a genetic marker of pseudopod formation, the morphological feature of α-motility, providing evidence for a widely distributed mode of cell crawling with a single evolutionary origin. PMID:28473602

Single Neuron Optimization as a Basis for Accurate Biophysical Modeling: The Case of Cerebellar Granule Cells.

PubMed

Masoli, Stefano; Rizza, Martina F; Sgritta, Martina; Van Geit, Werner; Schürmann, Felix; D'Angelo, Egidio

2017-01-01

In realistic neuronal modeling, once the ionic channel complement has been defined, the maximum ionic conductance (G i-max ) values need to be tuned in order to match the firing pattern revealed by electrophysiological recordings. Recently, selection/mutation genetic algorithms have been proposed to efficiently and automatically tune these parameters. Nonetheless, since similar firing patterns can be achieved through different combinations of G i-max values, it is not clear how well these algorithms approximate the corresponding properties of real cells. Here we have evaluated the issue by exploiting a unique opportunity offered by the cerebellar granule cell (GrC), which is electrotonically compact and has therefore allowed the direct experimental measurement of ionic currents. Previous models were constructed using empirical tuning of G i-max values to match the original data set. Here, by using repetitive discharge patterns as a template, the optimization procedure yielded models that closely approximated the experimental G i-max values. These models, in addition to repetitive firing, captured additional features, including inward rectification, near-threshold oscillations, and resonance, which were not used as features. Thus, parameter optimization using genetic algorithms provided an efficient modeling strategy for reconstructing the biophysical properties of neurons and for the subsequent reconstruction of large-scale neuronal network models.

Analysis of bone-cartilage-stromal progenitor populations in trauma induced and genetic models of heterotopic ossification

PubMed Central

Agarwal, Shailesh; Loder, Shawn; Li, Shuli; Shrestha, Swati; Li, Jon; Zhao, Bin; Mishina, Yuji; James, Aaron; Levi, Benjamin

2016-01-01

Heterotopic ossification (HO), the formation of extra-skeletal bone in soft tissues, is a pathologic process occurring after substantial burns or trauma, or in patients with type I bone morphogenetic protein (BMP) receptor hyperactivating mutations. Identifying the cells responsible for de novo bone formation during adulthood is of critical importance for therapeutic and regenerative purposes. Using a model of trauma-induced HO with hindlimb Achilles’ tenotomy and dorsal burn injury and a genetic non-trauma HO model (Nfatc1-Cre/caAcvr1fl/wt), we demonstrate enrichment of previously defined bone-cartilage-stromal progenitor cells (BCSP: AlphaV+/CD105+/Tie2-/CD45-/Thy1-/6C3-) at the site of HO formation when compared with marrow isolated from the ipsilateral hindlimb, or from tissue of the contralateral, uninjured hindlimb. Upon transplantation into tenotomy sites soon after injury, BCSPs isolated from neonatal mice or developing HO incorporate into the developing lesion in cartilage and bone and express chondrogenic and osteogenic transcription factors. Additionally, BCSPs isolated from developing HO similarly incorporate into new HO lesions upon transplantation. Finally, adventitial cells, but not pericytes, appear to play a supportive role in HO formation. Our findings indicate that BCSPs contribute to de novo bone formation during adulthood and may hold substantial regenerative potential. PMID:27068890

The interplay between genetic and bioelectrical signaling permits a spatial regionalisation of membrane potentials in model multicellular ensembles

PubMed Central

Cervera, Javier; Meseguer, Salvador; Mafe, Salvador

2016-01-01

The single cell-centred approach emphasises ion channels as specific proteins that determine individual properties, disregarding their contribution to multicellular outcomes. We simulate the interplay between genetic and bioelectrical signals in non-excitable cells from the local single-cell level to the long range multicellular ensemble. The single-cell genetic regulation is based on mean-field kinetic equations involving the mRNA and protein concentrations. The transcription rate factor is assumed to depend on the absolute value of the cell potential, which is dictated by the voltage-gated cell ion channels and the intercellular gap junctions. The interplay between genetic and electrical signals may allow translating single-cell states into multicellular states which provide spatio-temporal information. The model results have clear implications for biological processes: (i) bioelectric signals can override slightly different genetic pre-patterns; (ii) ensembles of cells initially at the same potential can undergo an electrical regionalisation because of persistent genetic differences between adjacent spatial regions; and (iii) shifts in the normal cell electrical balance could trigger significant changes in the genetic regulation. PMID:27731412

The interplay between genetic and bioelectrical signaling permits a spatial regionalisation of membrane potentials in model multicellular ensembles.

PubMed

Cervera, Javier; Meseguer, Salvador; Mafe, Salvador

2016-10-12

The single cell-centred approach emphasises ion channels as specific proteins that determine individual properties, disregarding their contribution to multicellular outcomes. We simulate the interplay between genetic and bioelectrical signals in non-excitable cells from the local single-cell level to the long range multicellular ensemble. The single-cell genetic regulation is based on mean-field kinetic equations involving the mRNA and protein concentrations. The transcription rate factor is assumed to depend on the absolute value of the cell potential, which is dictated by the voltage-gated cell ion channels and the intercellular gap junctions. The interplay between genetic and electrical signals may allow translating single-cell states into multicellular states which provide spatio-temporal information. The model results have clear implications for biological processes: (i) bioelectric signals can override slightly different genetic pre-patterns; (ii) ensembles of cells initially at the same potential can undergo an electrical regionalisation because of persistent genetic differences between adjacent spatial regions; and (iii) shifts in the normal cell electrical balance could trigger significant changes in the genetic regulation.

Genetic parameters of milk production traits and fatty acid contents in milk for Holstein cows in parity 1-3.

PubMed

Bastin, C; Soyeurt, H; Gengler, N

2013-04-01

The objective of this study was to estimate genetic parameters of milk, fat, and protein yields, fat and protein contents, somatic cell count, and 17 groups and individual milk fatty acid (FA) contents predicted by mid-infrared spectrometry for first-, second- and third-parity Holstein cows. Edited data included records collected in the Walloon region of Belgium from 37,768 cows in parity 1,22,566 cows in parity 2 and 8221 in parity 3. A total of 69 (23 traits for three parities) single-trait random regression animal test-day models were run. Approximate genetic correlations among traits were inferred from pairwise regressions among estimated breeding values of cow having observations. Heritability and genetic correlation estimates from this study reflected the origins of FA: de novo synthetized or originating from the diet and the body fat mobilization. Averaged daily heritabilities of FA contents in milk ranged between 0.18 and 0.47. Average daily genetic correlations (averaged across days in milk and parities) among groups and individual FA contents in milk ranged between 0.31 and 0.99. The genetic variability of FAs in combination with the moderate to high heritabilities indicated that FA contents in milk could be changed by genetic selection; however, desirable direction of change in these traits remains unclear and should be defined with respect to all issues of importance related to milk FA. © 2012 Blackwell Verlag GmbH.

Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

PubMed

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

2016-01-01

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

Myeloid-derived suppressor activity is mediated by monocytic lineages maintained by continuous inhibition of extrinsic and intrinsic death pathways.

PubMed

Haverkamp, Jessica M; Smith, Amber M; Weinlich, Ricardo; Dillon, Christopher P; Qualls, Joseph E; Neale, Geoffrey; Koss, Brian; Kim, Young; Bronte, Vincenzo; Herold, Marco J; Green, Douglas R; Opferman, Joseph T; Murray, Peter J

2014-12-18

Nonresolving inflammation expands a heterogeneous population of myeloid suppressor cells capable of inhibiting T cell function. This heterogeneity has confounded the functional dissection of individual myeloid subpopulations and presents an obstacle for antitumor immunity and immunotherapy. Using genetic manipulation of cell death pathways, we found the monocytic suppressor-cell subset, but not the granulocytic subset, requires continuous c-FLIP expression to prevent caspase-8-dependent, RIPK3-independent cell death. Development of the granulocyte subset requires MCL-1-mediated control of the intrinsic mitochondrial death pathway. Monocytic suppressors tolerate the absence of MCL-1 provided cytokines increase expression of the MCL-1-related protein A1. Monocytic suppressors mediate T cell suppression, whereas their granulocytic counterparts lack suppressive function. The loss of the granulocytic subset via conditional MCL-1 deletion did not alter tumor incidence implicating the monocytic compartment as the functionally immunosuppressive subset in vivo. Thus, death pathway modulation defines the development, survival, and function of myeloid suppressor cells. Copyright © 2014 Elsevier Inc. All rights reserved.

[Telomerase activity in uveal melanomas].

PubMed

Rohrbach, J M; Riedinger, C; Wild, M; Partsch, M

2000-05-01

The maximum number of cell divisions of a certain cell population is genetically fixed so that aging cells become non-dividing (senescent) at least. This replicative life span, also known as "Hayflick limit", is probably defined by a "critical" length of the telomeres. Telomeres are special DNA-sequences located at the four ends of the chromosomes which are shortened with each cell cycle. Cells of most, but not all malignant tumours have been shown to reactivate the enzyme telomerase so that telomeres can be reconstructed, "Hayflick limit" can be overcome, and unlimited cell division can be established. This study was undertaken to elucidate whether telomerase reactivation is used by uveal melanoma cells. Fresh tumour tissue was removed from 10 untreated uveal melanomas after enucleation. Telomerase activity was determined using a PCR ELISA according to the Telomeric Repeat Amplification Protocol (TRAP). Normal tissue of the skin and the conjunctiva served as control. Telomerase activity was detectable in 90% of the investigated uveal melanomas. All control specimens were telomerase negative. Uveal melanoma growth seems to depend on telomerase reactivation. Thus, telomerase inhibition could offer a new principle for uveal melanoma therapy in the future.

Targeted Disruption of Orchestration between Stroma and Tumor Cells in Pancreatic Cancer: Molecular Basis and Therapeutic Implications

PubMed Central

Kong, Xiangyu; Li, Lei; Li, Zhaoshen; Xie, Keping

2012-01-01

Pancreatic cancer is one of the most lethal malignancies, with a prominent desmoplastic reaction as the defining hallmark of the disease. The past several decades have seen dramatic progress in understanding of pancreatic cancer pathogenesis, including the identification of precursor lesions, sequential transformation from normal pancreas to invasive pancreatic cancer and corresponding signature genetic events, and the biological impact of those alterations on malignant behaviors. However, the current therapeutic strategies for epithelial tumor cells, which have exhibited potent antitumor activity in cell culture and animal models, have failed to have significant effects in the clinic. The desmoplastic stroma surrounding pancreatic cancer cells, which accounts for about 90% of a tumor’s mass, clearly is not a passive scaffold for cancer cells but an active contributor to carcinogenesis. Improved understanding of the dynamic interaction between cancer cells and their stroma will be important to designing new, effective therapeutic strategies for pancreatic cancer. This review focuses on the origination of stromal molecular and cellular components in pancreatic tumors, their biological effects on pancreatic cancer cells, and the orchestration between these two components. PMID:22749856

Changes in the Coding and Non-coding Transcriptome and DNA Methylome that Define the Schwann Cell Repair Phenotype after Nerve Injury.

PubMed

Arthur-Farraj, Peter J; Morgan, Claire C; Adamowicz, Martyna; Gomez-Sanchez, Jose A; Fazal, Shaline V; Beucher, Anthony; Razzaghi, Bonnie; Mirsky, Rhona; Jessen, Kristjan R; Aitman, Timothy J

2017-09-12

Repair Schwann cells play a critical role in orchestrating nerve repair after injury, but the cellular and molecular processes that generate them are poorly understood. Here, we perform a combined whole-genome, coding and non-coding RNA and CpG methylation study following nerve injury. We show that genes involved in the epithelial-mesenchymal transition are enriched in repair cells, and we identify several long non-coding RNAs in Schwann cells. We demonstrate that the AP-1 transcription factor C-JUN regulates the expression of certain micro RNAs in repair Schwann cells, in particular miR-21 and miR-34. Surprisingly, unlike during development, changes in CpG methylation are limited in injury, restricted to specific locations, such as enhancer regions of Schwann cell-specific genes (e.g., Nedd4l), and close to local enrichment of AP-1 motifs. These genetic and epigenomic changes broaden our mechanistic understanding of the formation of repair Schwann cell during peripheral nervous system tissue repair. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Analysis of cellular and humoral immune responses against cytomegalovirus in patients with autoimmune Addison's disease.

PubMed

Edvardsen, Kine; Hellesen, Alexander; Husebye, Eystein S; Bratland, Eirik

2016-03-09

Autoimmune Addison's disease (AAD) is caused by multiple genetic and environmental factors. Variants of genes encoding immunologically important proteins such as the HLA molecules are strongly associated with AAD, but any environmental risk factors have yet to be defined. We hypothesized that primary or reactivating infections with cytomegalovirus (CMV) could represent an environmental risk factor in AAD, and that CMV specific CD8(+) T cell responses may be dysregulated, possibly leading to a suboptimal control of CMV. In particular, the objective was to assess the HLA-B8 restricted CD8(+) T cell response to CMV since this HLA class I variant is a genetic risk factor for AAD. To examine the CD8(+) T cell response in detail, we analyzed the HLA-A2 and HLA-B8 restricted responses in AAD patients and healthy controls seropositive for CMV antibodies using HLA multimer technology, IFN-γ ELISpot and a CD107a based degranulation assay. No differences between patients and controls were found in functions or frequencies of CMV-specific T cells, regardless if the analyses were performed ex vivo or after in vitro stimulation and expansion. However, individual patients showed signs of reactivating CMV infection correlating with poor CD8(+) T cell responses to the virus, and a concomitant upregulation of interferon regulated genes in peripheral blood cells. Several recently diagnosed AAD patients also showed serological signs of ongoing primary CMV infection. CMV infection does not appear to be a major environmental risk factor in AAD, but may represent a precipitating factor in individual patients.

Deficiency in Protein Tyrosine Phosphatase PTP1B Shortens Lifespan and Leads to Development of Acute Leukemia.

PubMed

Le Sommer, Samantha; Morrice, Nicola; Pesaresi, Martina; Thompson, Dawn; Vickers, Mark A; Murray, Graeme I; Mody, Nimesh; Neel, Benjamin G; Bence, Kendra K; Wilson, Heather M; Delibegović, Mirela

2018-01-01

Protein tyrosine phosphatase PTP1B is a critical regulator of signaling pathways controlling metabolic homeostasis, cell proliferation, and immunity. In this study, we report that global or myeloid-specific deficiency of PTP1B in mice decreases lifespan. We demonstrate that myeloid-specific deficiency of PTP1B is sufficient to promote the development of acute myeloid leukemia. LysM-PTP1B -/- mice lacking PTP1B in the innate myeloid cell lineage displayed a dysregulation of bone marrow cells with a rapid decline in population at midlife and a concomitant increase in peripheral blood blast cells. This phenotype manifested further with extramedullary tumors, hepatic macrophage infiltration, and metabolic reprogramming, suggesting increased hepatic lipid metabolism prior to overt tumor development. Mechanistic investigations revealed an increase in anti-inflammatory M2 macrophage responses in liver and spleen, as associated with increased expression of arginase I and the cytokines IL10 and IL4. We also documented STAT3 hypersphosphorylation and signaling along with JAK-dependent upregulation of antiapoptotic proteins Bcl2 and BclXL. Our results establish a tumor suppressor role for PTP1B in the myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Significance: This study defines a tumor suppressor function for the protein tyrosine phosphatase PTP1B in myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Cancer Res; 78(1); 75-87. ©2017 AACR . ©2017 American Association for Cancer Research.

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

PubMed Central

Romaniuk, Joseph A. H.; Cegelski, Lynette

2015-01-01

The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms. PMID:26370936

Rare variants in RTEL1 are associated with familial interstitial pneumonia.

PubMed

Cogan, Joy D; Kropski, Jonathan A; Zhao, Min; Mitchell, Daphne B; Rives, Lynette; Markin, Cheryl; Garnett, Errine T; Montgomery, Keri H; Mason, Wendi R; McKean, David F; Powers, Julia; Murphy, Elissa; Olson, Lana M; Choi, Leena; Cheng, Dong-Sheng; Blue, Elizabeth Marchani; Young, Lisa R; Lancaster, Lisa H; Steele, Mark P; Brown, Kevin K; Schwarz, Marvin I; Fingerlin, Tasha E; Schwartz, David A; Lawson, William E; Loyd, James E; Zhao, Zhongming; Phillips, John A; Blackwell, Timothy S

2015-03-15

Up to 20% of cases of idiopathic interstitial pneumonia cluster in families, comprising the syndrome of familial interstitial pneumonia (FIP); however, the genetic basis of FIP remains uncertain in most families. To determine if new disease-causing rare genetic variants could be identified using whole-exome sequencing of affected members from FIP families, providing additional insights into disease pathogenesis. Affected subjects from 25 kindreds were selected from an ongoing FIP registry for whole-exome sequencing from genomic DNA. Candidate rare variants were confirmed by Sanger sequencing, and cosegregation analysis was performed in families, followed by additional sequencing of affected individuals from another 163 kindreds. We identified a potentially damaging rare variant in the gene encoding for regulator of telomere elongation helicase 1 (RTEL1) that segregated with disease and was associated with very short telomeres in peripheral blood mononuclear cells in 1 of 25 families in our original whole-exome sequencing cohort. Evaluation of affected individuals in 163 additional kindreds revealed another eight families (4.7%) with heterozygous rare variants in RTEL1 that segregated with clinical FIP. Probands and unaffected carriers of these rare variants had short telomeres (<10% for age) in peripheral blood mononuclear cells and increased T-circle formation, suggesting impaired RTEL1 function. Rare loss-of-function variants in RTEL1 represent a newly defined genetic predisposition for FIP, supporting the importance of telomere-related pathways in pulmonary fibrosis.

The Influenza A Virus Genotype Determines the Antiviral Function of NF-κB

PubMed Central

Dam, Sharmistha; Kracht, Michael; Pleschka, Stephan

2016-01-01

ABSTRACT The role of NF-κB in influenza A virus (IAV) infection does not reveal a coherent picture, as pro- and also antiviral functions of this transcription factor have been described. To address this issue, we used clustered regularly interspaced short palindromic repeat with Cas9 (CRISPR-Cas9)-mediated genome engineering to generate murine MLE-15 cells lacking two essential components of the NF-κB pathway. Cells devoid of either the central NF-κB essential modulator (NEMO) scaffold protein and thus defective in IκB kinase (IKK) activation or cells not expressing the NF-κB DNA-binding and transactivation subunit p65 were tested for propagation of the SC35 virus, which has an avian host range, and its mouse-adapted variant, SC35M. While NF-κB was not relevant for replication of SC35M, the absence of NF-κB activity increased replication of the nonadapted SC35 virus. This antiviral effect of NF-κB was most prominent upon infection of cells with low virus titers as they usually occur during the initiation phase of IAV infection. The defect in NF-κB signaling resulted in diminished IAV-triggered phosphorylation of interferon regulatory factor 3 (IRF3) and expression of the antiviral beta interferon (IFN-β) gene. To identify the viral proteins responsible for NF-κB dependency, reassortant viruses were generated by reverse genetics. SC35 viruses containing the SC35M segment encoding neuraminidase (NA) were completely inert to the inhibitory effect of NF-κB, emphasizing the importance of the viral genotype for susceptibility to the antiviral functions of NF-κB. IMPORTANCE This study addresses two different issues. First, we investigated the role of the host cell transcription factor NF-κB in IAV replication by genetic manipulation of IAVs by reverse genetics combined with targeted genome engineering of host cells using CRISPR-Cas9. The analysis of these two highly defined genetic systems indicated that the IAV genotype can influence whether NF-κB displays an antiviral function and thus might in part explain incoherent results from the literature. Second, we found that perturbation of NF-κB function greatly improved the growth of a nonadapted IAV, suggesting that NF-κB may contribute to the maintenance of the host species barrier. PMID:27356900

Metastatic pheochromocytoma: clinical, genetic, and histopathologic characteristics

PubMed Central

Zelinka, Tomáš; Musil, Zdeněk; Dušková, Jaroslava; Burton, Deborah; Merino, Maria J; Milosevic, Dragana; Widimský, Jiří; Pacak, Karel

2011-01-01

Background Pheochromocytomas are tumors arising from chromaffin tissue located in the adrenal medulla associated with typical symptoms and signs which may occasionally develop metastases, which are defined as the presence of tumor cells at sites where these cells are not found. This retrospective analysis was focused on clinical, genetic, and histopathologic characteristics of primary metastatic versus primary benign pheochromocytomas. Materials and methods We identified 41 subjects with metastatic pheochromocytoma and 108 subjects with apparently benign pheochromocytoma. We assessed dimension and biochemical profile of the primary tumor, age at presentation, and time to develop metastases. Results Subjects with metastatic pheochromocytoma presented at a significantly younger age (41.4±14.7 vs. 50.2±13.7 years; P<0.001), with larger primary tumors (8.38±3.27 cm vs. 6.18±2.75 cm; P<0.001) and secreted more frequently norepinephrine (95.1% vs. 83.3 %; P=0.046) compared to subjects with apparently benign pheochromocytomas. No significant differences were found in the incidence of genetic mutations in both groups of subjects (25.7 % in the metastatic group and 14.7 % in the benign group; P=0.13). From available histopathologic markers of potential malignancy, only necrosis occurred more frequently in subjects with metastatic pheochromocytoma (27.6 % vs. 0 %; P<0.001). The median time to develop metastases was 3.6 years with the longest interval 24 years. Conclusions In conclusion, regardless of a genetic background, the size of a primary pheochromocytoma and age of its first presentation are two independent risk factors associated with the development of metastatic disease. PMID:21692797

Molecular characterization of a strong candidate region for schizophrenia

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

Karayiorgou, M.; Housman, D.E.; Morrow, B.

Two lines of evidence point to a region on chromosome 22 as potentially involved in the etiology of schizophrenia: First, our own linkage data and second, observations that a greater than expected number of cases with the VCF (velo-cardio-facial) syndrome, a developmental syndrome due to microdeletions of the same genetic region, develop psychotic illness during adolescence. On the molecular genetic level, we are testing the hypothesis that the partial phenotypic overlap between schizophrenia and VCF may be due to overlapping genetic abnormalities. To that end, we have generated somatic cell hybrids from an initial group of nine VCF patients overmore » the age of 15 who underwent psychiatric evaluation. Three were assigned a DSM-III-R diagnosis of schizophrenia. Several hybrid cell lines were generated from each patient carrying either the deleted chromosome, or the intact chromosome, or both. We have analyzed these hybrids and the extent of their chromosome 22 deletions with 41 markers so far (21 polymorphic microsatellite markers and 20 STSs). One of these markers is COMT (catechol-O-methyltransferase) that could be considered a candidate for schizophrenia. We are searching for potential molecular genetic differences between the subgroup of VCF patients that do develop schizophrenia and the subgroup that do not. Our initial efforts concentrate on the possibility of correlation between the extent of the deletion and the schizophrenic phenotype. Results from our analysis so far will be presented. Our goal is to narrow and define more accurately the region potentially involved in the etiology of schizophrenia and successfully identify any gene(s) that may play a role.« less

Clinical and Molecular Features of Renal and Pheochromocytoma/Paraganglioma Tumor Association Syndrome (RAPTAS): Case Series and Literature Review.

PubMed

Casey, Ruth T; Warren, Anne Y; Martin, Jose Ezequiel; Challis, Benjamin G; Rattenberry, Eleanor; Whitworth, James; Andrews, Katrina A; Roberts, Thomas; Clark, Graeme R; West, Hannah; Smith, Philip S; Docquier, France M; Rodger, Fay; Murray, Vicki; Simpson, Helen L; Wallis, Yvonne; Giger, Olivier; Tran, Maxine; Tomkins, Susan; Stewart, Grant D; Park, Soo-Mi; Woodward, Emma R; Maher, Eamonn R

2017-11-01

The co-occurrence of pheochromocytoma (PC) and renal tumors was linked to the inherited familial cancer syndrome von Hippel-Lindau (VHL) disease more than six decades ago. Subsequently, other shared genetic causes of predisposition to renal tumors and to PC, paraganglioma (PGL), or head and neck paraganglioma (HNPGL) have been described, but case series of non-VHL-related cases of renal tumor and pheochromocytoma/paraganglioma tumor association syndrome (RAPTAS) are rare. To determine the clinical and molecular features of non-VHL RAPTAS by literature review and characterization of a case series. A review of the literature was performed and a retrospective study of referrals for investigation of genetic causes of RAPTAS. Literature review revealed evidence of an association, in addition to VHL disease, between germline mutations in SDHB, SDHC, SDHD, TMEM127, and MAX genes and RAPTAS [defined here as the co-occurrence of tumors from both classes (PC/PGL/HNPGL and renal tumors) in the same individual or in first-degree relatives]. In both the literature review and our case series of 22 probands with non-VHL RAPTAS, SDHB mutations were the most frequent cause of non-VHL RAPTAS. A genetic cause was identified in 36.3% (8/22) of kindreds. Renal tumors and PC/PGL/HNPGL tumors share common molecular features and their co-occurrence in an individual or family should prompt genetic investigations. We report a case of MAX-associated renal cell carcinoma and confirm the role of TMEM127 mutations with renal cell carcinoma predisposition. Copyright © 2017 Endocrine Society

The Next Generation of Orthotopic Thyroid Cancer Models: Immunocompetent Orthotopic Mouse Models of BRAFV600E-Positive Papillary and Anaplastic Thyroid Carcinoma

PubMed Central

Vanden Borre, Pierre; McFadden, David G.; Gunda, Viswanath; Sadow, Peter M.; Varmeh, Shohreh; Bernasconi, Maria; Jacks, Tyler

2014-01-01

Background: While the development of new treatments for aggressive thyroid cancer has advanced in the last 10 years, progress has trailed headways made with other malignancies. A lack of reliable authenticated human cell lines and reproducible animal models is one major roadblock to preclinical testing of novel therapeutics. Existing xenograft and orthotopic mouse models of aggressive thyroid cancer rely on the implantation of highly passaged human thyroid carcinoma lines in immunodeficient mice. Genetically engineered models of papillary and undifferentiated (anaplastic) thyroid carcinoma (PTC and ATC) are immunocompetent; however, slow and stochastic tumor development hinders high-throughput testing. Novel models of PTC and ATC in which tumors arise rapidly and synchronously in immunocompetent mice would facilitate the investigation of novel therapeutics and approaches. Methods: We characterized and utilized mouse cell lines derived from PTC and ATC tumors arising in genetically engineered mice with thyroid-specific expression of endogenous BrafV600E/WT and deletion of either Trp53 (p53) or Pten. These murine thyroid cancer cells were transduced with luciferase- and GFP-expressing lentivirus and implanted into the thyroid glands of immunocompetent syngeneic B6129SF1/J mice in which the growth characteristics were assessed. Results: Large locally aggressive thyroid tumors form within one week of implantation. Tumors recapitulate their histologic subtype, including well-differentiated PTC and ATC, and exhibit CD3+, CD8+, B220+, and CD163+ immune cell infiltration. Tumor progression can be followed in vivo using luciferase and ex vivo using GFP. Metastatic spread is not detected at early time points. Conclusions: We describe the development of the next generation of murine orthotopic thyroid cancer models. The implantation of genetically defined murine BRAF-mutated PTC and ATC cell lines into syngeneic mice results in rapid and synchronous tumor formation. This model allows for preclinical investigation of novel therapeutics and/or therapeutic combinations in the context of a functional immune system. PMID:24295207

Evidences Suggesting Involvement of Viruses in Oral Squamous Cell Carcinoma

PubMed Central

Gupta, Kanupriya; Metgud, Rashmi

2013-01-01

Oral cancer is one of the most common cancers and it constitutes a major health problem particularly in developing countries. Oral squamous cell carcinoma (OSCC) represents the most frequent of all oral neoplasms. Several risk factors have been well characterized to be associated with OSCC with substantial evidences. The etiology of OSCC is complex and involves many factors. The most clearly defined potential factors are smoking and alcohol, which substantially increase the risk of OSCC. However, despite this clear association, a substantial proportion of patients develop OSCC without exposure to them, emphasizing the role of other risk factors such as genetic susceptibility and oncogenic viruses. Some viruses are strongly associated with OSCC while the association of others is less frequent and may depend on cofactors for their carcinogenic effects. Therefore, the exact role of viruses must be evaluated with care in order to improve the diagnosis and treatment of OSCC. Although a viral association within a subset of OSCC has been shown, the molecular and histopathological characteristics of these tumors have yet to be clearly defined. PMID:24455418

Talking Glossary of Genetic Terms

MedlinePlus

... Y Z Test Your Knowledge Talking Glossary of Genetic Terms Designed to help learners at any level ... in a reference paper. The Talking Glossary of Genetic Terms The Human Genome Defined by Professionals at ...

A reporter mouse model for in vivo tracing and in vitro molecular studies of melanocytic lineage cells and their diseases.

PubMed

Crawford, Melissa; Leclerc, Valerie; Dagnino, Lina

2017-08-15

Alterations in melanocytic lineage cells give rise to a plethora of distinct human diseases, including neurocristopathies, cutaneous pigmentation disorders, loss of vision and hearing, and melanoma. Understanding the ontogeny and biology of melanocytic cells, as well as how they interact with their surrounding environment, are key steps in the development of therapies for diseases that involve this cell lineage. Efforts to culture and characterize primary melanocytes from normal or genetically engineered mouse models have at times yielded contrasting observations. This is due, in part, to differences in the conditions used to isolate, purify and culture these cells in individual studies. By breeding ROSA mT/mG and Tyr::CreER T2 mice, we generated animals in which melanocytic lineage cells are identified through expression of green fluorescent protein. We also used defined conditions to systematically investigate the proliferation and migration responses of primary melanocytes on various extracellular matrix (ECM) substrates. Under our culture conditions, mouse melanocytes exhibit doubling times in the range of 10 days, and retain exponential proliferative capacity for 50-60 days. In culture, these melanocytes showed distinct responses to different ECM substrates. Specifically, laminin-332 promoted cell spreading, formation of dendrites, random motility and directional migration. In contrast, low or intermediate concentrations of collagen I promoted adhesion and acquisition of a bipolar morphology, and interfered with melanocyte forward movements. Our systematic evaluation of primary melanocyte responses emphasizes the importance of clearly defining culture conditions for these cells. This, in turn, is essential for the interpretation of melanocyte responses to extracellular cues and to understand the molecular basis of disorders involving the melanocytic cell lineage. © 2017. Published by The Company of Biologists Ltd.

A reporter mouse model for in vivo tracing and in vitro molecular studies of melanocytic lineage cells and their diseases

PubMed Central

Crawford, Melissa; Leclerc, Valerie

2017-01-01

ABSTRACT Alterations in melanocytic lineage cells give rise to a plethora of distinct human diseases, including neurocristopathies, cutaneous pigmentation disorders, loss of vision and hearing, and melanoma. Understanding the ontogeny and biology of melanocytic cells, as well as how they interact with their surrounding environment, are key steps in the development of therapies for diseases that involve this cell lineage. Efforts to culture and characterize primary melanocytes from normal or genetically engineered mouse models have at times yielded contrasting observations. This is due, in part, to differences in the conditions used to isolate, purify and culture these cells in individual studies. By breeding ROSAmT/mG and Tyr::CreERT2 mice, we generated animals in which melanocytic lineage cells are identified through expression of green fluorescent protein. We also used defined conditions to systematically investigate the proliferation and migration responses of primary melanocytes on various extracellular matrix (ECM) substrates. Under our culture conditions, mouse melanocytes exhibit doubling times in the range of 10 days, and retain exponential proliferative capacity for 50-60 days. In culture, these melanocytes showed distinct responses to different ECM substrates. Specifically, laminin-332 promoted cell spreading, formation of dendrites, random motility and directional migration. In contrast, low or intermediate concentrations of collagen I promoted adhesion and acquisition of a bipolar morphology, and interfered with melanocyte forward movements. Our systematic evaluation of primary melanocyte responses emphasizes the importance of clearly defining culture conditions for these cells. This, in turn, is essential for the interpretation of melanocyte responses to extracellular cues and to understand the molecular basis of disorders involving the melanocytic cell lineage. PMID:28642245

TGF-β reduces DNA ds-break repair mechanisms to heighten genetic diversity and adaptability of CD44+/CD24- cancer cells.

PubMed

Pal, Debjani; Pertot, Anja; Shirole, Nitin H; Yao, Zhan; Anaparthy, Naishitha; Garvin, Tyler; Cox, Hilary; Chang, Kenneth; Rollins, Fred; Kendall, Jude; Edwards, Leyla; Singh, Vijay A; Stone, Gary C; Schatz, Michael C; Hicks, James; Hannon, Gregory J; Sordella, Raffaella

2017-01-16

Many lines of evidence have indicated that both genetic and non-genetic determinants can contribute to intra-tumor heterogeneity and influence cancer outcomes. Among the best described sub-population of cancer cells generated by non-genetic mechanisms are cells characterized by a CD44+/CD24- cell surface marker profile. Here, we report that human CD44+/CD24- cancer cells are genetically highly unstable because of intrinsic defects in their DNA-repair capabilities. In fact, in CD44+/CD24- cells, constitutive activation of the TGF-beta axis was both necessary and sufficient to reduce the expression of genes that are crucial in coordinating DNA damage repair mechanisms. Consequently, we observed that cancer cells that reside in a CD44+/CD24- state are characterized by increased accumulation of DNA copy number alterations, greater genetic diversity and improved adaptability to drug treatment. Together, these data suggest that the transition into a CD44+/CD24- cell state can promote intra-tumor genetic heterogeneity, spur tumor evolution and increase tumor fitness.

Genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia cell line

PubMed Central

STOCZYNSKA-FIDELUS, EWELINA; PIASKOWSKI, SYLWESTER; PAWLOWSKA, ROZA; SZYBKA, MALGORZATA; PECIAK, JOANNA; HULAS-BIGOSZEWSKA, KRYSTYNA; WINIECKA-KLIMEK, MARTA; RIESKE, PIOTR

2016-01-01

Thorough examination of genetic heterogeneity of cell lines is uncommon. In order to address this issue, the present study analyzed the genetic heterogeneity of RPMI-8402, a T-acute lymphoblastic leukemia (T-ALL) cell line. For this purpose, traditional techniques such as fluorescence in situ hybridization and immunocytochemistry were used, in addition to more advanced techniques, including cell sorting, Sanger sequencing and massive parallel sequencing. The results indicated that the RPMI-8402 cell line consists of several genetically different cell subpopulations. Furthermore, massive parallel sequencing of RPMI-8402 provided insight into the evolution of T-ALL carcinogenesis, since this cell line exhibited the genetic heterogeneity typical of T-ALL. Therefore, the use of cell lines for drug testing in future studies may aid the progress of anticancer drug research. PMID:26870252

Establishment and Characterization of a Highly Tumourigenic and Cancer Stem Cell Enriched Pancreatic Cancer Cell Line as a Well Defined Model System

PubMed Central

Fredebohm, Johannes; Boettcher, Michael; Eisen, Christian; Gaida, Matthias M.; Heller, Anette; Keleg, Shereen; Tost, Jörg; Greulich-Bode, Karin M.; Hotz-Wagenblatt, Agnes; Lathrop, Mark; Giese, Nathalia A.; Hoheisel, Jörg D.

2012-01-01

Standard cancer cell lines do not model the intratumoural heterogeneity situation sufficiently. Clonal selection leads to a homogeneous population of cells by genetic drift. Heterogeneity of tumour cells, however, is particularly critical for therapeutically relevant studies, since it is a prerequisite for acquiring drug resistance and reoccurrence of tumours. Here, we report the isolation of a highly tumourigenic primary pancreatic cancer cell line, called JoPaca-1 and its detailed characterization at multiple levels. Implantation of as few as 100 JoPaca-1 cells into immunodeficient mice gave rise to tumours that were histologically very similar to the primary tumour. The high heterogeneity of JoPaca-1 was reflected by diverse cell morphology and a substantial number of chromosomal aberrations. Comparative whole-genome sequencing of JoPaca-1 and BxPC-3 revealed mutations in genes frequently altered in pancreatic cancer. Exceptionally high expression of cancer stem cell markers and a high clonogenic potential in vitro and in vivo was observed. All of these attributes make this cell line an extremely valuable model to study the biology of and pharmaceutical effects on pancreatic cancer. PMID:23152778

Heritabilities and genetic correlations in the same traits across different strata of herds created according to continuous genomic, genetic, and phenotypic descriptors.

PubMed

Yin, Tong; König, Sven

2018-03-01

The most common approach in dairy cattle to prove genotype by environment interactions is a multiple-trait model application, and considering the same traits in different environments as different traits. We enhanced such concepts by defining continuous phenotypic, genetic, and genomic herd descriptors, and applying random regression sire models. Traits of interest were test-day traits for milk yield, fat percentage, protein percentage, and somatic cell score, considering 267,393 records from 32,707 first-lactation Holstein cows. Cows were born in the years 2010 to 2013, and kept in 52 large-scale herds from 2 federal states of north-east Germany. The average number of genotyped cows per herd (45,613 single nucleotide polymorphism markers per cow) was 133.5 (range: 45 to 415 genotyped cows). Genomic herd descriptors were (1) the level of linkage disequilibrium (r 2 ) within specific chromosome segments, and (2) the average allele frequency for single nucleotide polymorphisms in close distance to a functional mutation. Genetic herd descriptors were the (1) intra-herd inbreeding coefficient, and (2) the percentage of daughters from foreign sires. Phenotypic herd descriptors were (1) herd size, and (2) the herd mean for nonreturn rate. Most correlations among herd descriptors were close to 0, indicating independence of genomic, genetic, and phenotypic characteristics. Heritabilities for milk yield increased with increasing intra-herd linkage disequilibrium, inbreeding, and herd size. Genetic correlations in same traits between adjacent levels of herd descriptors were close to 1, but declined for descriptor levels in greater distance. Genetic correlation declines were more obvious for somatic cell score, compared with test-day traits with larger heritabilities (fat percentage and protein percentage). Also, for milk yield, alterations of herd descriptor levels had an obvious effect on heritabilities and genetic correlations. By trend, multiple trait model results (based on created discrete herd classes) confirmed the random regression estimates. Identified alterations of breeding values in dependency of herd descriptors suggest utilization of specific sires for specific herd structures, offering new possibilities to improve sire selection strategies. Regarding genomic selection designs and genetic gain transfer into commercial herds, cow herds for the utilization in cow training sets should reflect the genomic, genetic, and phenotypic pattern of the broad population. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Sampling scheme on genetic structure of tree species in fragmented tropical dry forest: an evaluation from landscape genetic simulations

Treesearch

Yessica Rico; Marie-Stephanie Samain

2017-01-01

Investigating how genetic variation is distributed across the landscape is fundamental to inform forest conservation and restoration. Detecting spatial genetic discontinuities has value for defining management units, germplasm collection, and target sites for reforestation; however, inappropriate sampling schemes can misidentify patterns of genetic structure....

MDM2 phenotypic and genotypic profiling, respective to TP53 genetic status, in diffuse large B-cell lymphoma patients treated with rituximab-CHOP immunochemotherapy: a report from the International DLBCL Rituximab-CHOP Consortium Program

PubMed Central

Xu-Monette, Zijun Y.; Møller, Michael B.; Tzankov, Alexander; Montes-Moreno, Santiago; Hu, Wenwei; Manyam, Ganiraju C.; Kristensen, Louise; Fan, Lei; Visco, Carlo; Dybkær, Karen; Chiu, April; Tam, Wayne; Zu, Youli; Bhagat, Govind; Richards, Kristy L.; Hsi, Eric D.; Choi, William W. L.; van Krieken, J. Han; Huang, Qin; Huh, Jooryung; Ai, Weiyun; Ponzoni, Maurilio; Ferreri, Andrés J. M.; Wu, Lin; Zhao, Xiaoying; Bueso-Ramos, Carlos E.; Wang, Sa A.; Go, Ronald S.; Li, Yong; Winter, Jane N.; Medeiros, L. Jeffrey

2013-01-01

MDM2 is a key negative regulator of the tumor suppressor p53, however, the prognostic significance of MDM2 overexpression in diffuse large B-cell lymphoma (DLBCL) has not been defined convincingly. In a p53 genetically–defined large cohort of de novo DLBCL patients treated with rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone (R-CHOP) chemotherapy, we assessed MDM2 and p53 expression by immunohistochemistry (n = 478), MDM2 gene amplification by fluorescence in situ hybridization (n = 364), and a single nucleotide polymorphism in the MDM2 promoter, SNP309, by SNP genotyping assay (n = 108). Our results show that MDM2 overexpression, unlike p53 overexpression, is not a significant prognostic factor in overall DLBCL. Both MDM2 and p53 overexpression do not predict for an adverse clinical outcome in patients with wild-type p53 but predicts for significantly poorer survival in patients with mutated p53. Variable p53 activities may ultimately determine the survival differences, as suggested by the gene expression profiling analysis. MDM2 amplification was observed in 3 of 364 (0.8%) patients with high MDM2 expression. The presence of SNP309 did not correlate with MDM2 expression and survival. This study indicates that evaluation of MDM2 and p53 expression correlating with TP53 genetic status is essential to assess their prognostic significance and is important for designing therapeutic strategies that target the MDM2-p53 interaction. PMID:23982177

Ancestry Dependent DNA Methylation and Influence of Maternal Nutrition

PubMed Central

Mozhui, Khyobeni; Smith, Alicia K.; Tylavsky, Frances A.

2015-01-01

There is extensive variation in DNA methylation between individuals and ethnic groups. These differences arise from a combination of genetic and non-genetic influences and potential modifiers include nutritional cues, early life experience, and social and physical environments. Here we compare genome-wide DNA methylation in neonatal cord blood from African American (AA; N = 112) and European American (EA; N = 91) participants of the CANDLE Study (Conditions Affecting Neurocognitive Development and Learning in Early Childhood). Our goal is to determine if there are replicable ancestry-specific methylation patterns that may implicate risk factors for diseases that have differential prevalence between populations. To identify the most robust ancestry-specific CpG sites, we replicate our results in lymphoblastoid cell lines from Yoruba African and CEPH European panels of HapMap. We also evaluate the influence of maternal nutrition—specifically, plasma levels of vitamin D and folate during pregnancy—on methylation in newborns. We define stable ancestry-dependent methylation of genes that include tumor suppressors and cell cycle regulators (e.g., APC, BRCA1, MCC). Overall, there is lower global methylation in African ancestral groups. Plasma levels of 25-hydroxy vitamin D are also considerably lower among AA mothers and about 60% of AA and 40% of EA mothers have concentrations below 20 ng/ml. Using a weighted correlation analysis, we define a network of CpG sites that is jointly modulated by ancestry and maternal vitamin D. Our results show that differences in DNA methylation patterns are remarkably stable and maternal micronutrients can exert an influence on the child epigenome. PMID:25742137

Defining the identity of human adipose-derived mesenchymal stem cells.

PubMed

Montelatici, Elisa; Baluce, Barbara; Ragni, Enrico; Lavazza, Cristiana; Parazzi, Valentina; Mazzola, Riccardo; Cantarella, Giovanna; Brambilla, Massimiliano; Giordano, Rosaria; Lazzari, Lorenza

2015-02-01

Adipose-derived mesenchymal stem cells (ADMSCs) are an ideal population for regenerative medical application. Both the isolation procedure and the culturing conditions are crucial steps, since low yield can limit further cell therapies, especially when minimal adipose tissue harvests are available for cell expansion. To date, a standardized procedure encompassing both isolation sites and expansion methods is missing, thus making the choice of the most appropriate conditions for the preparation of ADMSCs controversial, especially in view of the different applications needed. In this study, we compared the effects of three different commercial media (DMEM, aMEM, and EGM2), routinely used for ADMSCs expansion, and two supplements, FBS and human platelet lysate, recently proven to be an effective alternative to prevent xenogeneic antibody transfer and immune alloresponse in the host. Notably, all the conditions resulted in being safe for ADMSCs isolation and expansion with platelet lysate supplementation giving the highest isolation and proliferation rates, together with a commitment for osteogenic lineage. Then, we proved that the high ADMSC hematopoietic supportive potential is performed through a constant and abundant secretion of both GCSF and SCF. In conclusion, this study further expands the knowledge on ADMSCs, defining their identity definition and offers potential options for in vitro protocols for clinical production, especially related to HSC expansion without use of exogenous cytokines or genetic modifications.

Quantitative Detection of Low-Abundance Transcripts at Single-Cell Level in Human Epidermal Keratinocytes by Digital Droplet Reverse Transcription-Polymerase Chain Reaction.

PubMed

Auvré, Frédéric; Coutier, Julien; Martin, Michèle T; Fortunel, Nicolas O

2018-05-08

Genetic and epigenetic characterization of the large cellular diversity observed within tissues is essential to understanding the molecular networks that ensure the regulation of homeostasis, repair, and regeneration, but also pathophysiological processes. Skin is composed of multiple cell lineages and is therefore fully concerned by this complexity. Even within one particular lineage, such as epidermal keratinocytes, different immaturity statuses or differentiation stages are represented, which are still incompletely characterized. Accordingly, there is presently great demand for methods and technologies enabling molecular investigation at single-cell level. Also, most current methods used to analyze gene expression at RNA level, such as RT-qPCR, do not directly provide quantitative data, but rather comparative ratios between two conditions. A second important need in skin biology is thus to determine the number of RNA molecules in a given cell sample. Here, we describe a workflow that we have set up to meet these specific needs, by means of transcript quantification in cellular micro-samples using flow cytometry sorting and reverse transcription-digital droplet polymerase chain reaction. As a proof-of-principle, the workflow was tested for the detection of transcription factor transcripts expressed at low levels in keratinocyte precursor cells. A linear correlation was found between quantification values and keratinocyte input numbers in a low quantity range from 40 cells to 1 cell. Interpretable signals were repeatedly obtained from single-cell samples corresponding to estimated expression levels as low as 10-20 transcript copies per keratinocyte or less. The present workflow may have broad applications for the detection and quantification of low-abundance nucleic acid species in single cells, opening up perspectives for the study of cell-to-cell genetic and molecular heterogeneity. Interestingly, the process described here does not require internal references such as house-keeping gene expression, as it is initiated with defined cell numbers, precisely sorted by flow cytometry.

Titin Mutations in iPS cells Define Sarcomere Insufficiency as a Cause of Dilated Cardiomyopathy

PubMed Central

Hinson, John T.; Chopra, Anant; Nafissi, Navid; Polacheck, William J.; Benson, Craig C.; Swist, Sandra; Gorham, Joshua; Yang, Luhan; Schafer, Sebastian; Sheng, Calvin C.; Haghighi, Alireza; Homsy, Jason; Hubner, Norbert; Church, George; Cook, Stuart A.; Linke, Wolfgang A.; Chen, Christopher S.; Seidman, J. G.; Seidman, Christine E.

2015-01-01

Human mutations that truncate the massive sarcomere protein titin (TTNtv) are the most common genetic cause for dilated cardiomyopathy (DCM), a major cause of heart failure and premature death. Here we show that cardiac microtissues engineered from human induced pluripotent stem (iPS) cells are a powerful system for evaluating the pathogenicity of titin gene variants. We found that certain missense mutations, like TTNtv, diminish contractile performance and are pathogenic. By combining functional analyses with RNAseq, we explain why truncations in the A-band domain of TTN cause DCM while truncations in the I-band are better tolerated. Finally, we demonstrate that mutant titin protein in iPS-cardiomyocytes results in sarcomere insufficiency, impaired responses to mechanical and β-adrenergic stress, and attenuated growth factor and cell signaling activation. Our findings indicate that titin mutations cause DCM by disrupting critical linkages between sarcomerogenesis and adaptive remodelling. PMID:26315439

Evaluating Risks of Insertional Mutagenesis by DNA Transposons in Gene Therapy

PubMed Central

Hackett, Perry B.; Largaespada, David A.; Switzer, Kirsten C.; Cooper, Laurence J.N.

2013-01-01

Investigational therapy can be successfully undertaken using viral- and non-viral-mediated ex vivo gene transfer. Indeed, recent clinical trials have established the potential for genetically modified T cells to improve and restore health. Recently the Sleeping Beauty (SB) transposon/transposase system has been applied in clinical trials to stably insert a chimeric antigen receptor (CAR) to redirect T-cell specificity. We discuss the context in which the SB system can be harnessed for gene therapy and describe the human application of SB-modified CAR+ T cells. We have focused on theoretical issues relating to insertional mutagenesis in the context of human genomes that are naturally subjected to remobilization of transposons and the experimental evidence over the last decade of employing SB transposons for defining genes that induce cancer. These findings are put into the context of the use of SB transposons in the treatment of human disease. PMID:23313630

Eating on the fly: function and regulation of autophagy during cell growth, survival and death in Drosophila.

PubMed

Neufeld, Thomas P; Baehrecke, Eric H

2008-07-01

Significant progress has been made over recent years in defining the normal progression and regulation of autophagy, particularly in cultured mammalian cells and yeast model systems. However, apart from a few notable exceptions, our understanding of the physiological roles of autophagy has lagged behind these advances, and identification of components and features of autophagy unique to higher eukaryotes also remains a challenge. In this review we describe recent insights into the roles and control mechanisms of autophagy gained from in vivo studies in Drosophila. We focus on potential roles of autophagy in controlling cell growth and death, and describe how the regulation of autophagy has evolved to include metazoan-specific signaling pathways. We discuss genetic screening approaches that are being used to identify novel regulators and effectors of autophagy, and speculate about areas of research in this system likely to bear fruit in future studies.

Mechanisms of bacterial DNA replication restart

PubMed Central

Windgassen, Tricia A; Wessel, Sarah R; Bhattacharyya, Basudeb

2018-01-01

Abstract Multi-protein DNA replication complexes called replisomes perform the essential process of copying cellular genetic information prior to cell division. Under ideal conditions, replisomes dissociate only after the entire genome has been duplicated. However, DNA replication rarely occurs without interruptions that can dislodge replisomes from DNA. Such events produce incompletely replicated chromosomes that, if left unrepaired, prevent the segregation of full genomes to daughter cells. To mitigate this threat, cells have evolved ‘DNA replication restart’ pathways that have been best defined in bacteria. Replication restart requires recognition and remodeling of abandoned replication forks by DNA replication restart proteins followed by reloading of the replicative DNA helicase, which subsequently directs assembly of the remaining replisome subunits. This review summarizes our current understanding of the mechanisms underlying replication restart and the proteins that drive the process in Escherichia coli (PriA, PriB, PriC and DnaT). PMID:29202195

Cancer Stem Cell Theory and the Warburg Effect, Two Sides of the Same Coin?

PubMed Central

Pacini, Nicola; Borziani, Fabio

2014-01-01

Over the last 100 years, many studies have been performed to determine the biochemical and histopathological phenomena that mark the origin of neoplasms. At the end of the last century, the leading paradigm, which is currently well rooted, considered the origin of neoplasms to be a set of genetic and/or epigenetic mutations, stochastic and independent in a single cell, or rather, a stochastic monoclonal pattern. However, in the last 20 years, two important areas of research have underlined numerous limitations and incongruities of this pattern, the hypothesis of the so-called cancer stem cell theory and a revaluation of several alterations in metabolic networks that are typical of the neoplastic cell, the so-called Warburg effect. Even if this specific “metabolic sign” has been known for more than 85 years, only in the last few years has it been given more attention; therefore, the so-called Warburg hypothesis has been used in multiple and independent surveys. Based on an accurate analysis of a series of considerations and of biophysical thermodynamic events in the literature, we will demonstrate a homogeneous pattern of the cancer stem cell theory, of the Warburg hypothesis and of the stochastic monoclonal pattern; this pattern could contribute considerably as the first basis of the development of a new uniform theory on the origin of neoplasms. Thus, a new possible epistemological paradigm is represented; this paradigm considers the Warburg effect as a specific “metabolic sign” reflecting the stem origin of the neoplastic cell, where, in this specific metabolic order, an essential reason for the genetic instability that is intrinsic to the neoplastic cell is defined. PMID:24857919

Cancer stem cell theory and the warburg effect, two sides of the same coin?

PubMed

Pacini, Nicola; Borziani, Fabio

2014-05-19

Over the last 100 years, many studies have been performed to determine the biochemical and histopathological phenomena that mark the origin of neoplasms. At the end of the last century, the leading paradigm, which is currently well rooted, considered the origin of neoplasms to be a set of genetic and/or epigenetic mutations, stochastic and independent in a single cell, or rather, a stochastic monoclonal pattern. However, in the last 20 years, two important areas of research have underlined numerous limitations and incongruities of this pattern, the hypothesis of the so-called cancer stem cell theory and a revaluation of several alterations in metabolic networks that are typical of the neoplastic cell, the so-called Warburg effect. Even if this specific "metabolic sign" has been known for more than 85 years, only in the last few years has it been given more attention; therefore, the so-called Warburg hypothesis has been used in multiple and independent surveys. Based on an accurate analysis of a series of considerations and of biophysical thermodynamic events in the literature, we will demonstrate a homogeneous pattern of the cancer stem cell theory, of the Warburg hypothesis and of the stochastic monoclonal pattern; this pattern could contribute considerably as the first basis of the development of a new uniform theory on the origin of neoplasms. Thus, a new possible epistemological paradigm is represented; this paradigm considers the Warburg effect as a specific "metabolic sign" reflecting the stem origin of the neoplastic cell, where, in this specific metabolic order, an essential reason for the genetic instability that is intrinsic to the neoplastic cell is defined.

Cloned ferrets produced by somatic cell nuclear transfer

PubMed Central

Li, Ziyi; Sun, Xingshen; Chen, Juan; Liu, Xiaoming; Wisely, Samantha M.; Zhou, Qi; Renard, Jean-Paul; Leno, Gregory H.; Engelhardt, John F.

2007-01-01

Somatic cell nuclear transfer (SCNT) offers great potential for developing better animal models of human disease. The domestic ferret (Mustela putorius furo) is an ideal animal model for influenza infections and potentially other human respiratory diseases such as cystic fibrosis, where mouse models have failed to reproduce the human disease phenotype. Here, we report the successful production of live cloned, reproductively competent, ferrets using species-specific SCNT methodologies. Critical to developing a successful SCNT protocol for the ferret was the finding that hormonal treatment, normally used for superovulation, adversely affected the developmental potential of recipient oocytes. The onset of Oct4 expression was delayed and incomplete in parthenogenetically activated oocytes collected from hormone-treated females relative to oocytes collected from females naturally mated with vasectomized males. Stimulation induced by mating and in vitro oocyte maturation produced the optimal oocyte recipient for SCNT. Although nuclear injection and cell fusion produced mid-term fetuses at equivalent rates (~3–4%), only cell fusion gave rise to healthy surviving clones. Single cell fusion rates and the efficiency of SCNT were also enhanced by placing two somatic cells into the perivitelline space. These species-specific modifications facilitated the birth of live, healthy, and fertile cloned ferrets. The development of microsatellite genotyping for domestic ferrets confirmed that ferret clones were genetically derived from their respective somatic cells and unrelated to their surrogate mother. With this technology, it is now feasible to begin generating genetically defined ferrets for studying transmissible and inherited human lung diseases. Cloning of the domestic ferret may also aid in recovery and conservation of the endangered black-footed ferret and European mink. PMID:16584722

Beyond society: the evolution of organismality

PubMed Central

Queller, David C.; Strassmann, Joan E.

2009-01-01

The evolution of organismality is a social process. All organisms originated from groups of simpler units that now show high cooperation among the parts and are nearly free of conflicts. We suggest that this near-unanimous cooperation be taken as the defining trait of organisms. Consistency then requires that we accept some unconventional organisms, including some social insect colonies, some microbial groups and viruses, a few sexual partnerships and a number of mutualistic associations. Whether we call these organisms or not, a major task is to explain such cooperative entities, and our survey suggests that many of the traits commonly used to define organisms are not essential. These non-essential traits include physical contiguity, indivisibility, clonality or high relatedness, development from a single cell, short-term and long-term genetic cotransmission, germ–soma separation and membership in the same species. PMID:19805423

The underlying pathway structure of biochemical reaction networks

PubMed Central

Schilling, Christophe H.; Palsson, Bernhard O.

1998-01-01

Bioinformatics is yielding extensive, and in some cases complete, genetic and biochemical information about individual cell types and cellular processes, providing the composition of living cells and the molecular structure of its components. These components together perform integrated cellular functions that now need to be analyzed. In particular, the functional definition of biochemical pathways and their role in the context of the whole cell is lacking. In this study, we show how the mass balance constraints that govern the function of biochemical reaction networks lead to the translation of this problem into the realm of linear algebra. The functional capabilities of biochemical reaction networks, and thus the choices that cells can make, are reflected in the null space of their stoichiometric matrix. The null space is spanned by a finite number of basis vectors. We present an algorithm for the synthesis of a set of basis vectors for spanning the null space of the stoichiometric matrix, in which these basis vectors represent the underlying biochemical pathways that are fundamental to the corresponding biochemical reaction network. In other words, all possible flux distributions achievable by a defined set of biochemical reactions are represented by a linear combination of these basis pathways. These basis pathways thus represent the underlying pathway structure of the defined biochemical reaction network. This development is significant from a fundamental and conceptual standpoint because it yields a holistic definition of biochemical pathways in contrast to definitions that have arisen from the historical development of our knowledge about biochemical processes. Additionally, this new conceptual framework will be important in defining, characterizing, and studying biochemical pathways from the rapidly growing information on cellular function. PMID:9539712

Control of plant stem cell function by conserved interacting transcriptional regulators

PubMed Central

Zhou, Yun; Liu, Xing; Engstrom, Eric M.; Nimchuk, Zachary L.; Pruneda-Paz, Jose L.; Tarr, Paul T.; Yan, An; Kay, Steve A.; Meyerowitz, Elliot M.

2014-01-01

SUMMARY Plant stem cells in the shoot apical meristem (SAM) and root apical meristem (RAM) provide for postembryonic development of above-ground tissues and roots, respectively, while secondary vascular stem cells sustain vascular development1–4. WUSCHEL (WUS), a homeodomain transcription factor expressed in the rib meristem of the SAM, is a key regulatory factor controlling stem cell populations in the Arabidopsis SAM5–6 and is thought to establish the shoot stem cell niche via a feedback circuit with the CLAVATA3 (CLV3) peptide signaling pathway7. WUSCHEL-RELATED HOMEOBOX5 (WOX5), specifically expressed in root quiescent center (QC), defines QC identity and functions interchangeably with WUS in control of shoot and root stem cell niches8. WOX4, expressed in Arabidopsis procambial cells, defines the vascular stem cell niche9–11. WUS/WOX family proteins are evolutionarily and functionally conserved throughout the plant kingdom12 and emerge as key actors in the specification and maintenance of stem cells within all meristems13. However, the nature of the genetic regime in stem cell niches that centers on WOX gene function has been elusive, and molecular links underlying conserved WUS/WOX function in stem cell niches remain unknown. Here we demonstrate that the Arabidopsis HAIRY MERISTEM (HAM)family transcription regulators act as conserved interacting co-factors with WUS/WOX proteins. HAM and WUS share common targets in vivo and their physical interaction is important in driving downstream transcriptional programs and in promoting shoot stem cell proliferation. Differences in the overlapping expression patterns of WOX and HAM family members underlie the formation of diverse stem cell niche locations, and the HAM family is essential for all of these stem cell niches. These findings establish a new framework for the control of stem cell production during plant development. PMID:25363783

General Intelligence (g): Overview of a Complex Construct and Its Implications for Genetics Research.

PubMed

Plucker, Jonathan A; Shelton, Amy L

2015-01-01

Current technology has dramatically increased the prevalence of studies to establish the genetic correlates of a wide variety of human characteristics, including not only the physical attributes that determine what we look like and the risk of physiological disease but also the psychological and cognitive characteristics that often define who we are as individuals. Perhaps one of the most deeply personal and often controversial characteristics is the concept of general intelligence, known in the psychological literature as "g." As with the genetic study of any complex trait, the first step in studying the genetics of g is to carefully define the characteristic of interest. For g, this entails establishing what intelligence means and providing a clear operational definition for how it will be measured. In this paper, we provide a brief historical and theoretical overview of the construct of general intelligence, describe its relationship to the contemporary measurement of intelligence, and discuss these concepts in light of the challenges associated with defining g as a characteristic in the study of genetics. © 2015 The Hastings Center.

GENETICS AND POPULATION-LEVEL RISK ASSESSMENT

EPA Science Inventory

Genetic variation defines population structure and provides the mechanism for populations to adapt to novel stressors. Despite its fundamental importance in understanding populations, genetic information has been included rarely in models of population dynamics (endangered speci...

Defining the cellular lineage hierarchy in the interfollicular epidermis of adult skin.

PubMed

Sada, Aiko; Jacob, Fadi; Leung, Eva; Wang, Sherry; White, Brian S; Shalloway, David; Tumbar, Tudorita

2016-06-01

The interfollicular epidermis regenerates from heterogeneous basal skin cell populations that divide at different rates. It has previously been presumed that infrequently dividing basal cells known as label-retaining cells (LRCs) are stem cells, whereas non-LRCs are short-lived progenitors. Here we employ the H2B-GFP pulse-chase system in adult mouse skin and find that epidermal LRCs and non-LRCs are molecularly distinct and can be differentiated by Dlx1(CreER) and Slc1a3(CreER) genetic marking, respectively. Long-term lineage tracing and mathematical modelling of H2B-GFP dilution data show that LRCs and non-LRCs constitute two distinct stem cell populations with different patterns of proliferation, differentiation and upward cellular transport. During homeostasis, these populations are enriched in spatially distinct skin territories and can preferentially produce unique differentiated lineages. On wounding or selective killing, they can temporarily replenish each other's territory. These two discrete interfollicular stem cell populations are functionally interchangeable and intrinsically well adapted to thrive in distinct skin environments.

ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma

PubMed Central

Ndoye, Abibatou; Budina-Kolomets, Anna; Kugel, Curtis H.; Webster, Marie; Kaur, Amanpreet; Behera, Reeti; Rebecca, Vito; Li, Ling; Brafford, Patricia; Liu, Qin; Gopal, Y.N. Vashisht; Davies, Michael A.; Mills, Gordon B.; Xu, Xiaowei; Wu, Hong; Herlyn, Meenhard; Nicastri, Michael; Winkler, Jeffrey; Soengas, Maria S.; Amaravadi, Ravi; Murphy, Maureen; Weeraratna, Ashani T.

2017-01-01

Autophagy mediates resistance to various anticancer agents. In melanoma, resistance to targeted therapy has been linked to expression of Wnt5A, an intrinsic inhibitor of β-catenin, which also promotes invasion. In this study, we assessed the interplay between Wnt5A and autophagy by combining expression studies in human clinical biopsies with functional analyses in cell lines and mouse models. Melanoma cells with high Wnt5A and low β-catenin displayed increased basal autophagy. Genetic blockade of autophagy revealed an unexpected feedback loop whereby knocking down the autophagy factor ATG5 in Wnt5Ahigh cells decreased Wnt5A and increased β-catenin. To define the physiological relevance of this loop, melanoma cells with different Wnt status were treated in vitro and in vivo with the potent lysosomotropic compound Lys05. Wnt5Ahigh cells were less sensitive to Lys05 and could be reverted by inducing β-catenin activity. Our results suggest the efficacy of autophagy inhibitors might be improved by taking the Wnt signature of melanoma cells into account. PMID:28887323

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

PubMed Central

Dewari, Pooran Singh; Southgate, Benjamin; Mccarten, Katrina; Monogarov, German; O'Duibhir, Eoghan; Quinn, Niall; Tyrer, Ashley; Leitner, Marie-Christin; Plumb, Colin; Kalantzaki, Maria; Blin, Carla; Finch, Rebecca; Bressan, Raul Bardini; Morrison, Gillian; Jacobi, Ashley M; Behlke, Mark A; von Kriegsheim, Alex; Tomlinson, Simon; Krijgsveld, Jeroen

2018-01-01

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5–30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells. PMID:29638216

Differential Activation of Acid Sphingomyelinase and Ceramide Release Determines Invasiveness of Neisseria meningitidis into Brain Endothelial Cells

PubMed Central

Simonis, Alexander; Hebling, Sabrina; Gulbins, Erich; Schneider-Schaulies, Sibylle; Schubert-Unkmeir, Alexandra

2014-01-01

The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains. PMID:24945304

Overcoming reprogramming resistance of Fanconi anemia cells

PubMed Central

Müller, Lars U. W.; Milsom, Michael D.; Harris, Chad E.; Vyas, Rutesh; Brumme, Kristina M.; Parmar, Kalindi; Moreau, Lisa A.; Schambach, Axel; Park, In-Hyun; London, Wendy B.; Strait, Kelly; Schlaeger, Thorsten; DeVine, Alexander L.; Grassman, Elke; D'Andrea, Alan; Daley, George Q.

2012-01-01

Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal BM failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and protecting cells from the DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells, but previous attempts at gene therapy have failed largely because of inadequate numbers of hematopoietic stem cells available for gene correction. Induced pluripotent stem cells (iPSCs) constitute an alternate source of autologous cells that are amenable to ex vivo expansion, genetic correction, and molecular characterization. In the present study, we demonstrate that reprogramming leads to activation of the FA pathway, increased DNA double-strand breaks, and senescence. We also demonstrate that defects in the FA DNA-repair pathway decrease the reprogramming efficiency of murine and human primary cells. FA pathway complementation reduces senescence and restores the reprogramming efficiency of somatic FA cells to normal levels. Disease-specific iPSCs derived in this fashion maintain a normal karyotype and are capable of hematopoietic differentiation. These data define the role of the FA pathway in reprogramming and provide a strategy for future translational applications of patient-specific FA iPSCs. PMID:22371882

Thyroid Hormone Differentially Modulates Warburg Phenotype in Breast Cancer Cells

PubMed Central

Suhane, Sonal; Ramanujan, V Krishnan

2011-01-01

Sustenance of cancer cells in vivo critically depends on a variety of genetic and metabolic adaptations. Aerobic glycolysis or Warburg effect has been a defining biochemical hallmark of transformed cells for more than five decades although a clear molecular basis of this observation is emerging only in recent years. In this study, we present our findings that thyroid hormone exerts its non-genomic and genomic actions in two model human breast cancer cell lines differentially. By laying a clear foundation for experimentally monitoring the Warburg phenotype in living cancer cells, we demonstrate that thyroid hormone-induced modulation of bioenergetic profiles in these two model cell lines depends on the degree of Warburg phenotype that they display. Further we also show that thyroid hormone can sensitize mitochondria in aggressive, triple-negative breast cancer cells favorably to increase the chemotherapeutic efficacy in these cells. Even though the role of thyroid hormone in modulating mitochondrial metabolism has been known, the current study accentuates the critical role it plays in modulating Warburg phenotype in breast cancer cells. The clinical significance of this finding is the possibility to devise strategies for metabolically modulating aggressive triple-negative tumors so as to enhance their chemosensitivity in vivo. PMID:21945435

Collective Genetic Interaction Effects and the Role of Antigen Presenting Cells in Autoimmune Diseases

DTIC Science & Technology

2017-01-12

RESEARCH ARTICLE Collective Genetic Interaction Effects and the Role of Antigen-Presenting Cells in Autoimmune Diseases Hyung Jun Woo*, Chenggang Yu...autoimmunity. Genetic predispositions center around the major histocompatibility complex (MHC) class II loci involved in antigen presentation, the key...helper and regulatory T cells showing strong dis- ease-associated interactions with B cells. Our results provide direct genetic evidence point- ing to

Period doubling induced by thermal noise amplification in genetic circuits

PubMed Central

Ruocco, G.; Fratalocchi, A.

2014-01-01

Rhythms of life are dictated by oscillations, which take place in a wide rage of biological scales. In bacteria, for example, oscillations have been proven to control many fundamental processes, ranging from gene expression to cell divisions. In genetic circuits, oscillations originate from elemental block such as autorepressors and toggle switches, which produce robust and noise-free cycles with well defined frequency. In some circumstances, the oscillation period of biological functions may double, thus generating bistable behaviors whose ultimate origin is at the basis of intense investigations. Motivated by brain studies, we here study an “elemental” genetic circuit, where a simple nonlinear process interacts with a noisy environment. In the proposed system, nonlinearity naturally arises from the mechanism of cooperative stability, which regulates the concentration of a protein produced during a transcription process. In this elemental model, bistability results from the coherent amplification of environmental fluctuations due to a stochastic resonance of nonlinear origin. This suggests that the period doubling observed in many biological functions might result from the intrinsic interplay between nonlinearity and thermal noise. PMID:25404210

Francis Galton: and eugenics today.

PubMed Central

Galton, D J; Galton, C J

1998-01-01

Eugenics can be defined as the use of science applied to the qualitative and quantitative improvement of the human genome. The subject was initiated by Francis Galton with considerable support from Charles Darwin in the latter half of the 19th century. Its scope has increased enormously since the recent revolution in molecular genetics. Genetic files can be easily obtained for individuals either antenatally or at birth; somatic gene therapy has been introduced for some rare inborn errors of metabolism; and gene manipulation of human germ-line cells will no doubt occur in the near future to generate organs for transplantation. The past history of eugenics has been appalling, with gross abuses in the USA between 1931 and 1945 when compulsory sterilization was practised; and in Germany between 1933 and 1945 when mass extermination and compulsory sterilization were performed. To prevent such abuses in the future statutory bodies, such as a genetics commission, should be established to provide guidance and rules of conduct for use of the new information and technologies as applied to the human genome. PMID:9602996

Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

PubMed Central

Vizeacoumar, Franco J.; van Dyk, Nydia; S.Vizeacoumar, Frederick; Cheung, Vincent; Li, Jingjing; Sydorskyy, Yaroslav; Case, Nicolle; Li, Zhijian; Datti, Alessandro; Nislow, Corey; Raught, Brian; Zhang, Zhaolei; Frey, Brendan; Bloom, Kerry

2010-01-01

We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions. PMID:20065090

Period doubling induced by thermal noise amplification in genetic circuits.

PubMed

Ruocco, G; Fratalocchi, A

2014-11-18

Rhythms of life are dictated by oscillations, which take place in a wide rage of biological scales. In bacteria, for example, oscillations have been proven to control many fundamental processes, ranging from gene expression to cell divisions. In genetic circuits, oscillations originate from elemental block such as autorepressors and toggle switches, which produce robust and noise-free cycles with well defined frequency. In some circumstances, the oscillation period of biological functions may double, thus generating bistable behaviors whose ultimate origin is at the basis of intense investigations. Motivated by brain studies, we here study an "elemental" genetic circuit, where a simple nonlinear process interacts with a noisy environment. In the proposed system, nonlinearity naturally arises from the mechanism of cooperative stability, which regulates the concentration of a protein produced during a transcription process. In this elemental model, bistability results from the coherent amplification of environmental fluctuations due to a stochastic resonance of nonlinear origin. This suggests that the period doubling observed in many biological functions might result from the intrinsic interplay between nonlinearity and thermal noise.

Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer.

PubMed

Mouradov, Dmitri; Sloggett, Clare; Jorissen, Robert N; Love, Christopher G; Li, Shan; Burgess, Antony W; Arango, Diego; Strausberg, Robert L; Buchanan, Daniel; Wormald, Samuel; O'Connor, Liam; Wilding, Jennifer L; Bicknell, David; Tomlinson, Ian P M; Bodmer, Walter F; Mariadason, John M; Sieber, Oliver M

2014-06-15

Human colorectal cancer cell lines are used widely to investigate tumor biology, experimental therapy, and biomarkers. However, to what extent these established cell lines represent and maintain the genetic diversity of primary cancers is uncertain. In this study, we profiled 70 colorectal cancer cell lines for mutations and DNA copy number by whole-exome sequencing and SNP microarray analyses, respectively. Gene expression was defined using RNA-Seq. Cell line data were compared with those published for primary colorectal cancers in The Cancer Genome Atlas. Notably, we found that exome mutation and DNA copy-number spectra in colorectal cancer cell lines closely resembled those seen in primary colorectal tumors. Similarities included the presence of two hypermutation phenotypes, as defined by signatures for defective DNA mismatch repair and DNA polymerase ε proofreading deficiency, along with concordant mutation profiles in the broadly altered WNT, MAPK, PI3K, TGFβ, and p53 pathways. Furthermore, we documented mutations enriched in genes involved in chromatin remodeling (ARID1A, CHD6, and SRCAP) and histone methylation or acetylation (ASH1L, EP300, EP400, MLL2, MLL3, PRDM2, and TRRAP). Chromosomal instability was prevalent in nonhypermutated cases, with similar patterns of chromosomal gains and losses. Although paired cell lines derived from the same tumor exhibited considerable mutation and DNA copy-number differences, in silico simulations suggest that these differences mainly reflected a preexisting heterogeneity in the tumor cells. In conclusion, our results establish that human colorectal cancer lines are representative of the main subtypes of primary tumors at the genomic level, further validating their utility as tools to investigate colorectal cancer biology and drug responses. ©2014 American Association for Cancer Research.

Single tube genotyping of sickle cell anaemia using PCR-based SNP analysis

PubMed Central

Waterfall, Christy M.; Cobb, Benjamin D.

2001-01-01

Allele-specific amplification (ASA) is a generally applicable technique for the detection of known single nucleotide polymorphisms (SNPs), deletions, insertions and other sequence variations. Conventionally, two reactions are required to determine the zygosity of DNA in a two-allele system, along with significant upstream optimisation to define the specific test conditions. Here, we combine single tube bi-directional ASA with a ‘matrix-based’ optimisation strategy, speeding up the whole process in a reduced reaction set. We use sickle cell anaemia as our model SNP system, a genetic disease that is currently screened using ASA methods. Discriminatory conditions were rapidly optimised enabling the unambiguous identification of DNA from homozygous sickle cell patients (HbS/S), heterozygous carriers (HbA/S) or normal DNA in a single tube. Simple downstream mathematical analyses based on product yield across the optimisation set allow an insight into the important aspects of priming competition and component interactions in this competitive PCR. This strategy can be applied to any polymorphism, defining specific conditions using a multifactorial approach. The inherent simplicity and low cost of this PCR-based method validates bi-directional ASA as an effective tool in future clinical screening and pharmacogenomic research where more expensive fluorescence-based approaches may not be desirable. PMID:11726702

Single tube genotyping of sickle cell anaemia using PCR-based SNP analysis.

PubMed

Waterfall, C M; Cobb, B D

2001-12-01

Allele-specific amplification (ASA) is a generally applicable technique for the detection of known single nucleotide polymorphisms (SNPs), deletions, insertions and other sequence variations. Conventionally, two reactions are required to determine the zygosity of DNA in a two-allele system, along with significant upstream optimisation to define the specific test conditions. Here, we combine single tube bi-directional ASA with a 'matrix-based' optimisation strategy, speeding up the whole process in a reduced reaction set. We use sickle cell anaemia as our model SNP system, a genetic disease that is currently screened using ASA methods. Discriminatory conditions were rapidly optimised enabling the unambiguous identification of DNA from homozygous sickle cell patients (HbS/S), heterozygous carriers (HbA/S) or normal DNA in a single tube. Simple downstream mathematical analyses based on product yield across the optimisation set allow an insight into the important aspects of priming competition and component interactions in this competitive PCR. This strategy can be applied to any polymorphism, defining specific conditions using a multifactorial approach. The inherent simplicity and low cost of this PCR-based method validates bi-directional ASA as an effective tool in future clinical screening and pharmacogenomic research where more expensive fluorescence-based approaches may not be desirable.

Natural genetic variation profoundly regulates gene expression in immune cells and dictates susceptibility to CNS autoimmunity

PubMed Central

Bearoff, Frank; del Rio, Roxana; Case, Laure K.; Dragon, Julie A.; Nguyen-Vu, Trang; Lin, Chin-Yo; Blankenhorn, Elizabeth P.; Teuscher, Cory; Krementsov, Dimitry N.

2016-01-01

Regulation of gene expression in immune cells is known to be under genetic control, and likely contributes to susceptibility to autoimmune diseases, such as multiple sclerosis (MS). How this occurs in concert across multiple immune cell types is poorly understood. Using a mouse model that harnesses the genetic diversity of wild-derived mice, more accurately reflecting genetically diverse human populations, we provide an extensive characterization of the genetic regulation of gene expression in five different naïve immune cell types relevant to MS. The immune cell transcriptome is shown to be under profound genetic control, exhibiting diverse patterns: global, cell-specific, and sex-specific. Bioinformatic analysis of the genetically-controlled transcript networks reveals reduced cell type-specificity and inflammatory activity in wild-derived PWD/PhJ mice, compared with the conventional laboratory strain C57BL/6J. Additionally, candidate MS-GWAS genes were significantly enriched among transcripts overrepresented in C57BL/6J cells compared to PWD. These expression level differences correlate with robust differences in susceptibility to experimental autoimmune encephalomyelitis, the principal model of MS, and skewing of the encephalitogenic T cell responses. Taken together, our results provide functional insights into the genetic regulation of the immune transcriptome, and shed light on how this in turn contributes to susceptibility to autoimmune disease. PMID:27653816

Celebrating the 30th anniversary of genetic epidemiology: how to define our scope?

PubMed

Ziegler, Andreas; König, Inke R

2014-07-01

We review the scope of the scientific discipline genetic epidemiology by considering the steps of genetic epidemiologic research. Starting from the classical definition of genetic epidemiology as provided by Morton and Chung [1978, ISBN-13: 9780125080507], we propose a slightly modernized definition of the term genetic epidemiology. © 2014 WILEY PERIODICALS, INC.

Knowledge-making distinctions in synthetic biology.

PubMed

O'Malley, Maureen A; Powell, Alexander; Davies, Jonathan F; Calvert, Jane

2008-01-01

Synthetic biology is an increasingly high-profile area of research that can be understood as encompassing three broad approaches towards the synthesis of living systems: DNA-based device construction, genome-driven cell engineering and protocell creation. Each approach is characterized by different aims, methods and constructs, in addition to a range of positions on intellectual property and regulatory regimes. We identify subtle but important differences between the schools in relation to their treatments of genetic determinism, cellular context and complexity. These distinctions tie into two broader issues that define synthetic biology: the relationships between biology and engineering, and between synthesis and analysis. These themes also illuminate synthetic biology's connections to genetic and other forms of biological engineering, as well as to systems biology. We suggest that all these knowledge-making distinctions in synthetic biology raise fundamental questions about the nature of biological investigation and its relationship to the construction of biological components and systems. (c) 2007 Wiley Periodicals, Inc.

KATP Channels in the Cardiovascular System

PubMed Central

Foster, Monique N.; Coetzee, William A.

2015-01-01

KATP channels are integral to the functions of many cells and tissues. The use of electrophysiological methods has allowed for a detailed characterization of KATP channels in terms of their biophysical properties, nucleotide sensitivities, and modification by pharmacological compounds. However, even though they were first described almost 25 years ago (Noma 1983, Trube and Hescheler 1984), the physiological and pathophysiological roles of these channels, and their regulation by complex biological systems, are only now emerging for many tissues. Even in tissues where their roles have been best defined, there are still many unanswered questions. This review aims to summarize the properties, molecular composition, and pharmacology of KATP channels in various cardiovascular components (atria, specialized conduction system, ventricles, smooth muscle, endothelium, and mitochondria). We will summarize the lessons learned from available genetic mouse models and address the known roles of KATP channels in cardiovascular pathologies and how genetic variation in KATP channel genes contribute to human disease. PMID:26660852